atpγs  (Jena Bioscience)


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    Name:
    ATPγS
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    Catalog Number:
    nu-406-25
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    Nucleotides Nucleosides
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    25 mg
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    Structured Review

    Jena Bioscience atpγs
    α3ΔN proteasomes showed enhanced proteolytic activity compared with wild-type proteasomes. ( a ) Purified CP and RP from the wt and α3ΔN cell lines. ( b ) suc-LLVY-AMC assay using purified CP. α3ΔN CP showed ∼three-fold higher suc-LLVY-AMC hydrolysis activity than wt CP. ( c ) The three different proteolytic sites in the purified CP were measured by using fluorogenic peptide substrates suc-LLVY-AMC (for chymotrypsin-like activity), Boc-LRR-AMC (for trypsin-like) and Z-LLE-AMC (caspase-like). ( d ) Same as b , except 26S proteasomes were used to measure suc-LLVY-AMC hydrolysis activity in the presence and absence of <t>ATPγS,</t> a slowly hydrolyzable analogue of ATP. ( e ) Michaelis–Menten plot, K M , and k cat values of wt and α3ΔN 26S proteasomes with ATPγS on concentration-dependent suc-LLVY-AMC cleavage for 15 min. The data were fit to a hyperbolic curve by nonlinear regression ( R 2 > 0.98) to calculate the enzyme kinetic data. The graphs shown are representative of at least three independent determinations and each data point is the mean±s.d. ( f ) Reconstitution of the holoenzymes using wt RP and wt or α3ΔN CP in various molar ratios. ( g ) Ub-Sic1 PY degradation assay using wt and α3ΔN 26S proteasomes. Reactions incubated with Ub-Sic1 PY and purified proteasomes for the indicated times were analysed by SDS–PAGE/IB using anti-T7 for Sic1, anti-α3 and anti-flag antibodies. ( h ) Quantification of Ub-Sic1 PY proteins in the degradation assay.

    https://www.bioz.com/result/atpγs/product/Jena Bioscience
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    atpγs - by Bioz Stars, 2021-03
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    Images

    1) Product Images from "Open-gate mutants of the mammalian proteasome show enhanced ubiquitin-conjugate degradation"

    Article Title: Open-gate mutants of the mammalian proteasome show enhanced ubiquitin-conjugate degradation

    Journal: Nature Communications

    doi: 10.1038/ncomms10963

    α3ΔN proteasomes showed enhanced proteolytic activity compared with wild-type proteasomes. ( a ) Purified CP and RP from the wt and α3ΔN cell lines. ( b ) suc-LLVY-AMC assay using purified CP. α3ΔN CP showed ∼three-fold higher suc-LLVY-AMC hydrolysis activity than wt CP. ( c ) The three different proteolytic sites in the purified CP were measured by using fluorogenic peptide substrates suc-LLVY-AMC (for chymotrypsin-like activity), Boc-LRR-AMC (for trypsin-like) and Z-LLE-AMC (caspase-like). ( d ) Same as b , except 26S proteasomes were used to measure suc-LLVY-AMC hydrolysis activity in the presence and absence of ATPγS, a slowly hydrolyzable analogue of ATP. ( e ) Michaelis–Menten plot, K M , and k cat values of wt and α3ΔN 26S proteasomes with ATPγS on concentration-dependent suc-LLVY-AMC cleavage for 15 min. The data were fit to a hyperbolic curve by nonlinear regression ( R 2 > 0.98) to calculate the enzyme kinetic data. The graphs shown are representative of at least three independent determinations and each data point is the mean±s.d. ( f ) Reconstitution of the holoenzymes using wt RP and wt or α3ΔN CP in various molar ratios. ( g ) Ub-Sic1 PY degradation assay using wt and α3ΔN 26S proteasomes. Reactions incubated with Ub-Sic1 PY and purified proteasomes for the indicated times were analysed by SDS–PAGE/IB using anti-T7 for Sic1, anti-α3 and anti-flag antibodies. ( h ) Quantification of Ub-Sic1 PY proteins in the degradation assay.
    Figure Legend Snippet: α3ΔN proteasomes showed enhanced proteolytic activity compared with wild-type proteasomes. ( a ) Purified CP and RP from the wt and α3ΔN cell lines. ( b ) suc-LLVY-AMC assay using purified CP. α3ΔN CP showed ∼three-fold higher suc-LLVY-AMC hydrolysis activity than wt CP. ( c ) The three different proteolytic sites in the purified CP were measured by using fluorogenic peptide substrates suc-LLVY-AMC (for chymotrypsin-like activity), Boc-LRR-AMC (for trypsin-like) and Z-LLE-AMC (caspase-like). ( d ) Same as b , except 26S proteasomes were used to measure suc-LLVY-AMC hydrolysis activity in the presence and absence of ATPγS, a slowly hydrolyzable analogue of ATP. ( e ) Michaelis–Menten plot, K M , and k cat values of wt and α3ΔN 26S proteasomes with ATPγS on concentration-dependent suc-LLVY-AMC cleavage for 15 min. The data were fit to a hyperbolic curve by nonlinear regression ( R 2 > 0.98) to calculate the enzyme kinetic data. The graphs shown are representative of at least three independent determinations and each data point is the mean±s.d. ( f ) Reconstitution of the holoenzymes using wt RP and wt or α3ΔN CP in various molar ratios. ( g ) Ub-Sic1 PY degradation assay using wt and α3ΔN 26S proteasomes. Reactions incubated with Ub-Sic1 PY and purified proteasomes for the indicated times were analysed by SDS–PAGE/IB using anti-T7 for Sic1, anti-α3 and anti-flag antibodies. ( h ) Quantification of Ub-Sic1 PY proteins in the degradation assay.

