mgtnp atp  (Jena Bioscience)


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  • 93
    Name:
    TNP ATP
    Description:

    Catalog Number:
    NU-221L
    Price:
    369.6
    Category:
    Nucleotides Nucleosides
    Size:
    5 x 200 µl
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    Structured Review

    Jena Bioscience mgtnp atp
    Examples of fluorescence stopped-flow traces showing the kinetics of <t>MgTNP-ATP</t> binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

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    Images

    1) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    2) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    3) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    4) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    5) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    6) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    7) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    8) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    9) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    10) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    11) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    12) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    13) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    14) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    15) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    16) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    17) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    18) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    19) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    20) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    21) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    22) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    23) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    24) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    25) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    26) Product Images from "Nucleotide inhibition of the pancreatic ATP-sensitive K+ channel explored with patch-clamp fluorometry"

    Article Title: Nucleotide inhibition of the pancreatic ATP-sensitive K+ channel explored with patch-clamp fluorometry

    Journal: bioRxiv

    doi: 10.1101/803999

    Simultaneous measurements of nucleotide binding and channel current. A. Brightfieid and fluorescence images of a patch pipette and excised, inside out patch expressing Kir6.2*-GFP + SUR1, with the location of the centre of the spectrometer slit overlaid as a white, vertical line. B. Current (left) and spectra (right) acquired from the same excised, inside-out patch exposed to TNP-ATP and coloured according to concentration. C. Concentration-response (n = 9) for TNP-ATP inhibition of Kir6.2*-GFP + SUR1 currents ( I / I max ) and for quenching of ANAP fluorescence ( F / F max ). Both current inhibition and fluorescence quenching were fit to the Hill equation. Current inhibition: IC 50 = 6.23μΜ, h = 0.92, I max = 0.96, fluorescence quenching: EC 50 = 77.7 μΜ, h = 0.87, E max = 1.00. D. The same data as in C fit to an MWC-type model. Solid curves represent the median fit; shaded areas represent the 95% quantile interval. Values for the fits are reported in the text and in Table 3 . E. MWC-type model for inhibition of K ATP by nucleotides. Open subunits are shown as circles; closed are shown as squares. Nucleotide-bound subunits are represented by filled symbols. L, D , and K A are defined in the text. F. Posterior probability distributions for the MWC-type model generated by MCMC fits to the data in C overlaid on the prior probability distribution (dashed line) for each parameter.
    Figure Legend Snippet: Simultaneous measurements of nucleotide binding and channel current. A. Brightfieid and fluorescence images of a patch pipette and excised, inside out patch expressing Kir6.2*-GFP + SUR1, with the location of the centre of the spectrometer slit overlaid as a white, vertical line. B. Current (left) and spectra (right) acquired from the same excised, inside-out patch exposed to TNP-ATP and coloured according to concentration. C. Concentration-response (n = 9) for TNP-ATP inhibition of Kir6.2*-GFP + SUR1 currents ( I / I max ) and for quenching of ANAP fluorescence ( F / F max ). Both current inhibition and fluorescence quenching were fit to the Hill equation. Current inhibition: IC 50 = 6.23μΜ, h = 0.92, I max = 0.96, fluorescence quenching: EC 50 = 77.7 μΜ, h = 0.87, E max = 1.00. D. The same data as in C fit to an MWC-type model. Solid curves represent the median fit; shaded areas represent the 95% quantile interval. Values for the fits are reported in the text and in Table 3 . E. MWC-type model for inhibition of K ATP by nucleotides. Open subunits are shown as circles; closed are shown as squares. Nucleotide-bound subunits are represented by filled symbols. L, D , and K A are defined in the text. F. Posterior probability distributions for the MWC-type model generated by MCMC fits to the data in C overlaid on the prior probability distribution (dashed line) for each parameter.

    Techniques Used: Binding Assay, Fluorescence, Transferring, Expressing, Concentration Assay, Inhibition, Generated

