mgtnp atp  (Jena Bioscience)


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    Name:
    TNP ATP
    Description:

    Catalog Number:
    nu-221l
    Price:
    352.15
    Applications:
    Agonistic ligand, mainly for nucleoside receptor A1 Nucleoside-triphosphates can be converted by different membrane-bound phosphatases into nucleosides acting as nucleoside receptor ligands. The ester form is protected during uptake and transport and can be well-directed released through activation.
    Purity:
    ≥ 95 % (HPLC)
    Category:
    Nucleotides Nucleosides
    Buy from Supplier


    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 ).

    https://www.bioz.com/result/mgtnp atp/product/Jena Bioscience
    Average 94 stars, based on 94 article reviews
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    mgtnp atp - by Bioz Stars, 2020-09
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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 "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

    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 "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: eLife

    doi: 10.7554/eLife.52775

    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% quantiles, 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 plateaus 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% quantiles, 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 plateaus of current at saturating concentrations of TNP-ATP than either of the other two models.

    Techniques Used: Construct, Binding Assay

    Bleaching correction for PCF experiments. ( A ) Raw ANAP fluorescence intensities (left) and corrected ANAP fluorescence intensities (right) from a representative PCF experiment with Kir6.2*-GFP + SUR1 plotted against the exposure time. The fit to Equation 1 is shown as a black dashed line. To minimise artifacts from our bleaching corrections we performed experiments from both high-to-low and low-to-high TNP-ATP concentrations. ( B ) The parameters from fits to Equation 1 for each PCF experiment in Figure 2 are shown individually. ( C ) The fraction of ANAP fluorescence intensity remaining at the end of each PCF experiment in Figure 2 is shown by dividing the raw ANAP intensity of the last exposure by that of the first. The mean fractional remaining intensity of 0.34 is shown as a horizontal blue line.
    Figure Legend Snippet: Bleaching correction for PCF experiments. ( A ) Raw ANAP fluorescence intensities (left) and corrected ANAP fluorescence intensities (right) from a representative PCF experiment with Kir6.2*-GFP + SUR1 plotted against the exposure time. The fit to Equation 1 is shown as a black dashed line. To minimise artifacts from our bleaching corrections we performed experiments from both high-to-low and low-to-high TNP-ATP concentrations. ( B ) The parameters from fits to Equation 1 for each PCF experiment in Figure 2 are shown individually. ( C ) The fraction of ANAP fluorescence intensity remaining at the end of each PCF experiment in Figure 2 is shown by dividing the raw ANAP intensity of the last exposure by that of the first. The mean fractional remaining intensity of 0.34 is shown as a horizontal blue line.

    Techniques Used: Fluorescence

    SUR1 affects the apparent affinity for 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): E C 50 = 37.6 μ M , h = 0.83 , E m ⁢ a ⁢ x = 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) with or without co-expression of SUR1, measured in excised, inside-out patches. Kir6.2-GFP + SUR1: E C 50 = 1.17 μ M , h = 1.14 , E m ⁢ a ⁢ x = 0.97 , n = 7; Kir6.2-GFP (no SUR1): E C 50 = 273 μ M , h = 1.09 , E m ⁢ a ⁢ x = 1.00 , n = 3.
    Figure Legend Snippet: SUR1 affects the apparent affinity for 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): E C 50 = 37.6 μ M , h = 0.83 , E m ⁢ a ⁢ x = 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) with or without co-expression of SUR1, measured in excised, inside-out patches. Kir6.2-GFP + SUR1: E C 50 = 1.17 μ M , h = 1.14 , E m ⁢ a ⁢ x = 0.97 , n = 7; Kir6.2-GFP (no SUR1): E C 50 = 273 μ M , h = 1.09 , E m ⁢ a ⁢ x = 1.00 , n = 3.

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

    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: I C 50 = 62.7 μ M , h = 1.28 , I m ⁢ a ⁢ x = 0.99 , n = 3; Kir6.2*-GFP + SUR1: I C 50 = 79.5 μ M , h = 1.42 , I m ⁢ a ⁢ x = 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): I C 50 = 1.17 μ M , h = 1.14 , I m ⁢ a ⁢ x = 0.97 , n = 7, Kir6.2*-GFP + SUR1 (TNP-ATP): I C 50 = 6.23 μ M , h = 0.92 , I m ⁢ a ⁢ x = 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 100 µM 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: I C 50 = 62.7 μ M , h = 1.28 , I m ⁢ a ⁢ x = 0.99 , n = 3; Kir6.2*-GFP + SUR1: I C 50 = 79.5 μ M , h = 1.42 , I m ⁢ a ⁢ x = 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): I C 50 = 1.17 μ M , h = 1.14 , I m ⁢ a ⁢ x = 0.97 , n = 7, Kir6.2*-GFP + SUR1 (TNP-ATP): I C 50 = 6.23 μ M , h = 0.92 , I m ⁢ a ⁢ x = 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 100 µM 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

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    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 fit of the intermediate phase data to gave values of k2 and k-2 that are 5.9 and 6.6 % of those of the corresponding rate constants for the fast phase (k1 and k-1 respectively), leading to similar values of the dissociation constants for the enzyme.MgTNP-ATP complex formed in each phase, K1 (fast phase) and K2 (intermediate phase), which are also similar to the Ka described above for the activation of the enzyme by MgTNP-ATP. and the solid line represents a fit to ..