    Techniques Used: Activity Assay, Purification, Ub-AMC Assay, Concentration Assay, Degradation Assay, Incubation, SDS Page

    2) Product Images from "Long-range allosteric effects - How adenine nucleotides shape the conformational landscape of the Ski2-like RNA helicase, Brr2"

    Article Title: Long-range allosteric effects - How adenine nucleotides shape the conformational landscape of the Ski2-like RNA helicase, Brr2

    Journal: bioRxiv

    doi: 10.1101/2021.01.28.428616

    Kinetics of mant-ADP and mant -ATPγS interaction with hBrr2 NC and hBrr2CC. A and B . Time courses of 5 μM mant -ADP ( A ) or man t-ATPγS ( B ) binding to hBrr2 NC (1; 0.4 μM) and hBrr2CC (2; 0.2 μM) measured by FRET from Trp to mant . Controls correspond to binding experiments with unlabeled ADP or ATPγS (3 and 4). C and D . Individual nucleotide binding traces were fitted to single exponentials and the dependencies of the apparent rate constants on nucleotide concentration for hBrr2 NC ( C ) and hBrr2CC ( D ) were fitted by a linear equation, k app = k 1 [mant-nucleotide]+ k- 1 , in which k 1 is derived from the slope and k- 1 from the Y-axis intercept. Closed circles, mant -ADP; open circles, mant -ATPγS. Values represent means ± SD of at least three independent measurements. E and F . Dissociation of 5 μM mant -ADP ( E ) or mant -ATPγS ( F ) from hBrr2 NC (1; 0.4 μM hBrr2 NC ) and hBrr2 CC (2; 0.2 μM) in the presence of the respective unlabeled nucleotide in excess (100 μM). Control experiments (3) were carried out in the absence of unlabeled nucleotide (curves shown are for hBrr2 CC ).
    Figure Legend Snippet: Kinetics of mant-ADP and mant -ATPγS interaction with hBrr2 NC and hBrr2CC. A and B . Time courses of 5 μM mant -ADP ( A ) or man t-ATPγS ( B ) binding to hBrr2 NC (1; 0.4 μM) and hBrr2CC (2; 0.2 μM) measured by FRET from Trp to mant . Controls correspond to binding experiments with unlabeled ADP or ATPγS (3 and 4). C and D . Individual nucleotide binding traces were fitted to single exponentials and the dependencies of the apparent rate constants on nucleotide concentration for hBrr2 NC ( C ) and hBrr2CC ( D ) were fitted by a linear equation, k app = k 1 [mant-nucleotide]+ k- 1 , in which k 1 is derived from the slope and k- 1 from the Y-axis intercept. Closed circles, mant -ADP; open circles, mant -ATPγS. Values represent means ± SD of at least three independent measurements. E and F . Dissociation of 5 μM mant -ADP ( E ) or mant -ATPγS ( F ) from hBrr2 NC (1; 0.4 μM hBrr2 NC ) and hBrr2 CC (2; 0.2 μM) in the presence of the respective unlabeled nucleotide in excess (100 μM). Control experiments (3) were carried out in the absence of unlabeled nucleotide (curves shown are for hBrr2 CC ).

    Techniques Used: Binding Assay, Concentration Assay, Derivative Assay

    Kinetics of mant-ADP and mant -ATPγS interaction with hBrr2 FL and hBrr2 T1 . A and B . Time courses of 5 μM mant -ADP ( A ) or man t-ATPγS ( B ) binding to hBrr2 FL (1; 0.2 μM) and hBrr2 T1 (2; 0.2 μM) measured by FRET from Trp to mant . Controls were performed with unlabeled ADP or ATPγS (3-4). C and D . Individual nucleotide binding traces were fitted to double exponential equations, and the dependence of the apparent rate constants of the NC (circles) and CC (diamonds) on nucleotide concentration were fitted by a linear equation. Open symbols, hBrr2 FL ; closed symbols, hBrr2 T1 . The cassettes of hBrr2 FL and hBrr2 T1 bind nucleotides with different velocities as observed for the variants containing either one of the cassettes, hBrr2 NC and hBrr2 CC . While the CC binds mant -ADP and mant -ATPγS with similar rates, the NC in hBrr2 T1 binds mant -ATPγS faster than the NC in hBrr2 FL . Values represent means ± SD of at least three independent measurements. E and F . Dissociation of 5 μM mant-ADP ( E ) or mant -ATPγS ( F ) from hBrr2 FL (1; 0.2 μM) and hBrr2 T1 (2; 0.2 μM) in the presence of the respective unlabeled nucleotide in excess (100 μM). Control experiments (3-4) were carried out in the absence of unlabeled nucleotide.
    Figure Legend Snippet: Kinetics of mant-ADP and mant -ATPγS interaction with hBrr2 FL and hBrr2 T1 . A and B . Time courses of 5 μM mant -ADP ( A ) or man t-ATPγS ( B ) binding to hBrr2 FL (1; 0.2 μM) and hBrr2 T1 (2; 0.2 μM) measured by FRET from Trp to mant . Controls were performed with unlabeled ADP or ATPγS (3-4). C and D . Individual nucleotide binding traces were fitted to double exponential equations, and the dependence of the apparent rate constants of the NC (circles) and CC (diamonds) on nucleotide concentration were fitted by a linear equation. Open symbols, hBrr2 FL ; closed symbols, hBrr2 T1 . The cassettes of hBrr2 FL and hBrr2 T1 bind nucleotides with different velocities as observed for the variants containing either one of the cassettes, hBrr2 NC and hBrr2 CC . While the CC binds mant -ADP and mant -ATPγS with similar rates, the NC in hBrr2 T1 binds mant -ATPγS faster than the NC in hBrr2 FL . Values represent means ± SD of at least three independent measurements. E and F . Dissociation of 5 μM mant-ADP ( E ) or mant -ATPγS ( F ) from hBrr2 FL (1; 0.2 μM) and hBrr2 T1 (2; 0.2 μM) in the presence of the respective unlabeled nucleotide in excess (100 μM). Control experiments (3-4) were carried out in the absence of unlabeled nucleotide.