    Kir6.2-C166S disrupts current inhibition, not nucleotide binding. A. Cartoon (from PDB accession #6BAA) showing the location of Kir6.2-C166 (purple) relative to the inhibitory nucleotide binding site (TNP-ATP from PDB accession #5XW6 shown in red), W311 is shown as blue spheres. B. Concentration dependence of TNP-ATP binding to unroofed membrane fragments expressing Kir6.2*-C166S-GFP + SUR1 shown in green, expressed as quenching of ANAP fluorescence. The Hill fits shown previously for Kir6.2*-GFP + SUR1 and Kir6.2*-G334D-GFP are shown in blue and orange dashed curves, respectively. Kir6.2*-C166S-GFP + SUR1: £C 50 = 32.0 μΜ, h = 0.92, E max = 0.96, n = 12, C. Representative current and fluorescence traces recorded simultaneously from an excised patch expressing Kir6.2*-C166S-GFP + SUR1, Exposure to different concentrations of TNP-ATP are shown by colour, D. Concentration-response (n = 6) for TNP-ATP inhibition of Kir6,2*-C166S-GFP + SUR1 currents ( I / I max ) and for quenching of ANAP fluorescence ( F / F max ). Data were fit with the MWC-type model. Solid curves represent the median fits and shaded areas indicate the 95% quantile intervals. Dashed curves represent the previous median fits of the MWC-type model to the Kir6.2*-GFP + SUR1 data from Figure 2D , Parameter estimates are reported in Table 3 . E. Posterior probability distributions for the full MWC-type model fit to Kir6.2*-C166S-GFP + SUR1 or Kir6.2*-GFP + SUR1 (data from Figure 2F ) overlaid on the prior probability distribution.
    Figure Legend Snippet: Kir6.2-C166S disrupts current inhibition, not nucleotide binding. A. Cartoon (from PDB accession #6BAA) showing the location of Kir6.2-C166 (purple) relative to the inhibitory nucleotide binding site (TNP-ATP from PDB accession #5XW6 shown in red), W311 is shown as blue spheres. B. Concentration dependence of TNP-ATP binding to unroofed membrane fragments expressing Kir6.2*-C166S-GFP + SUR1 shown in green, expressed as quenching of ANAP fluorescence. The Hill fits shown previously for Kir6.2*-GFP + SUR1 and Kir6.2*-G334D-GFP are shown in blue and orange dashed curves, respectively. Kir6.2*-C166S-GFP + SUR1: £C 50 = 32.0 μΜ, h = 0.92, E max = 0.96, n = 12, C. Representative current and fluorescence traces recorded simultaneously from an excised patch expressing Kir6.2*-C166S-GFP + SUR1, Exposure to different concentrations of TNP-ATP are shown by colour, D. Concentration-response (n = 6) for TNP-ATP inhibition of Kir6,2*-C166S-GFP + SUR1 currents ( I / I max ) and for quenching of ANAP fluorescence ( F / F max ). Data were fit with the MWC-type model. Solid curves represent the median fits and shaded areas indicate the 95% quantile intervals. Dashed curves represent the previous median fits of the MWC-type model to the Kir6.2*-GFP + SUR1 data from Figure 2D , Parameter estimates are reported in Table 3 . E. Posterior probability distributions for the full MWC-type model fit to Kir6.2*-C166S-GFP + SUR1 or Kir6.2*-GFP + SUR1 (data from Figure 2F ) overlaid on the prior probability distribution.

    Techniques Used: Inhibition, Binding Assay, Concentration Assay, Expressing, Fluorescence

    SUR1-K205 modulates both nucleotide affinity and inhibition of Kir6.2. A. H ydrophobic surface representation of Kir6.2 (yellow, PDB accession #6BAA) and SUR1 (blue, PDB accession #6PZI), Residue K205 on SUR1 is highlighted in pink. As this residue was built as an alanine in the structure, we used the mutagenesis tool in PyMol to insert the native lysine residue. A docked TNP-ATP molecule is shown in red, B. Representative current and fluorescence traces acquired simultaneously from excised patchesexpressing Kir6.2*-GFP with SUR1-K205Aor SUR1-K205E. C,D. Concentration-response for TNP-ATP inhibition of currents ( I / I max ) and for quenching of ANAP fluorescence ( F / F max ) in excised inside-out membrane patches expressing Kir6.2*-GFP + SUR1-K205A (C, n = 9) or Kir6.2*-GFP + SUR1-K205E (D, n = 9), Data were fit to the MWC-type model. Solid curves represent the median fits and shaded areas indicate the 95% quantile intervals. Fits to Kir6.2*-GFP + wild-type SUR1 are shown as dashed curves, E. Posterior probability distributions for the full MWC-type model fit to Kir6.2*-GFP co-expressed with wild-type SUR1 (fits from Figure 2 ), SUR1-K205A and SUR1-K205E overlaid on the prior probability distribution.
    Figure Legend Snippet: SUR1-K205 modulates both nucleotide affinity and inhibition of Kir6.2. A. H ydrophobic surface representation of Kir6.2 (yellow, PDB accession #6BAA) and SUR1 (blue, PDB accession #6PZI), Residue K205 on SUR1 is highlighted in pink. As this residue was built as an alanine in the structure, we used the mutagenesis tool in PyMol to insert the native lysine residue. A docked TNP-ATP molecule is shown in red, B. Representative current and fluorescence traces acquired simultaneously from excised patchesexpressing Kir6.2*-GFP with SUR1-K205Aor SUR1-K205E. C,D. Concentration-response for TNP-ATP inhibition of currents ( I / I max ) and for quenching of ANAP fluorescence ( F / F max ) in excised inside-out membrane patches expressing Kir6.2*-GFP + SUR1-K205A (C, n = 9) or Kir6.2*-GFP + SUR1-K205E (D, n = 9), Data were fit to the MWC-type model. Solid curves represent the median fits and shaded areas indicate the 95% quantile intervals. Fits to Kir6.2*-GFP + wild-type SUR1 are shown as dashed curves, E. Posterior probability distributions for the full MWC-type model fit to Kir6.2*-GFP co-expressed with wild-type SUR1 (fits from Figure 2 ), SUR1-K205A and SUR1-K205E overlaid on the prior probability distribution.