    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 solid line represents a fit of the data to and the values of KT , nt and kcat2 for the activation by MgTNP-ATP are similar to those obtained from the fit of the data in , i.e. 21 ± 3 μM, 2.3 ± 0.6 and 1.9 ± 0.1 s-1 . (see Materials and Methods). (b) Activation of pyruvate carboxylase by acetyl CoA at different fixed concentrations of MgTNP-ATP: (■) 0 μM; (▲) 10 μM; (▼) 15 μM; (◆) 25 μM; (●) 50 μM; (□) 75 μM. .. Solid lines represent a global non-linear least squares regression fit of the data to =0.98) (see Materials and Methods). (c) Replot of the data in the absence of acetyl CoA from (b), showing the activation of the enzyme by MgTNP-ATP in the absence of acetyl CoA The solid line represents a fit of the data to shows the dependence of enzymic activity on acetyl CoA concentration for a mutant enzyme in which an R472S mutation has been made.

    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 kinetics of binding of MgTNP-ATP to pyruvate carboxylase were determined by monitoring the associated increase in fluorescence with time. .. The process is triphasic, where the data for the dependence of kobs of the fast phase on [MgTNP-ATP] gave equally good fits to equations describing the binding of one or two molecules of 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: .. The process is triphasic, where the data for the dependence of kobs of the fast phase on [MgTNP-ATP] gave equally good fits to equations describing the binding of one or two molecules of MgTNP-ATP. .. The data for dependence of kobs of the intermediate phase on [MgTNP-ATP] gave the better fits to equations describing two different models.

    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: .. One possibility is that MgTNP-ATP and acetyl CoA are competing for the same allosteric binding sites on the enzyme. .. Based on the structure determined in the presence of the ethyl CoA analogue of acetyl CoA, the allosteric site of RePC there appears to be capable of accommodating the trinitrophenyl group of MgTNP-ATP since there is only one major hydrogen bonding interaction between Asn1055 and the 3′-ribosyl oxygen of acetyl CoA, located 3.8 Å away (see ) [ ].

    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: .. Although we cannot definitely rule out the possibility that MgTNP-ATP binds at the acetyl CoA binding site, but in a different orientation to acetyl CoA, a more likely explanation is that MgTNP-ATP is binding to a different site, with there being allosteric interactions between these two sites. .. Given that MgTNP-ATP is primarily regarded as an MgATP analogue and has the capability of activating the enzyme, we sought evidence that MgATP itself could activate the enzyme.

    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: .. The kinetics of binding of MgTNP-ATP to pyruvate carboxylase were determined by monitoring the associated increase in fluorescence with time. .. The process is triphasic, where the data for the dependence of kobs of the fast phase on [MgTNP-ATP] gave equally good fits to equations describing the binding of one or two molecules of 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: .. In the first model, two molecules of MgTNP-ATP bind in the fast phase, followed by a rate-limiting first-order conformational change in the intermediate phase that leads to an increase in fluorescence, either directly or by the rapid association of more MgTNP-ATP ( ). .. In the second model, the binding of one molecule of MgTNP-ATP in the fast phase is followed by another in the intermediate phase ( ).

    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: .. The absorbance of the solution in the compartment without enzyme was then measured at 408 nm and compared to the initial absorbance to calciulate the change in concentration of MgTNP-ATP. ..

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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 ).
    Mgtnp Atp, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 94/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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: The absorbance of the solution in the compartment without enzyme was then measured at 408 nm and compared to the initial absorbance to calciulate the change in concentration of MgTNP-ATP.

    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: The absorbance of the solution in the compartment without enzyme was then measured at 408 nm and compared to the initial absorbance to calciulate the change in concentration of MgTNP-ATP.

    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: The absorbance of the solution in the compartment without enzyme was then measured at 408 nm and compared to the initial absorbance to calciulate the change in concentration of MgTNP-ATP.

    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: The absorbance of the solution in the compartment without enzyme was then measured at 408 nm and compared to the initial absorbance to calciulate the change in concentration of MgTNP-ATP.

    Techniques: Fluorescence, Flow Cytometry, Mass Spectrometry