    Techniques Used: Binding Assay, Concentration Assay

    Effects of hBrr2 T1 mutations on the kinetics of mant -ADP and mant -ATPγS binding. A and B . Apparent rate constants ( k app NC and k app CC ) of mant -ATPγS binding to the respective cassettes in hBrr2 T1 and variants thereof. Binding of mant -ATPγS to either NC or CC is affected by all inter-cassette mutations tested. S1087L in the N-terminal HB domain originates from a retinitis pigmentosa-linked mutation of hBrr2, and was carried along as a negative control. C and D . Apparent rate constants ( k app NC and k app CC ) of mant -ADP binding to the respective cassettes of hBrr2 T1 and variants thereof. While most inter-cassette mutants bind mant -ADP at similar rates as hBrr2 T1 , mant -ADP binding is almost completely abrogated at both NC and CC in R603A (red). hBrr2 T1 , black; S1087L (control with residue exchange in the N-terminal HB ratchet helix), blue; R603A (cassette interface NC), red; R637A (cassette interface NC), light gray; PPP1296-8AAA (linker), magenta; H1548A (cassette interface CC), dark gray. Coloring as in A and B . S1087L in the N-terminal HB domain originates from a retinitis pigmentosa-linked mutation of hBrr2, and was carried along as a negative control. E and F . Apparent rate constants ( k app NC ) of mant -ADP and mant -ATPγS binding to the NC of hBrr2 T1 (closed circles) and its GK1355-6QE variant (altered CC nucleotide binding pocket; open circles). The time courses of nucleotide binding to GK1355-6QE were fitted by a single exponential indicating no detectable nucleotide binding at the CC, as expected due to the two residue exchanges in the CC binding pocket. As GK1355-6QE only has an intact NC nucleotide binding pocket, only the hBrr2 T1 k app NC is shown for comparison. GK1355-6QE shows reduced rates for nucleotide binding at the NC, suggesting long range modulation of NC nucleotide binding by nucleotide binding at the CC in hBrr2 T1 . Values represent means ± SD of at least three independent measurements.
    Figure Legend Snippet: Effects of hBrr2 T1 mutations on the kinetics of mant -ADP and mant -ATPγS binding. A and B . Apparent rate constants ( k app NC and k app CC ) of mant -ATPγS binding to the respective cassettes in hBrr2 T1 and variants thereof. Binding of mant -ATPγS to either NC or CC is affected by all inter-cassette mutations tested. S1087L in the N-terminal HB domain originates from a retinitis pigmentosa-linked mutation of hBrr2, and was carried along as a negative control. C and D . Apparent rate constants ( k app NC and k app CC ) of mant -ADP binding to the respective cassettes of hBrr2 T1 and variants thereof. While most inter-cassette mutants bind mant -ADP at similar rates as hBrr2 T1 , mant -ADP binding is almost completely abrogated at both NC and CC in R603A (red). hBrr2 T1 , black; S1087L (control with residue exchange in the N-terminal HB ratchet helix), blue; R603A (cassette interface NC), red; R637A (cassette interface NC), light gray; PPP1296-8AAA (linker), magenta; H1548A (cassette interface CC), dark gray. Coloring as in A and B . S1087L in the N-terminal HB domain originates from a retinitis pigmentosa-linked mutation of hBrr2, and was carried along as a negative control. E and F . Apparent rate constants ( k app NC ) of mant -ADP and mant -ATPγS binding to the NC of hBrr2 T1 (closed circles) and its GK1355-6QE variant (altered CC nucleotide binding pocket; open circles). The time courses of nucleotide binding to GK1355-6QE were fitted by a single exponential indicating no detectable nucleotide binding at the CC, as expected due to the two residue exchanges in the CC binding pocket. As GK1355-6QE only has an intact NC nucleotide binding pocket, only the hBrr2 T1 k app NC is shown for comparison. GK1355-6QE shows reduced rates for nucleotide binding at the NC, suggesting long range modulation of NC nucleotide binding by nucleotide binding at the CC in hBrr2 T1 . Values represent means ± SD of at least three independent measurements.

    Techniques Used: Binding Assay, Mutagenesis, Negative Control, Variant Assay

    Nucleotide specificity of the hBrr2 cassettes. A and B . Time courses of mant -nucleotide binding to 0.5 μM nucleotide-free hBrr2 NC ( A ) and hBrr2 CC ( B ) measured by FRET from Trp to mant. 1, mant -ADP (5 μM); 2, mant -ATPγS (5 μM); 3, mant -ATP (5 μM); 4, mant -AMPPNP (5 μM); 5, mant -GDP (5 μM); 6, mant -GTP (5 μM); 7, mant -GTPγS (5 μM). The hBrr2 cassettes bind mant -ADP and mant -ATPγS but do not interact with mant -AMPPNP or mant -G nucleotides.
    Figure Legend Snippet: Nucleotide specificity of the hBrr2 cassettes. A and B . Time courses of mant -nucleotide binding to 0.5 μM nucleotide-free hBrr2 NC ( A ) and hBrr2 CC ( B ) measured by FRET from Trp to mant. 1, mant -ADP (5 μM); 2, mant -ATPγS (5 μM); 3, mant -ATP (5 μM); 4, mant -AMPPNP (5 μM); 5, mant -GDP (5 μM); 6, mant -GTP (5 μM); 7, mant -GTPγS (5 μM). The hBrr2 cassettes bind mant -ADP and mant -ATPγS but do not interact with mant -AMPPNP or mant -G nucleotides.

    Techniques Used: Binding Assay

    Distances of Trp residues to mant moieties. A and B . Comparison of the distances of the mant moieties in mant -ADP ( A ) or mant -ATPγS ( B ) to the nearest Trp residues around the NC (left) and CC (right) nucleotide binding pockets. Dashed lines, distances between the C8 atom of the mant -nucleotides to the Ca atoms of the respective Trp residues.
    Figure Legend Snippet: Distances of Trp residues to mant moieties. A and B . Comparison of the distances of the mant moieties in mant -ADP ( A ) or mant -ATPγS ( B ) to the nearest Trp residues around the NC (left) and CC (right) nucleotide binding pockets. Dashed lines, distances between the C8 atom of the mant -nucleotides to the Ca atoms of the respective Trp residues.