    Techniques Used: Inhibition, Mutagenesis, Fluorescence, Concentration Assay, Expressing

    SUR.1 affects the apparent affinity for and kinetics of nucleotide binding to Kir6.2. A. Concentration dependence of TNP-ATP binding to unroofed membrane fragments expressing Kir6.2*-GFP without SUR1 (brown), expressed as quenching of ANAP fluorescence. The smooth curve is a descriptive Hill fit Kir6.2*-GFP (no SUR1): EC 50 = 37.6 μΜ, h = 0.83, E max = 0.92, n = 14. The Hill fit to Kir6.2*-GFP + SUR1 is shown as a blue dashed curve. B. Concentration-response curve for TNP-ATP inhibition of Kir6.2-GFP (no ANAP label) without or without co-expression of SUR1, measured in excised, inside-out patches. Kir6.2-GFP + SUR1 : EC 50 = 1.17μΜ, h = 1.14, E max = 0.97, n = 7; Kir6.2-GFP (no SUR1): EC 50 = 273 μΜ, h = 1.09, E max = 1.00, n = 3. C. Time-courses of TNP-ATP binding and unbinding to Kir6.2*-GFP expressed in unroofed membrane fragments in the presence or absence of SUR1. Data are displayed as 1 -F/F max so that upward deflections indicate binding and downward deflections indicate unbinding. Small data points represent individual experiments. Overlaid are larger points representing the mean ±standard error at each time point. The smooth curves are single exponential fits to the wash-on or wash-off of a given concentration of TNP-ATP. D. Rate constants ( k obs and k off ) from the exponential fits as in C are plotted as functions of the TNP-ATP concentration. Linear fits to were performed using equation 3 . Kir6.2*-GFP (no SUR1): k on = 5560 M −1 s −1 ±2180M −1 s −1 , k off = 0.24 s −1 ± 0.13 s −1 , n = 2-6 per concentration. Kir6.2*-GFP + SUR1 : k on = 5640 M −1 s −1 ± 812 M −1 s −1 , k off = 0.18s −1 ± 0.05 s −1 , n = 3-4 per concentration. Dashed lines indicate the mean rates measured for wash-off experiments (koff) from all test concentrations combined. Kir6.2*-GFP (no SUR1): k off = 0.36s −1 ± 0,04s −1 . Kir6.2*-GFP + SUR1 : k off = 0.16s −1 ± 0.0004s −1 .
    Figure Legend Snippet: SUR.1 affects the apparent affinity for and kinetics of nucleotide binding to Kir6.2. A. Concentration dependence of TNP-ATP binding to unroofed membrane fragments expressing Kir6.2*-GFP without SUR1 (brown), expressed as quenching of ANAP fluorescence. The smooth curve is a descriptive Hill fit Kir6.2*-GFP (no SUR1): EC 50 = 37.6 μΜ, h = 0.83, E max = 0.92, n = 14. The Hill fit to Kir6.2*-GFP + SUR1 is shown as a blue dashed curve. B. Concentration-response curve for TNP-ATP inhibition of Kir6.2-GFP (no ANAP label) without or without co-expression of SUR1, measured in excised, inside-out patches. Kir6.2-GFP + SUR1 : EC 50 = 1.17μΜ, h = 1.14, E max = 0.97, n = 7; Kir6.2-GFP (no SUR1): EC 50 = 273 μΜ, h = 1.09, E max = 1.00, n = 3. C. Time-courses of TNP-ATP binding and unbinding to Kir6.2*-GFP expressed in unroofed membrane fragments in the presence or absence of SUR1. Data are displayed as 1 -F/F max so that upward deflections indicate binding and downward deflections indicate unbinding. Small data points represent individual experiments. Overlaid are larger points representing the mean ±standard error at each time point. The smooth curves are single exponential fits to the wash-on or wash-off of a given concentration of TNP-ATP. D. Rate constants ( k obs and k off ) from the exponential fits as in C are plotted as functions of the TNP-ATP concentration. Linear fits to were performed using equation 3 . Kir6.2*-GFP (no SUR1): k on = 5560 M −1 s −1 ±2180M −1 s −1 , k off = 0.24 s −1 ± 0.13 s −1 , n = 2-6 per concentration. Kir6.2*-GFP + SUR1 : k on = 5640 M −1 s −1 ± 812 M −1 s −1 , k off = 0.18s −1 ± 0.05 s −1 , n = 3-4 per concentration. Dashed lines indicate the mean rates measured for wash-off experiments (koff) from all test concentrations combined. Kir6.2*-GFP (no SUR1): k off = 0.36s −1 ± 0,04s −1 . Kir6.2*-GFP + SUR1 : k off = 0.16s −1 ± 0.0004s −1 .

