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. 1992 Oct 1;287(Pt 1):101–106. doi: 10.1042/bj2870101

Estimation of the rate constants associated with the inhibitory effect of okadaic acid on type 2A protein phosphatase by time-course analysis.

A Takai 1, Y Ohno 1, T Yasumoto 1, G Mieskes 1
PMCID: PMC1133129  PMID: 1329723

Abstract

As is often the case with tightly binding inhibitors, okadaic acid produces its inhibitory effect on type 2A protein phosphatase (PP2A) in a time-dependent manner. We measured the rate constants associated with the binding of okadaic acid to PP2A by analysing the time-course of the reduction of the p-nitrophenyl phosphate (pNPP) phosphatase activity of the enzyme after application of okadaic acid. The rate constants for dissociation of okadaic acid from PP2A were also estimated from the time-course of the recovery of the activity from inhibition by okadaic acid after addition of a mouse IgG1 monoclonal antibody raised against the inhibitor. Our results show that the rate constants for the binding of okadaic acid and PP2A are of the order of 10(7) M-1.s-1, a typical value for reactions involving relatively large molecules, whereas those for their dissociation are in the range 10(-4)-10(-3) s-1. The very low values of the latter seems to be the determining factor for the exceedingly high affinity of okadaic acid for PP2A. The dissociation constants for the interaction of okadaic acid with the free enzyme and the enzyme-substrate complex, estimated as the ratio of the rate constants, are both in the range 30-40 pM, in agreement with the results of previous dose-inhibition analyses.