    Techniques Used: Binding Assay

    3) Product Images from "Protein translocation by the SecA ATPase occurs by a power‐stroke mechanism"

    Article Title: Protein translocation by the SecA ATPase occurs by a power‐stroke mechanism

    Journal: The EMBO Journal

    doi: 10.15252/embj.2018101140

    Clamp dynamics and comparison with the two‐helix finger FRET traces of clamp movements in the presence of different nucleotides were used to determine the number of states best fit by a Markov model. Transition density plot of idealized ATP FRET states obtained in (A). Representative traces obtained with ATPγS. The upper FRET trace was calculated from the middle traces obtained by exciting the donor fluorophore and measuring both donor (green) and acceptor (red) fluorescence. The lowest trace was obtained by exciting the acceptor fluorophore directly. The arrow indicates a bleaching event. Distribution of FRET values determined from 315 traces as in (C) fit with a Gaussian model (black curve). Comparison of high and low FRET state occupancy in ADP•P i and ADP•V i for the clamp and THF. The distributions of dwell times of the low FRET states observed in ATP were fit with a single exponential (1,539 low FRET states). The inset shows average dwell time and error, defined as the standard error based on the number of traces. As in (F), but with high FRET (1,773 high FRET states). Comparison of dwell times of the high FRET states for the two‐helix finger (THF) and clamp for different fluorophore positions. Errors as in (G) with significance based on two‐sample Kolmogorov–Smirnov tests with a 1% threshold. n.s. P = 0.012 (left), n.s. P = 0.681 (right); * P
    Figure Legend Snippet: Clamp dynamics and comparison with the two‐helix finger FRET traces of clamp movements in the presence of different nucleotides were used to determine the number of states best fit by a Markov model. Transition density plot of idealized ATP FRET states obtained in (A). Representative traces obtained with ATPγS. The upper FRET trace was calculated from the middle traces obtained by exciting the donor fluorophore and measuring both donor (green) and acceptor (red) fluorescence. The lowest trace was obtained by exciting the acceptor fluorophore directly. The arrow indicates a bleaching event. Distribution of FRET values determined from 315 traces as in (C) fit with a Gaussian model (black curve). Comparison of high and low FRET state occupancy in ADP•P i and ADP•V i for the clamp and THF. The distributions of dwell times of the low FRET states observed in ATP were fit with a single exponential (1,539 low FRET states). The inset shows average dwell time and error, defined as the standard error based on the number of traces. As in (F), but with high FRET (1,773 high FRET states). Comparison of dwell times of the high FRET states for the two‐helix finger (THF) and clamp for different fluorophore positions. Errors as in (G) with significance based on two‐sample Kolmogorov–Smirnov tests with a 1% threshold. n.s. P = 0.012 (left), n.s. P = 0.681 (right); * P

    Techniques Used: Fluorescence

    4) Product Images from "Subunit Interactions and Cooperativity in the Microtubule-severing AAA ATPase Spastin *"

    Article Title: Subunit Interactions and Cooperativity in the Microtubule-severing AAA ATPase Spastin *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.291898

    Effect of ATPγS. A , a logarithmic plot of the wild type ATPase activity ( black , with fit to a Hill function as in A ) is compared with the turnover in the presence of 0.2 and 0.5 m m ATPγS. It shows an increase of the ATP concentration
    Figure Legend Snippet: Effect of ATPγS. A , a logarithmic plot of the wild type ATPase activity ( black , with fit to a Hill function as in A ) is compared with the turnover in the presence of 0.2 and 0.5 m m ATPγS. It shows an increase of the ATP concentration

    Techniques Used: Activity Assay, Concentration Assay

    Related Articles

    other:

    Article Title: TraG-Like Proteins of Type IV Secretion Systems: Functional Dissection of the Multiple Activities of TraG (RP4) and TrwB (R388)
    Article Snippet: The following reagents were obtained from the suppliers indicated: Ni-nitrilotriacetic acid (NTA) Superflow (Qiagen), Superdex 200 columns and radioactive nucleotides (Amersham Pharmacia Biotech), nucleotides (Roche Molecular Biochemicals or Sigma), 2′,3′- O -(2,4,6-trinitrophenyl)ATP, disodium salt (TNP-ATP) and TNP-ADP (Molecular Probes), adenosine-5′-(γ-thio)-triphosphate, sodium salt (ATPγS) and adenosine-5-[(β,γ)-imido]triphosphate, triethylammonium salt (AppNp) (Jena Bioscience), enzymes (New England Biolabs), and Brij 58 and Triton X-100 (Sigma).

    Article Title: Long-range allosteric effects - How adenine nucleotides shape the conformational landscape of the Ski2-like RNA helicase, Brr2
    Article Snippet: Crystals were soaked for 1 h in 10 mM ADP or ATPγS, or in 1 mM mant -ADP or mant-ATPγS (Jena Bioscience) in reservoir solution.

    High Performance Liquid Chromatography:

    Article Title: Subunit Interactions and Cooperativity in the Microtubule-severing AAA ATPase Spastin *
    Article Snippet: .. The purity of ATPγS (Jena Biosciences) was checked by HPLC for contaminations with ADP, which is converted to ATP in coupled enzymatic assays and would disturb the analysis. .. 20 μl of a 1 m m nucleotide solution was applied to a reversed phase C18 column (Gemini-NX, 3 μm, 100A, 100 × 4.60 mm) and eluted with 10 m m tetrabutyl ammonium chloride, 10 m m K2 HPO4 , 25% acetonitrile, pH 7.0, at a flow rate of 1 ml/min.

    Incubation:

    Article Title: Plant Aurora kinases interact with and phosphorylate transcription factors.
    Article Snippet: Aurora kinase (AUR) is a well-known mitotic serine/threonine kinase that regulates centromere formation, chromosome segregation, and cytokinesis in eukaryotes. .. Aurora kinase (AUR) is a well-known mitotic serine/threonine kinase that regulates centromere formation, chromosome segregation, and cytokinesis in eukaryotes. ..

    Article Title: Protein translocation by the SecA ATPase occurs by a power‐stroke mechanism
    Article Snippet: Reactions were incubated for 10 min at 37°C while shaking at 650 rpm in a Thermomixer R (Eppendorf, Germany). .. When indicated, 1 mM ADP•BeFx , 1 mM ADP and Vi (sodium orthovanadate, New England Biolabs), 5 mM ATPγS (Jena Bioscience, Jena, Germany), or 1 mM ADP and Pi were added after the initial 10 min, along with 1 U of hexokinase (Roche Applied Science, Germany) and 20 mM glucose, and incubated for an additional 5 min. ..