    Techniques Used: Binding Assay, Concentration Assay, Expressing, Fluorescence, Inhibition

    A FRET assay to measure nucleotide binding to Kir6.2. A. Cartoon illustrating the topology of K ATP , The inhibitory nucleotide-binding site on Kir6.2 is shown in red; the stimulatory nucleotide-binding sites on SUR1 are shown in green. The three transmembrane domains of SUR1 are designated TMDO, TMD1, and TMD2. The loop connecting TMDO to TMD1 is designated LO. The nucleotide binding domains of SUR1 are labelled NBD1 and NBD2. B. Chemical structures of ANAP and TNP-ATP. The fluorescent moieties are highlighted. C. Side view of the structure of the cytosolic domains of Kir6.2 (PDB accession #6BAA)and one SUR1 subunit (PDB accession #6PZI). TNP-ATP (red, from PDB accession #5XW6) was docked into the nucleotide-binding site of Kir6.2 and positioned in NBS1 of SUR1 (green, from PDB accession #3AR7) by alignment as described in Materials and Methods. Distances from the native tryptophan at position 311 in Kir6.2 to the fluorescent moieties of the TNP-ATPs are displayed in A. D. Theoretical FRET efficiency between ANAP and TNP-ATP as a function of distance, calculated from the Forster equation. The distances and corresponding FRET efficiencies between ANAP at position 311 and TNP-ATP bound to Kir6.2 (E K j r ) and SUR1 (E SUR ) are indicated. E. Spectral images acquired from an unroofed membrane expressing Kir6.2*-GFP + SUR1 and exposed to increasing concentrations of TNP-ATP. The y-dimension in each image represents distance. The x-dimension represents wavelength. F. Line-averaged, background-subtracted spectra from E displayed with increasing concentrations of TNP-ATP coloured from purple to orange. The three fluorophores have distinct peaks: ANAP at 472 nm, GFP at 508 nm, and TNP-ATP at 561 nm. The shaded rectangle indicates the wavelength range used to measure ANAP intensity. G. Concentration-response relationship for binding of TNP-ATP to Kir6.2*-GFP + SUR1 in unroofed membranes. Data were plotted as F / F max , where F max is the fluorescence intensity in the absence of nucleotide. The smooth curve is a descriptive Hill fit. EC 50 = 25.6 μΜ, h = 0.82, E max = 0.93, n = 18. H. Concentration-response relationship for binding of TNP-ATP to Kir6.2*-G334D-GFP + SUR1 in unroofed membranes. The dashed blue curve is the fit from G. The orange curve is a descriptive Hill fit to the G334D data. EC 50 = 493 μΜ, h = 2.63, E max = 0.08, n = 9. The inset shows the location of G334D (green) in relation to the inhibitory ATP binding site on Kir6.2 (PDB accession #6BAA). TNP-ATP (PDB accession #5XW6) shown in red sticks.
    Figure Legend Snippet: A FRET assay to measure nucleotide binding to Kir6.2. A. Cartoon illustrating the topology of K ATP , The inhibitory nucleotide-binding site on Kir6.2 is shown in red; the stimulatory nucleotide-binding sites on SUR1 are shown in green. The three transmembrane domains of SUR1 are designated TMDO, TMD1, and TMD2. The loop connecting TMDO to TMD1 is designated LO. The nucleotide binding domains of SUR1 are labelled NBD1 and NBD2. B. Chemical structures of ANAP and TNP-ATP. The fluorescent moieties are highlighted. C. Side view of the structure of the cytosolic domains of Kir6.2 (PDB accession #6BAA)and one SUR1 subunit (PDB accession #6PZI). TNP-ATP (red, from PDB accession #5XW6) was docked into the nucleotide-binding site of Kir6.2 and positioned in NBS1 of SUR1 (green, from PDB accession #3AR7) by alignment as described in Materials and Methods. Distances from the native tryptophan at position 311 in Kir6.2 to the fluorescent moieties of the TNP-ATPs are displayed in A. D. Theoretical FRET efficiency between ANAP and TNP-ATP as a function of distance, calculated from the Forster equation. The distances and corresponding FRET efficiencies between ANAP at position 311 and TNP-ATP bound to Kir6.2 (E K j r ) and SUR1 (E SUR ) are indicated. E. Spectral images acquired from an unroofed membrane expressing Kir6.2*-GFP + SUR1 and exposed to increasing concentrations of TNP-ATP. The y-dimension in each image represents distance. The x-dimension represents wavelength. F. Line-averaged, background-subtracted spectra from E displayed with increasing concentrations of TNP-ATP coloured from purple to orange. The three fluorophores have distinct peaks: ANAP at 472 nm, GFP at 508 nm, and TNP-ATP at 561 nm. The shaded rectangle indicates the wavelength range used to measure ANAP intensity. G. Concentration-response relationship for binding of TNP-ATP to Kir6.2*-GFP + SUR1 in unroofed membranes. Data were plotted as F / F max , where F max is the fluorescence intensity in the absence of nucleotide. The smooth curve is a descriptive Hill fit. EC 50 = 25.6 μΜ, h = 0.82, E max = 0.93, n = 18. H. Concentration-response relationship for binding of TNP-ATP to Kir6.2*-G334D-GFP + SUR1 in unroofed membranes. The dashed blue curve is the fit from G. The orange curve is a descriptive Hill fit to the G334D data. EC 50 = 493 μΜ, h = 2.63, E max = 0.08, n = 9. The inset shows the location of G334D (green) in relation to the inhibitory ATP binding site on Kir6.2 (PDB accession #6BAA). TNP-ATP (PDB accession #5XW6) shown in red sticks.

