A Colorimetric Assay for the Determination of Acid Nucleoside Triphosphatase Activity

Kirchgesser, Michael; Dahlmann, N

doi:10.1515/cclm.1990.28.6.407

Cited by 7 publications

(7 citation statements)

References 3 publications

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“…2F, Ca 2ϩ -ATPase activity in SR fractions was determined by measuring the amount of P i released after the addition of ATP using the malachite green ATPase method (20). Briefly, as described above, after incubation (30 min, 30°C) without or with 250 units of rPKAc, SR vesicles were isolated and suspended in sucrose/K-PIPES buffer.…”

Section: Camentioning

confidence: 99%

Regulation of Sarcoplasmic Reticulum Ca2+ ATPase 2 (SERCA2) Activity by Phosphodiesterase 3A (PDE3A) in Human Myocardium

Ahmad

Shen

Vandeput

et al. 2015

Journal of Biological Chemistry

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Background: PDE3A is part of a SERCA2 signaling complex in cardiac myocytes. Results: PDE3, not PDE4, regulates the activation of SERCA2 by PKA in human myocardium; phosphorylation of PDE3A1 at Ser-292/Ser-293 promotes its integration into the SERCA2 signaling complex. Conclusion: PDE3A1 regulates cAMP-mediated control of SERCA2 through its phosphorylation-dependent interaction with SERCA2. Significance: Targeted disruption of the PDE3A1-SERCA2 interaction may provide a new therapeutic approach for heart failure.

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Section: Camentioning

confidence: 99%

Regulation of Sarcoplasmic Reticulum Ca2+ ATPase 2 (SERCA2) Activity by Phosphodiesterase 3A (PDE3A) in Human Myocardium

Ahmad

Shen

Vandeput

et al. 2015

Journal of Biological Chemistry

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“…The protein concentrations were determined by the Amido Black method (25). Ca 2ϩ -ATPase activity in SR vesicles was assayed by malachite green ATPase method (26) in the presence of a Ca 2ϩ ionophore (1 M ionomycin) and the absence or presence of SIN-1. Mg 2ϩ -ATPase remaining in the SR preparations was subtracted from the total ATPase by adding 1 mM EGTA to assay media.…”

Section: Materials-[mentioning

confidence: 99%

Classes of Thiols That Influence the Activity of the Skeletal Muscle Calcium Release Channel

Sun

et al. 2001

Journal of Biological Chemistry

150

133

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The skeletal muscle Ca 2؉ release channel/ryanodine receptor (RyR1) is a prototypic redox-responsive ion channel. Nearly half of the 101 cysteines per RyR1 subunit are kept in a reduced (free thiol) state under conditions comparable with resting muscle. Here we assessed the effects of physiological determinants of cellular redox state (oxygen tension, reduced (GSH) or oxidized (GSSG) glutathione, and NO/O 2 . (released by 3-morpholinosydnonimine)) on RyR1 redox state and activity. Oxidation of ϳ10 RyR1 thiols (from ϳ48 to ϳ38 thiols/RyR1 subunit) had little effect on channel activity. Channel activity increased reversibly as the number of thiols was further reduced to ϳ23/subunit, whereas more extensive oxidation (to ϳ13 thiols/subunit) inactivated the channel irreversibly. Neither S-nitrosylation nor tyrosine nitration contributed to these effects. The results identify at least three functional classes of RyR1 thiols and suggest that 1) the channel may be protected from oxidation by a large reservoir of functionally inert thiols, 2) the channel may be designed to respond to moderate oxidative stress by a change in activation setpoint, and 3) the channel is susceptible to oxidative injury under more extensive conditions.

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“…Colorimetric assays such as the Fiske-Subbarow and malachite green tests for the release of inorganic phosphate are widely used for measuring ATPase activity and other processes involving free phosphate (3,10). Here we discuss an automated, high-throughput screen designed to obtain efficient and effective small-molecule inhibitors of apyrase from a library of compounds.…”

Section: Introductionmentioning

confidence: 99%

Automated Colorimetric Screen for Apyrase Inhibitors

et al. 2002

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Apyrases are enzymes that efficiently hydrolyze ATP and ADP and may operate both inside and outside the cell. Although apyrases are important to a variety of cellular mechanisms and uses in industry, there are no available apyrase-specific inhibitors. Colorimetric assays based on the Fiske-Subbarow method for measuring inorganic phosphate are able to detect the release of inorganic phosphate from ATP and other nucleotides. We found that this type of assay could be automated and used to screen for apyrase-inhibiting compounds by assaying for a reduction in released phosphate in the presence of potential inhibitors. The automation of this assay allowed for the successful screening of a commercially available compound library. Several low molecular weight compounds were identified that, when used at micromolar concentrations, effectively inhibited apyrase activity.

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A Colorimetric Assay for the Determination of Acid Nucleoside Triphosphatase Activity

Cited by 7 publications

References 3 publications

Regulation of Sarcoplasmic Reticulum Ca2+ ATPase 2 (SERCA2) Activity by Phosphodiesterase 3A (PDE3A) in Human Myocardium

Regulation of Sarcoplasmic Reticulum Ca2+ ATPase 2 (SERCA2) Activity by Phosphodiesterase 3A (PDE3A) in Human Myocardium

Classes of Thiols That Influence the Activity of the Skeletal Muscle Calcium Release Channel

Automated Colorimetric Screen for Apyrase Inhibitors

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