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    Jena Bioscience atpγs
    α3ΔN proteasomes showed enhanced proteolytic activity compared with wild-type proteasomes. ( a ) Purified CP and RP from the wt and α3ΔN cell lines. ( b ) suc-LLVY-AMC assay using purified CP. α3ΔN CP showed ∼three-fold higher suc-LLVY-AMC hydrolysis activity than wt CP. ( c ) The three different proteolytic sites in the purified CP were measured by using fluorogenic peptide substrates suc-LLVY-AMC (for chymotrypsin-like activity), Boc-LRR-AMC (for trypsin-like) and Z-LLE-AMC (caspase-like). ( d ) Same as b , except 26S proteasomes were used to measure suc-LLVY-AMC hydrolysis activity in the presence and absence of <t>ATPγS,</t> a slowly hydrolyzable analogue of ATP. ( e ) Michaelis–Menten plot, K M , and k cat values of wt and α3ΔN 26S proteasomes with ATPγS on concentration-dependent suc-LLVY-AMC cleavage for 15 min. The data were fit to a hyperbolic curve by nonlinear regression ( R 2 > 0.98) to calculate the enzyme kinetic data. The graphs shown are representative of at least three independent determinations and each data point is the mean±s.d. ( f ) Reconstitution of the holoenzymes using wt RP and wt or α3ΔN CP in various molar ratios. ( g ) Ub-Sic1 PY degradation assay using wt and α3ΔN 26S proteasomes. Reactions incubated with Ub-Sic1 PY and purified proteasomes for the indicated times were analysed by SDS–PAGE/IB using anti-T7 for Sic1, anti-α3 and anti-flag antibodies. ( h ) Quantification of Ub-Sic1 PY proteins in the degradation assay.
    Atpγs, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/atpγs/product/Jena Bioscience
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    Price from $9.99 to $1999.99
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    α3ΔN proteasomes showed enhanced proteolytic activity compared with wild-type proteasomes. ( a ) Purified CP and RP from the wt and α3ΔN cell lines. ( b ) suc-LLVY-AMC assay using purified CP. α3ΔN CP showed ∼three-fold higher suc-LLVY-AMC hydrolysis activity than wt CP. ( c ) The three different proteolytic sites in the purified CP were measured by using fluorogenic peptide substrates suc-LLVY-AMC (for chymotrypsin-like activity), Boc-LRR-AMC (for trypsin-like) and Z-LLE-AMC (caspase-like). ( d ) Same as b , except 26S proteasomes were used to measure suc-LLVY-AMC hydrolysis activity in the presence and absence of ATPγS, a slowly hydrolyzable analogue of ATP. ( e ) Michaelis–Menten plot, K M , and k cat values of wt and α3ΔN 26S proteasomes with ATPγS on concentration-dependent suc-LLVY-AMC cleavage for 15 min. The data were fit to a hyperbolic curve by nonlinear regression ( R 2 > 0.98) to calculate the enzyme kinetic data. The graphs shown are representative of at least three independent determinations and each data point is the mean±s.d. ( f ) Reconstitution of the holoenzymes using wt RP and wt or α3ΔN CP in various molar ratios. ( g ) Ub-Sic1 PY degradation assay using wt and α3ΔN 26S proteasomes. Reactions incubated with Ub-Sic1 PY and purified proteasomes for the indicated times were analysed by SDS–PAGE/IB using anti-T7 for Sic1, anti-α3 and anti-flag antibodies. ( h ) Quantification of Ub-Sic1 PY proteins in the degradation assay.

    Journal: Nature Communications

    Article Title: Open-gate mutants of the mammalian proteasome show enhanced ubiquitin-conjugate degradation

    doi: 10.1038/ncomms10963

    Figure Lengend Snippet: α3ΔN proteasomes showed enhanced proteolytic activity compared with wild-type proteasomes. ( a ) Purified CP and RP from the wt and α3ΔN cell lines. ( b ) suc-LLVY-AMC assay using purified CP. α3ΔN CP showed ∼three-fold higher suc-LLVY-AMC hydrolysis activity than wt CP. ( c ) The three different proteolytic sites in the purified CP were measured by using fluorogenic peptide substrates suc-LLVY-AMC (for chymotrypsin-like activity), Boc-LRR-AMC (for trypsin-like) and Z-LLE-AMC (caspase-like). ( d ) Same as b , except 26S proteasomes were used to measure suc-LLVY-AMC hydrolysis activity in the presence and absence of ATPγS, a slowly hydrolyzable analogue of ATP. ( e ) Michaelis–Menten plot, K M , and k cat values of wt and α3ΔN 26S proteasomes with ATPγS on concentration-dependent suc-LLVY-AMC cleavage for 15 min. The data were fit to a hyperbolic curve by nonlinear regression ( R 2 > 0.98) to calculate the enzyme kinetic data. The graphs shown are representative of at least three independent determinations and each data point is the mean±s.d. ( f ) Reconstitution of the holoenzymes using wt RP and wt or α3ΔN CP in various molar ratios. ( g ) Ub-Sic1 PY degradation assay using wt and α3ΔN 26S proteasomes. Reactions incubated with Ub-Sic1 PY and purified proteasomes for the indicated times were analysed by SDS–PAGE/IB using anti-T7 for Sic1, anti-α3 and anti-flag antibodies. ( h ) Quantification of Ub-Sic1 PY proteins in the degradation assay.

    Article Snippet: Sources of major biochemical reagents are as follows: PS-341 (LC Laboratories, USA); epoxomicin and Ub-VS (Boston Biochem, USA); ATP (Calbiochem, USA); ATPγS (Jena Bioscience, Germany); ubiquitin (Sigma); MG132 (Bachem); suc-LLVY-AMC (Bachem); Z-LLE-AMC (Enzo Life Sciences); Boc-LRR-AMC (Enzo Life Sciences).

    Techniques: Activity Assay, Purification, Ub-AMC Assay, Concentration Assay, Degradation Assay, Incubation, SDS Page

    Displacement of bound TNP-ATP by other nucleotides, DNA, and inorganic salts. The fluorescence of TNP-ATP-protein mixtures was measured before and after addition of the indicated compounds. The percent fluorescence reduction was set as a measure of the displacement of bound TNP-ATP, i.e., as a measure of competition for the binding site or inhibition of nucleotide binding. Concentrations were as follows: TraGΔ2 or TrwBΔ1, 10 μM; TNP-ATP, 50 μM; nucleotides, K x H y PO 4 , AppNp, Na 2 SO 4 , MgSO 4 , and MgCl 2 , 5 mM; Na 4 P 2 O 7 and ATPγS, 2.5 mM; ssDNA, 18 nM; dsDNA, 15 nM.