    Techniques Used: Binding Assay, Expressing, Concentration Assay, Fluorescence

    Comparing the ability of each model to explain the data. Fits for each construct with each model (MWC-type, single-binding, negative-cooperativity) are displayed with the solid curve representing the median fit, the shaded area representing the 95% quartiles, and the dashed curve representing the median fit if the L parameter is fixed (to 6.0 for Kir6.2*-C166S-GFP + SUR1 and to 0.8 for the other three constructs). As the two fits were very similar, the dashed curve mostly overlays the solid curve. The most notable differences between the fits are that the negative cooperativity model allows for non-sigmoidal curves, and the single-binding model predicts much larger pedestals of current at saturating concentrations of TNP-ATP than either of the other two models.
    Figure Legend Snippet: Comparing the ability of each model to explain the data. Fits for each construct with each model (MWC-type, single-binding, negative-cooperativity) are displayed with the solid curve representing the median fit, the shaded area representing the 95% quartiles, and the dashed curve representing the median fit if the L parameter is fixed (to 6.0 for Kir6.2*-C166S-GFP + SUR1 and to 0.8 for the other three constructs). As the two fits were very similar, the dashed curve mostly overlays the solid curve. The most notable differences between the fits are that the negative cooperativity model allows for non-sigmoidal curves, and the single-binding model predicts much larger pedestals of current at saturating concentrations of TNP-ATP than either of the other two models.