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Selected References

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  1. Bialojan C., Takai A. Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics. Biochem J. 1988 Nov 15;256(1):283–290. doi: 10.1042/bj2560283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. CLELAND W. W. The kinetics of enzyme-catalyzed reactions with two or more substrates or products. II. Inhibition: nomenclature and theory. Biochim Biophys Acta. 1963 Feb 12;67:173–187. doi: 10.1016/0006-3002(63)91815-8. [DOI] [PubMed] [Google Scholar]
  3. Cayla X., Goris J., Hermann J., Jessus C., Hendrix P., Merlevede W. Phosphotyrosyl phosphatase activity of the polycation-stimulated protein phosphatases and involvement of dephosphorylation in cell cycle regulation. Adv Enzyme Regul. 1990;30:265–285. doi: 10.1016/0065-2571(90)90022-t. [DOI] [PubMed] [Google Scholar]
  4. Cha S., Agarwal R. P., Parks R. E., Jr Tight-binding inhibitors-II. Non-steady state nature of inhibition of milk xanthine oxidase by allopurinol and alloxanthine and of human erythrocytic adenosine deaminase by coformycin. Biochem Pharmacol. 1975 Dec 1;24(23):2187–2197. doi: 10.1016/0006-2952(75)90051-9. [DOI] [PubMed] [Google Scholar]
  5. Cha S. Tight-binding inhibitors--III. A new approach for the determination of competition between tight-binding inhibitors and substrates--inhibition of adenosine deaminase by coformycin. Biochem Pharmacol. 1976 Dec 15;25(24):2695–2702. doi: 10.1016/0006-2952(76)90259-8. [DOI] [PubMed] [Google Scholar]
  6. Cha S. Tight-binding inhibitors-I. Kinetic behavior. Biochem Pharmacol. 1975 Dec 1;24(23):2177–2185. doi: 10.1016/0006-2952(75)90050-7. [DOI] [PubMed] [Google Scholar]
  7. Cohen P., Holmes C. F., Tsukitani Y. Okadaic acid: a new probe for the study of cellular regulation. Trends Biochem Sci. 1990 Mar;15(3):98–102. doi: 10.1016/0968-0004(90)90192-e. [DOI] [PubMed] [Google Scholar]
  8. Cohen P. The structure and regulation of protein phosphatases. Annu Rev Biochem. 1989;58:453–508. doi: 10.1146/annurev.bi.58.070189.002321. [DOI] [PubMed] [Google Scholar]
  9. Cornish-Bowden A., Porter W. R., Trager W. F. Evaluation of distribution-free confidence limits for enzyme kinetic parameters. J Theor Biol. 1978 Sep 21;74(2):163–175. doi: 10.1016/0022-5193(78)90069-3. [DOI] [PubMed] [Google Scholar]
  10. Goris J., Hermann J., Hendrix P., Ozon R., Merlevede W. Okadaic acid, a specific protein phosphatase inhibitor, induces maturation and MPF formation in Xenopus laevis oocytes. FEBS Lett. 1989 Mar 13;245(1-2):91–94. doi: 10.1016/0014-5793(89)80198-x. [DOI] [PubMed] [Google Scholar]
  11. Haystead T. A., Sim A. T., Carling D., Honnor R. C., Tsukitani Y., Cohen P., Hardie D. G. Effects of the tumour promoter okadaic acid on intracellular protein phosphorylation and metabolism. Nature. 1989 Jan 5;337(6202):78–81. doi: 10.1038/337078a0. [DOI] [PubMed] [Google Scholar]
  12. Henderson P. J. A linear equation that describes the steady-state kinetics of enzymes and subcellular particles interacting with tightly bound inhibitors. Biochem J. 1972 Apr;127(2):321–333. doi: 10.1042/bj1270321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hescheler J., Mieskes G., Rüegg J. C., Takai A., Trautwein W. Effects of a protein phosphatase inhibitor, okadaic acid, on membrane currents of isolated guinea-pig cardiac myocytes. Pflugers Arch. 1988 Aug;412(3):248–252. doi: 10.1007/BF00582504. [DOI] [PubMed] [Google Scholar]
  14. Ishihara H., Martin B. L., Brautigan D. L., Karaki H., Ozaki H., Kato Y., Fusetani N., Watabe S., Hashimoto K., Uemura D. Calyculin A and okadaic acid: inhibitors of protein phosphatase activity. Biochem Biophys Res Commun. 1989 Mar 31;159(3):871–877. doi: 10.1016/0006-291x(89)92189-x. [DOI] [PubMed] [Google Scholar]
  15. MacKintosh C., Beattie K. A., Klumpp S., Cohen P., Codd G. A. Cyanobacterial microcystin-LR is a potent and specific inhibitor of protein phosphatases 1 and 2A from both mammals and higher plants. FEBS Lett. 1990 May 21;264(2):187–192. doi: 10.1016/0014-5793(90)80245-e. [DOI] [PubMed] [Google Scholar]
  16. Porter W. R., Trager W. F. Improved non-parametric statistical methods for the estimation of Michaelis-Menten kinetic parameters by the direct linear plot. Biochem J. 1977 Feb 1;161(2):293–302. doi: 10.1042/bj1610293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Takai A., Mieskes G. Inhibitory effect of okadaic acid on the p-nitrophenyl phosphate phosphatase activity of protein phosphatases. Biochem J. 1991 Apr 1;275(Pt 1):233–239. doi: 10.1042/bj2750233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Takai A., Murata M., Torigoe K., Isobe M., Mieskes G., Yasumoto T. Inhibitory effect of okadaic acid derivatives on protein phosphatases. A study on structure-affinity relationship. Biochem J. 1992 Jun 1;284(Pt 2):539–544. doi: 10.1042/bj2840539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Takai A. Okadaic acid. Protein phosphatase inhibition and muscle contractile effects. J Muscle Res Cell Motil. 1988 Dec;9(6):563–565. doi: 10.1007/BF01738761. [DOI] [PubMed] [Google Scholar]
  20. Tung H. Y., Resink T. J., Hemmings B. A., Shenolikar S., Cohen P. The catalytic subunits of protein phosphatase-1 and protein phosphatase 2A are distinct gene products. Eur J Biochem. 1984 Feb 1;138(3):635–641. doi: 10.1111/j.1432-1033.1984.tb07962.x. [DOI] [PubMed] [Google Scholar]
  21. Usagawa T., Nishimura M., Itoh Y., Uda T., Yasumoto T. Preparation of monoclonal antibodies against okadaic acid prepared from the sponge Halichondria okadai. Toxicon. 1989;27(12):1323–1330. doi: 10.1016/0041-0101(89)90064-0. [DOI] [PubMed] [Google Scholar]
  22. Williams J. W., Morrison J. F., Duggleby R. G. Methotrexate, a high-affinity pseudosubstrate of dihydrofolate reductase. Biochemistry. 1979 Jun 12;18(12):2567–2573. doi: 10.1021/bi00579a021. [DOI] [PubMed] [Google Scholar]
  23. Williams J. W., Morrison J. F. The kinetics of reversible tight-binding inhibition. Methods Enzymol. 1979;63:437–467. doi: 10.1016/0076-6879(79)63019-7. [DOI] [PubMed] [Google Scholar]

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