    Journal: Journal of Bacteriology

    Article Title: TraG-Like Proteins of Type IV Secretion Systems: Functional Dissection of the Multiple Activities of TraG (RP4) and TrwB (R388)

    doi: 10.1128/JB.185.15.4371-4381.2003

    Figure Lengend Snippet: Displacement of bound TNP-ATP by other nucleotides, DNA, and inorganic salts. The fluorescence of TNP-ATP-protein mixtures was measured before and after addition of the indicated compounds. The percent fluorescence reduction was set as a measure of the displacement of bound TNP-ATP, i.e., as a measure of competition for the binding site or inhibition of nucleotide binding. Concentrations were as follows: TraGΔ2 or TrwBΔ1, 10 μM; TNP-ATP, 50 μM; nucleotides, K x H y PO 4 , AppNp, Na 2 SO 4 , MgSO 4 , and MgCl 2 , 5 mM; Na 4 P 2 O 7 and ATPγS, 2.5 mM; ssDNA, 18 nM; dsDNA, 15 nM.

    Article Snippet: The following reagents were obtained from the suppliers indicated: Ni-nitrilotriacetic acid (NTA) Superflow (Qiagen), Superdex 200 columns and radioactive nucleotides (Amersham Pharmacia Biotech), nucleotides (Roche Molecular Biochemicals or Sigma), 2′,3′- O -(2,4,6-trinitrophenyl)ATP, disodium salt (TNP-ATP) and TNP-ADP (Molecular Probes), adenosine-5′-(γ-thio)-triphosphate, sodium salt (ATPγS) and adenosine-5-[(β,γ)-imido]triphosphate, triethylammonium salt (AppNp) (Jena Bioscience), enzymes (New England Biolabs), and Brij 58 and Triton X-100 (Sigma).

    Techniques: Fluorescence, Binding Assay, Inhibition

    Kinetics of mant-ADP and mant -ATPγS interaction with hBrr2 NC and hBrr2CC. A and B . Time courses of 5 μM mant -ADP ( A ) or man t-ATPγS ( B ) binding to hBrr2 NC (1; 0.4 μM) and hBrr2CC (2; 0.2 μM) measured by FRET from Trp to mant . Controls correspond to binding experiments with unlabeled ADP or ATPγS (3 and 4). C and D . Individual nucleotide binding traces were fitted to single exponentials and the dependencies of the apparent rate constants on nucleotide concentration for hBrr2 NC ( C ) and hBrr2CC ( D ) were fitted by a linear equation, k app = k 1 [mant-nucleotide]+ k- 1 , in which k 1 is derived from the slope and k- 1 from the Y-axis intercept. Closed circles, mant -ADP; open circles, mant -ATPγS. Values represent means ± SD of at least three independent measurements. E and F . Dissociation of 5 μM mant -ADP ( E ) or mant -ATPγS ( F ) from hBrr2 NC (1; 0.4 μM hBrr2 NC ) and hBrr2 CC (2; 0.2 μM) in the presence of the respective unlabeled nucleotide in excess (100 μM). Control experiments (3) were carried out in the absence of unlabeled nucleotide (curves shown are for hBrr2 CC ).

    Journal: bioRxiv

    Article Title: Long-range allosteric effects - How adenine nucleotides shape the conformational landscape of the Ski2-like RNA helicase, Brr2

    doi: 10.1101/2021.01.28.428616

    Figure Lengend Snippet: Kinetics of mant-ADP and mant -ATPγS interaction with hBrr2 NC and hBrr2CC. A and B . Time courses of 5 μM mant -ADP ( A ) or man t-ATPγS ( B ) binding to hBrr2 NC (1; 0.4 μM) and hBrr2CC (2; 0.2 μM) measured by FRET from Trp to mant . Controls correspond to binding experiments with unlabeled ADP or ATPγS (3 and 4). C and D . Individual nucleotide binding traces were fitted to single exponentials and the dependencies of the apparent rate constants on nucleotide concentration for hBrr2 NC ( C ) and hBrr2CC ( D ) were fitted by a linear equation, k app = k 1 [mant-nucleotide]+ k- 1 , in which k 1 is derived from the slope and k- 1 from the Y-axis intercept. Closed circles, mant -ADP; open circles, mant -ATPγS. Values represent means ± SD of at least three independent measurements. E and F . Dissociation of 5 μM mant -ADP ( E ) or mant -ATPγS ( F ) from hBrr2 NC (1; 0.4 μM hBrr2 NC ) and hBrr2 CC (2; 0.2 μM) in the presence of the respective unlabeled nucleotide in excess (100 μM). Control experiments (3) were carried out in the absence of unlabeled nucleotide (curves shown are for hBrr2 CC ).

    Article Snippet: Crystals were soaked for 1 h in 10 mM ADP or ATPγS, or in 1 mM mant -ADP or mant-ATPγS (Jena Bioscience) in reservoir solution.

    Techniques: Binding Assay, Concentration Assay, Derivative Assay

    Kinetics of mant-ADP and mant -ATPγS interaction with hBrr2 FL and hBrr2 T1 . A and B . Time courses of 5 μM mant -ADP ( A ) or man t-ATPγS ( B ) binding to hBrr2 FL (1; 0.2 μM) and hBrr2 T1 (2; 0.2 μM) measured by FRET from Trp to mant . Controls were performed with unlabeled ADP or ATPγS (3-4). C and D . Individual nucleotide binding traces were fitted to double exponential equations, and the dependence of the apparent rate constants of the NC (circles) and CC (diamonds) on nucleotide concentration were fitted by a linear equation. Open symbols, hBrr2 FL ; closed symbols, hBrr2 T1 . The cassettes of hBrr2 FL and hBrr2 T1 bind nucleotides with different velocities as observed for the variants containing either one of the cassettes, hBrr2 NC and hBrr2 CC . While the CC binds mant -ADP and mant -ATPγS with similar rates, the NC in hBrr2 T1 binds mant -ATPγS faster than the NC in hBrr2 FL . Values represent means ± SD of at least three independent measurements. E and F . Dissociation of 5 μM mant-ADP ( E ) or mant -ATPγS ( F ) from hBrr2 FL (1; 0.2 μM) and hBrr2 T1 (2; 0.2 μM) in the presence of the respective unlabeled nucleotide in excess (100 μM). Control experiments (3-4) were carried out in the absence of unlabeled nucleotide.