    Techniques Used: Construct, Binding Assay

    Kir6.2*-GFP is functionally similar to Kir6.2-GFP. A. Concentration-response curve for ATP inhibition of Kir6.2-GFP + SUR1 or Kir6.2*-GFP + SUR1, measured in excised, inside-out patches. The smooth curves are descriptive Hill fits to the data, Kir6.2-GFP + SUR1: IC 50 = 62.7 μΜ, h = 1.28, I max = 0.99, n = 3; Kir6,2*-GFP + SUR1: IC 50 = 79.5 μΜ, h = 1.42, I max = 1.00, n = 4. B, C. Concentration-response relationships for current inhibition in excised, inside-out patches expressing Kir6.2-GFP + SUR1 (C) or Kir6.2*-GFP + SUR1 (D) exposed to either ATP or TNP-ATP. The smooth curves are descriptive Hill fits to the data, Kir6.2-GFP + SUR1 (TNP-ATP): IC 50 = 1,17μΜ, h = 1.14, I max = 0.97, n = 7, Kir6.2*-GFP + SUR1 (TNP-ATP): IC 50 = 6.23 μΜ, h = 0.92, I max = 0.96, n = 9. Data and fits for inhibition of Kir6.2*-GFP + SUR1 by TNP-ATP are the same as in Figure 2 . D. Fractional current inhibition by 100μΜ tolbutamide measured in excised, inside out patches. Data were normalised to the average current in control solution before and after tolbutamide exposure. Each data point represents an individual patch. Kir6.2-GFP without SUR1, n = 5; Kir6.2-GFP + SUR1, n = 3; Kir6,2*-GFP + SUR1, n =4.
    Figure Legend Snippet: Kir6.2*-GFP is functionally similar to Kir6.2-GFP. A. Concentration-response curve for ATP inhibition of Kir6.2-GFP + SUR1 or Kir6.2*-GFP + SUR1, measured in excised, inside-out patches. The smooth curves are descriptive Hill fits to the data, Kir6.2-GFP + SUR1: IC 50 = 62.7 μΜ, h = 1.28, I max = 0.99, n = 3; Kir6,2*-GFP + SUR1: IC 50 = 79.5 μΜ, h = 1.42, I max = 1.00, n = 4. B, C. Concentration-response relationships for current inhibition in excised, inside-out patches expressing Kir6.2-GFP + SUR1 (C) or Kir6.2*-GFP + SUR1 (D) exposed to either ATP or TNP-ATP. The smooth curves are descriptive Hill fits to the data, Kir6.2-GFP + SUR1 (TNP-ATP): IC 50 = 1,17μΜ, h = 1.14, I max = 0.97, n = 7, Kir6.2*-GFP + SUR1 (TNP-ATP): IC 50 = 6.23 μΜ, h = 0.92, I max = 0.96, n = 9. Data and fits for inhibition of Kir6.2*-GFP + SUR1 by TNP-ATP are the same as in Figure 2 . D. Fractional current inhibition by 100μΜ tolbutamide measured in excised, inside out patches. Data were normalised to the average current in control solution before and after tolbutamide exposure. Each data point represents an individual patch. Kir6.2-GFP without SUR1, n = 5; Kir6.2-GFP + SUR1, n = 3; Kir6,2*-GFP + SUR1, n =4.

    Techniques Used: Concentration Assay, Inhibition, Expressing

    27) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    28) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    29) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    30) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    31) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    32) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    33) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    34) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    35) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    36) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    37) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    38) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    39) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    40) Product Images from "Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA"

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2011.03.006

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Techniques Used: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.
    Figure Legend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Techniques Used: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.
    Figure Legend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Techniques Used: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.
    Figure Legend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Techniques Used: Fluorescence, Flow Cytometry, Mass Spectrometry

    Related Articles

    Mutagenesis:

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA
    Article Snippet: This mutation greatly disrupts the binding of acetyl CoA, raising its Ka by more than 200 fold. .. However, the mutation has a relatively small effect on the KT for MgTNP-ATP. .. If MgTNP-ATP was binding in the acetyl CoA binding site in the same orientation as acetyl CoA, one would expect MgTNP-ATP binding to be also severely disrupted by the R472S mutation.

    Activation Assay:

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA
    Article Snippet: .. Activation of pyruvate carboxylation by either acetyl CoA or MgTNP-ATP was analysed by nonlinear least-squares regression fitting of the data to , which was derived for the reaction scheme shown in . kobs = (kcat1 + kcat2 ([A] / Ka )n ) / (1 + ([A] / Ka )n ) (i) where: kcat1 is the catalytic rate constant of the reaction in the absence of either acetyl CoA or MgTNP-ATP; kcat2 is the catalytic rate constant of the reaction in the presence of saturating concentrations of acetyl CoA or MgTNP-ATP; Ka is the activation constant for acetyl CoA or MgTNP-ATP; n is the Hill coefficient of cooperativity; [A] is the concentration of either acetyl CoA or MgTNP-ATP. .. The activation of pyruvate carboxylation activity in the presence of varying concentrations of acetyl CoA at different fixed concentrations of MgTNP-ATP was analysed by non-linear least-squares regression fitting of the data to derived from the reaction scheme shown in . kobs = (kcat1 + kcat2 ([AcCoA] / Ka )na + kcat3 ([MgTNP−ATP] / KT )nt ) / (1 + ([AcCoA] / Ka )na + ([MgTNP−ATP] / KT )nt ) (ii) where: kcat1 is the catalytic rate constant of the reaction in the absence of both acetyl CoA and MgTNP-ATP; kcat 2 is the catalytic rate constant of the reaction at saturating concentrations of acetyl CoA; kcat3 is the catalytic rate constant of the reaction in the presence of saturating concentrations of MgTNP-ATP; Ka is the activation constant for acetyl CoA; KT is the activation constant for MgTNP-ATP; na is the Hill coefficient for acetyl CoA activation; nt is the hill coefficient for MgTNP-ATP activation; [AcCoA] is the concentration of acetyl CoA.

    Derivative Assay:

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA
    Article Snippet: .. Activation of pyruvate carboxylation by either acetyl CoA or MgTNP-ATP was analysed by nonlinear least-squares regression fitting of the data to , which was derived for the reaction scheme shown in . kobs = (kcat1 + kcat2 ([A] / Ka )n ) / (1 + ([A] / Ka )n ) (i) where: kcat1 is the catalytic rate constant of the reaction in the absence of either acetyl CoA or MgTNP-ATP; kcat2 is the catalytic rate constant of the reaction in the presence of saturating concentrations of acetyl CoA or MgTNP-ATP; Ka is the activation constant for acetyl CoA or MgTNP-ATP; n is the Hill coefficient of cooperativity; [A] is the concentration of either acetyl CoA or MgTNP-ATP. .. The activation of pyruvate carboxylation activity in the presence of varying concentrations of acetyl CoA at different fixed concentrations of MgTNP-ATP was analysed by non-linear least-squares regression fitting of the data to derived from the reaction scheme shown in . kobs = (kcat1 + kcat2 ([AcCoA] / Ka )na + kcat3 ([MgTNP−ATP] / KT )nt ) / (1 + ([AcCoA] / Ka )na + ([MgTNP−ATP] / KT )nt ) (ii) where: kcat1 is the catalytic rate constant of the reaction in the absence of both acetyl CoA and MgTNP-ATP; kcat 2 is the catalytic rate constant of the reaction at saturating concentrations of acetyl CoA; kcat3 is the catalytic rate constant of the reaction in the presence of saturating concentrations of MgTNP-ATP; Ka is the activation constant for acetyl CoA; KT is the activation constant for MgTNP-ATP; na is the Hill coefficient for acetyl CoA activation; nt is the hill coefficient for MgTNP-ATP activation; [AcCoA] is the concentration of acetyl CoA.

    Concentration Assay:

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA
    Article Snippet: .. Activation of pyruvate carboxylation by either acetyl CoA or MgTNP-ATP was analysed by nonlinear least-squares regression fitting of the data to , which was derived for the reaction scheme shown in . kobs = (kcat1 + kcat2 ([A] / Ka )n ) / (1 + ([A] / Ka )n ) (i) where: kcat1 is the catalytic rate constant of the reaction in the absence of either acetyl CoA or MgTNP-ATP; kcat2 is the catalytic rate constant of the reaction in the presence of saturating concentrations of acetyl CoA or MgTNP-ATP; Ka is the activation constant for acetyl CoA or MgTNP-ATP; n is the Hill coefficient of cooperativity; [A] is the concentration of either acetyl CoA or MgTNP-ATP. .. The activation of pyruvate carboxylation activity in the presence of varying concentrations of acetyl CoA at different fixed concentrations of MgTNP-ATP was analysed by non-linear least-squares regression fitting of the data to derived from the reaction scheme shown in . kobs = (kcat1 + kcat2 ([AcCoA] / Ka )na + kcat3 ([MgTNP−ATP] / KT )nt ) / (1 + ([AcCoA] / Ka )na + ([MgTNP−ATP] / KT )nt ) (ii) where: kcat1 is the catalytic rate constant of the reaction in the absence of both acetyl CoA and MgTNP-ATP; kcat 2 is the catalytic rate constant of the reaction at saturating concentrations of acetyl CoA; kcat3 is the catalytic rate constant of the reaction in the presence of saturating concentrations of MgTNP-ATP; Ka is the activation constant for acetyl CoA; KT is the activation constant for MgTNP-ATP; na is the Hill coefficient for acetyl CoA activation; nt is the hill coefficient for MgTNP-ATP activation; [AcCoA] is the concentration of acetyl CoA.

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA
    Article Snippet: Measurement of the binding stoichiometry was attempted by two methods, the first was equilibrium dialysis in which a dialysis apparatus was used that had two-compartments, separated by a semi-permeable membrane that prevented movement of the enzyme between the compartments but allowed the free movement of MgTNP-ATP. .. Both compartments initially contained the same concentration of MgTNP-ATP but only one compartment contained pyruvate carboxylase at concentrations up to 18 μM. .. The solutions in both compartments contained 0.1 M Tris-Cl,, pH 7.8 and 20 mM NaHCO3 and were stirred with magnetic stirrer bars for 30 min at 30°C (sufficient to equilibrate from a situation where MgTNP-ATP was only present in one compartment, with buffer in the other).