    Journal: bioRxiv

    Article Title: Long-range allosteric effects - How adenine nucleotides shape the conformational landscape of the Ski2-like RNA helicase, Brr2

    doi: 10.1101/2021.01.28.428616

    Figure Lengend Snippet: Kinetics of mant-ADP and mant -ATPγS interaction with hBrr2 FL and hBrr2 T1 . A and B . Time courses of 5 μM mant -ADP ( A ) or man t-ATPγS ( B ) binding to hBrr2 FL (1; 0.2 μM) and hBrr2 T1 (2; 0.2 μM) measured by FRET from Trp to mant . Controls were performed with unlabeled ADP or ATPγS (3-4). C and D . Individual nucleotide binding traces were fitted to double exponential equations, and the dependence of the apparent rate constants of the NC (circles) and CC (diamonds) on nucleotide concentration were fitted by a linear equation. Open symbols, hBrr2 FL ; closed symbols, hBrr2 T1 . The cassettes of hBrr2 FL and hBrr2 T1 bind nucleotides with different velocities as observed for the variants containing either one of the cassettes, hBrr2 NC and hBrr2 CC . While the CC binds mant -ADP and mant -ATPγS with similar rates, the NC in hBrr2 T1 binds mant -ATPγS faster than the NC in hBrr2 FL . Values represent means ± SD of at least three independent measurements. E and F . Dissociation of 5 μM mant-ADP ( E ) or mant -ATPγS ( F ) from hBrr2 FL (1; 0.2 μM) and hBrr2 T1 (2; 0.2 μM) in the presence of the respective unlabeled nucleotide in excess (100 μM). Control experiments (3-4) were carried out in the absence of unlabeled nucleotide.

    Article Snippet: Crystals were soaked for 1 h in 10 mM ADP or ATPγS, or in 1 mM mant -ADP or mant-ATPγS (Jena Bioscience) in reservoir solution.

    Techniques: Binding Assay, Concentration Assay

    Effects of hBrr2 T1 mutations on the kinetics of mant -ADP and mant -ATPγS binding. A and B . Apparent rate constants ( k app NC and k app CC ) of mant -ATPγS binding to the respective cassettes in hBrr2 T1 and variants thereof. Binding of mant -ATPγS to either NC or CC is affected by all inter-cassette mutations tested. S1087L in the N-terminal HB domain originates from a retinitis pigmentosa-linked mutation of hBrr2, and was carried along as a negative control. C and D . Apparent rate constants ( k app NC and k app CC ) of mant -ADP binding to the respective cassettes of hBrr2 T1 and variants thereof. While most inter-cassette mutants bind mant -ADP at similar rates as hBrr2 T1 , mant -ADP binding is almost completely abrogated at both NC and CC in R603A (red). hBrr2 T1 , black; S1087L (control with residue exchange in the N-terminal HB ratchet helix), blue; R603A (cassette interface NC), red; R637A (cassette interface NC), light gray; PPP1296-8AAA (linker), magenta; H1548A (cassette interface CC), dark gray. Coloring as in A and B . S1087L in the N-terminal HB domain originates from a retinitis pigmentosa-linked mutation of hBrr2, and was carried along as a negative control. E and F . Apparent rate constants ( k app NC ) of mant -ADP and mant -ATPγS binding to the NC of hBrr2 T1 (closed circles) and its GK1355-6QE variant (altered CC nucleotide binding pocket; open circles). The time courses of nucleotide binding to GK1355-6QE were fitted by a single exponential indicating no detectable nucleotide binding at the CC, as expected due to the two residue exchanges in the CC binding pocket. As GK1355-6QE only has an intact NC nucleotide binding pocket, only the hBrr2 T1 k app NC is shown for comparison. GK1355-6QE shows reduced rates for nucleotide binding at the NC, suggesting long range modulation of NC nucleotide binding by nucleotide binding at the CC in hBrr2 T1 . Values represent means ± SD of at least three independent measurements.

    Journal: bioRxiv

    Article Title: Long-range allosteric effects - How adenine nucleotides shape the conformational landscape of the Ski2-like RNA helicase, Brr2

    doi: 10.1101/2021.01.28.428616

    Figure Lengend Snippet: Effects of hBrr2 T1 mutations on the kinetics of mant -ADP and mant -ATPγS binding. A and B . Apparent rate constants ( k app NC and k app CC ) of mant -ATPγS binding to the respective cassettes in hBrr2 T1 and variants thereof. Binding of mant -ATPγS to either NC or CC is affected by all inter-cassette mutations tested. S1087L in the N-terminal HB domain originates from a retinitis pigmentosa-linked mutation of hBrr2, and was carried along as a negative control. C and D . Apparent rate constants ( k app NC and k app CC ) of mant -ADP binding to the respective cassettes of hBrr2 T1 and variants thereof. While most inter-cassette mutants bind mant -ADP at similar rates as hBrr2 T1 , mant -ADP binding is almost completely abrogated at both NC and CC in R603A (red). hBrr2 T1 , black; S1087L (control with residue exchange in the N-terminal HB ratchet helix), blue; R603A (cassette interface NC), red; R637A (cassette interface NC), light gray; PPP1296-8AAA (linker), magenta; H1548A (cassette interface CC), dark gray. Coloring as in A and B . S1087L in the N-terminal HB domain originates from a retinitis pigmentosa-linked mutation of hBrr2, and was carried along as a negative control. E and F . Apparent rate constants ( k app NC ) of mant -ADP and mant -ATPγS binding to the NC of hBrr2 T1 (closed circles) and its GK1355-6QE variant (altered CC nucleotide binding pocket; open circles). The time courses of nucleotide binding to GK1355-6QE were fitted by a single exponential indicating no detectable nucleotide binding at the CC, as expected due to the two residue exchanges in the CC binding pocket. As GK1355-6QE only has an intact NC nucleotide binding pocket, only the hBrr2 T1 k app NC is shown for comparison. GK1355-6QE shows reduced rates for nucleotide binding at the NC, suggesting long range modulation of NC nucleotide binding by nucleotide binding at the CC in hBrr2 T1 . Values represent means ± SD of at least three independent measurements.