    Binding Assay:

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA
    Article Snippet: The lack of kinetic evidence supporting the binding of MgTNP-ATP to the MgATP substrate binding binding sites in the catalytically active subunits indicates that the small decrease in fluorescence observed on the addition of 2.5 mM MgATP is most likely due to conformational changes leading to either a change in the environment of the remaining bound MgTNP-ATP or to a small degree of displacement of the fluorophore. .. Further, the fact that some MgTNP-ATP remained bound to the enzyme in the presence of saturating concentrations of both acetyl CoA and MgATP strongly suggests that there is another binding site on the enzyme from which MgTNP-ATP cannot be displaced by these ligands. .. The kinetics of binding of MgTNP-ATP to pyruvate carboxylase were determined by monitoring the associated increase in fluorescence with time.

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA
    Article Snippet: In ( and ), k2 is a first-order rate constant describing a conformational change leading to an increase in fluorescence of the enzyme.MgTNP-ATP complex whilst k-2 is the reverse first-order rate constant. .. In ( and ), k2 is the second-order rate constant for further binding of MgTNP-ATP and k-2 is the dissociation constant of this complex, whilst in ( and ) k2 is a third-order rate constant for the further binding of two molecules of MgTNP-ATP. .. Thus , and describe reactions in which a single MgTNP-ATP molecule binds in the fast phase, followed by either a conformational change, binding of a further MgTNP-ATP or binding of a further two MgTNP-ATP molecules respectively, This also applies to , and except that in the fast phase two molecules of MgTNP-ATP bind.

    other:

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA
    Article Snippet: The apparent first order rate constants for the fast and intermediate phases increased with increasing [MgTNP-ATP] and had phases of approximately equal amplitudes.

    Fluorescence:

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA
    Article Snippet: Apparent kcat values were calculated by dividing the measured reaction velocity by the biotin concentration of the RePC used in the assay. .. Fluorescence emission spectra of MgTNP-ATP were recorded on a Cary spectrofluorometer with an excitation wavelength of 408 nm and emissions recorded between 500 and 600 nm. .. Experiments were performed at 30°C in 0.1 M Tris-Cl, pH7.8 and 20 mM NaHCO3 , with 5 μM MgTNP-ATP.

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA
    Article Snippet: Addition of 2.5 mM MgATP resulted in a further decrease in fluorescence of approximately 13%, with a smaller red-shift to 550 nm (spectrum (iv)). .. Again the residual fluorescence is higher (164%) than that of the solution of MgTNP-ATP alone, suggesting that even in the presence of both saturating acetyl CoA and MgATP, some of the MgTNP-ATP still remains bound to the enzyme. .. Fluorescence stopped-flow experiments were used to measure the kinetics of MgTNP-ATP binding to the wild-type enzyme. shows stopped-flow traces of the time-courses of MgTNP-ATP binding to the enzyme. show a fast phase and an intermediate phase of the reaction of approximately equal amplitudes that are essentially complete in about 30 msec and 1 sec respectively.

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    Jena Bioscience mgtnp atp
    Examples of fluorescence stopped-flow traces showing the kinetics of <t>MgTNP-ATP</t> binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
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    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Journal: Archives of biochemistry and biophysics

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    doi: 10.1016/j.abb.2011.03.006

    Figure Lengend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Article Snippet: Fluorescence emission spectra of MgTNP-ATP were recorded on a Cary spectrofluorometer with an excitation wavelength of 408 nm and emissions recorded between 500 and 600 nm.

    Techniques: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Journal: Archives of biochemistry and biophysics

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    doi: 10.1016/j.abb.2011.03.006

    Figure Lengend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Article Snippet: Fluorescence emission spectra of MgTNP-ATP were recorded on a Cary spectrofluorometer with an excitation wavelength of 408 nm and emissions recorded between 500 and 600 nm.

    Techniques: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Journal: Archives of biochemistry and biophysics

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    doi: 10.1016/j.abb.2011.03.006

    Figure Lengend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Article Snippet: Fluorescence emission spectra of MgTNP-ATP were recorded on a Cary spectrofluorometer with an excitation wavelength of 408 nm and emissions recorded between 500 and 600 nm.

    Techniques: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Journal: Archives of biochemistry and biophysics

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    doi: 10.1016/j.abb.2011.03.006

    Figure Lengend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Article Snippet: Fluorescence emission spectra of MgTNP-ATP were recorded on a Cary spectrofluorometer with an excitation wavelength of 408 nm and emissions recorded between 500 and 600 nm.

    Techniques: Fluorescence, Flow Cytometry, Mass Spectrometry