    Article Snippet: Crystals were soaked for 1 h in 10 mM ADP or ATPγS, or in 1 mM mant -ADP or mant-ATPγS (Jena Bioscience) in reservoir solution.

    Techniques: Binding Assay, Mutagenesis, Negative Control, Variant Assay

    Nucleotide specificity of the hBrr2 cassettes. A and B . Time courses of mant -nucleotide binding to 0.5 μM nucleotide-free hBrr2 NC ( A ) and hBrr2 CC ( B ) measured by FRET from Trp to mant. 1, mant -ADP (5 μM); 2, mant -ATPγS (5 μM); 3, mant -ATP (5 μM); 4, mant -AMPPNP (5 μM); 5, mant -GDP (5 μM); 6, mant -GTP (5 μM); 7, mant -GTPγS (5 μM). The hBrr2 cassettes bind mant -ADP and mant -ATPγS but do not interact with mant -AMPPNP or mant -G nucleotides.

    Journal: bioRxiv

    Article Title: Long-range allosteric effects - How adenine nucleotides shape the conformational landscape of the Ski2-like RNA helicase, Brr2

    doi: 10.1101/2021.01.28.428616

    Figure Lengend Snippet: Nucleotide specificity of the hBrr2 cassettes. A and B . Time courses of mant -nucleotide binding to 0.5 μM nucleotide-free hBrr2 NC ( A ) and hBrr2 CC ( B ) measured by FRET from Trp to mant. 1, mant -ADP (5 μM); 2, mant -ATPγS (5 μM); 3, mant -ATP (5 μM); 4, mant -AMPPNP (5 μM); 5, mant -GDP (5 μM); 6, mant -GTP (5 μM); 7, mant -GTPγS (5 μM). The hBrr2 cassettes bind mant -ADP and mant -ATPγS but do not interact with mant -AMPPNP or mant -G nucleotides.

    Article Snippet: Crystals were soaked for 1 h in 10 mM ADP or ATPγS, or in 1 mM mant -ADP or mant-ATPγS (Jena Bioscience) in reservoir solution.

    Techniques: Binding Assay

    Distances of Trp residues to mant moieties. A and B . Comparison of the distances of the mant moieties in mant -ADP ( A ) or mant -ATPγS ( B ) to the nearest Trp residues around the NC (left) and CC (right) nucleotide binding pockets. Dashed lines, distances between the C8 atom of the mant -nucleotides to the Ca atoms of the respective Trp residues.

    Journal: bioRxiv

    Article Title: Long-range allosteric effects - How adenine nucleotides shape the conformational landscape of the Ski2-like RNA helicase, Brr2

    doi: 10.1101/2021.01.28.428616

    Figure Lengend Snippet: Distances of Trp residues to mant moieties. A and B . Comparison of the distances of the mant moieties in mant -ADP ( A ) or mant -ATPγS ( B ) to the nearest Trp residues around the NC (left) and CC (right) nucleotide binding pockets. Dashed lines, distances between the C8 atom of the mant -nucleotides to the Ca atoms of the respective Trp residues.

    Article Snippet: Crystals were soaked for 1 h in 10 mM ADP or ATPγS, or in 1 mM mant -ADP or mant-ATPγS (Jena Bioscience) in reservoir solution.

    Techniques: Binding Assay

    Clamp dynamics and comparison with the two‐helix finger FRET traces of clamp movements in the presence of different nucleotides were used to determine the number of states best fit by a Markov model. Transition density plot of idealized ATP FRET states obtained in (A). Representative traces obtained with ATPγS. The upper FRET trace was calculated from the middle traces obtained by exciting the donor fluorophore and measuring both donor (green) and acceptor (red) fluorescence. The lowest trace was obtained by exciting the acceptor fluorophore directly. The arrow indicates a bleaching event. Distribution of FRET values determined from 315 traces as in (C) fit with a Gaussian model (black curve). Comparison of high and low FRET state occupancy in ADP•P i and ADP•V i for the clamp and THF. The distributions of dwell times of the low FRET states observed in ATP were fit with a single exponential (1,539 low FRET states). The inset shows average dwell time and error, defined as the standard error based on the number of traces. As in (F), but with high FRET (1,773 high FRET states). Comparison of dwell times of the high FRET states for the two‐helix finger (THF) and clamp for different fluorophore positions. Errors as in (G) with significance based on two‐sample Kolmogorov–Smirnov tests with a 1% threshold. n.s. P = 0.012 (left), n.s. P = 0.681 (right); * P

    Journal: The EMBO Journal

    Article Title: Protein translocation by the SecA ATPase occurs by a power‐stroke mechanism

    doi: 10.15252/embj.2018101140

    Figure Lengend Snippet: Clamp dynamics and comparison with the two‐helix finger FRET traces of clamp movements in the presence of different nucleotides were used to determine the number of states best fit by a Markov model. Transition density plot of idealized ATP FRET states obtained in (A). Representative traces obtained with ATPγS. The upper FRET trace was calculated from the middle traces obtained by exciting the donor fluorophore and measuring both donor (green) and acceptor (red) fluorescence. The lowest trace was obtained by exciting the acceptor fluorophore directly. The arrow indicates a bleaching event. Distribution of FRET values determined from 315 traces as in (C) fit with a Gaussian model (black curve). Comparison of high and low FRET state occupancy in ADP•P i and ADP•V i for the clamp and THF. The distributions of dwell times of the low FRET states observed in ATP were fit with a single exponential (1,539 low FRET states). The inset shows average dwell time and error, defined as the standard error based on the number of traces. As in (F), but with high FRET (1,773 high FRET states). Comparison of dwell times of the high FRET states for the two‐helix finger (THF) and clamp for different fluorophore positions. Errors as in (G) with significance based on two‐sample Kolmogorov–Smirnov tests with a 1% threshold. n.s. P = 0.012 (left), n.s. P = 0.681 (right); * P

    Article Snippet: When indicated, 1 mM ADP•BeFx , 1 mM ADP and Vi (sodium orthovanadate, New England Biolabs), 5 mM ATPγS (Jena Bioscience, Jena, Germany), or 1 mM ADP and Pi were added after the initial 10 min, along with 1 U of hexokinase (Roche Applied Science, Germany) and 20 mM glucose, and incubated for an additional 5 min.

    Techniques: Fluorescence