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. 2002 Jan 15;361(Pt 2):355–361. doi: 10.1042/0264-6021:3610355

Pre-steady-state phosphorylation and dephosphorylation of detergent-purified plasma-membrane Ca2+-ATPase.

Luis M Bredeston 1, Alcides F Rega 1
PMCID: PMC1222315  PMID: 11772407

Abstract

Pre-steady-state phosphorylation and dephosphorylation of purified and phospholipid-depleted plasma-membrane Ca(2+)-ATPase (PMCA) solubilized in the detergent polyoxyethylene 10 lauryl ether were studied at 25 degrees C. The time course of phosphorylation with ATP of the enzyme associated with Ca(2+), probably the true phosphorylation reaction, showed a fast phase (k(app) near 400 s(-1)) followed by a slow phase (k(app)=23 s(-1)). With asolectin or acidic phosphatidylinositol, the concentration of phosphoenzyme (EP) increased at as high a rate as before, passed through a maximum at 4 ms and stabilized at a steady level that was approx. half that without lipids. Calmodulin (CaM) did not change the rate of the fast phase, accelerated the slow phase (k(app)=93 s(-1)) and increased [EP] with small changes in the shape of the time course. Dephosphorylation was slow (k(app)=30 s(-1)) and insensitive to CaM. Asolectin accelerated dephosphorylation, which followed biexponential kinetics with fast (k(app)=220 s(-1)) and slow (k(app)=20 s(-1)) components. CaM stimulated the fast component by nearly 50%. The results show that the behaviour of the PMCA is complex, and suggest that acidic phospholipids and CaM activate PMCA through different mechanisms. Acceleration of dephosphorylation seems relevant during activation of the PMCA by acidic phospholipids.

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Selected References

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  1. Adamo H. P., Rega A. F., Garrahan P. J. Pre-steady-state phosphorylation of the human red cell Ca2+-ATPase. J Biol Chem. 1988 Nov 25;263(33):17548–17554. [PubMed] [Google Scholar]
  2. Adamo H. P., Rega A. F., Garrahan P. J. The E2 in equilibrium E1 transition of the Ca2(+)-ATPase from plasma membranes studied by phosphorylation. J Biol Chem. 1990 Mar 5;265(7):3789–3792. [PubMed] [Google Scholar]
  3. Bredeston L. M., Rega A. F. Phosphatidylcholine makes specific activity of the purified Ca(2+)-ATPase from plasma membranes independent of enzyme concentration. Biochim Biophys Acta. 1999 Aug 20;1420(1-2):57–62. doi: 10.1016/s0005-2736(99)00084-x. [DOI] [PubMed] [Google Scholar]
  4. Brodin P., Falchetto R., Vorherr T., Carafoli E. Identification of two domains which mediate the binding of activating phospholipids to the plasma-membrane Ca2+ pump. Eur J Biochem. 1992 Mar 1;204(2):939–946. doi: 10.1111/j.1432-1033.1992.tb16715.x. [DOI] [PubMed] [Google Scholar]
  5. Caride A. J., Rega A. F., Garrahan P. J. The reaction of Mg2+ with the Ca2+-ATPase from human red cell membranes and its modification by Ca2+. Biochim Biophys Acta. 1986 Dec 16;863(2):165–177. doi: 10.1016/0005-2736(86)90256-7. [DOI] [PubMed] [Google Scholar]
  6. Castello P. R., Caride A. J., González Flecha F. L., Fernández H. N., Rossi J. P., Delfino J. M. Identification of transmembrane domains of the red cell calcium pump with a new photoactivatable phospholipidic probe. Biochem Biophys Res Commun. 1994 May 30;201(1):194–200. doi: 10.1006/bbrc.1994.1688. [DOI] [PubMed] [Google Scholar]
  7. Filoteo A. G., Enyedi A., Penniston J. T. The lipid-binding peptide from the plasma membrane Ca2+ pump binds calmodulin, and the primary calmodulin-binding domain interacts with lipid. J Biol Chem. 1992 Jun 15;267(17):11800–11805. [PubMed] [Google Scholar]
  8. Froehlich J. P., Taniguchi K., Fendler K., Mahaney J. E., Thomas D. D., Albers R. W. Complex kinetic behavior in the Na,K- and Ca-ATPases. Evidence for subunit-subunit interactions and energy conservation during catalysis. Ann N Y Acad Sci. 1997 Nov 3;834:280–296. doi: 10.1111/j.1749-6632.1997.tb52259.x. [DOI] [PubMed] [Google Scholar]
  9. Froehlich J. P., Taylor E. W. Transient state kinetic studies of sarcoplasmic reticulum adenosine triphosphatase. J Biol Chem. 1975 Mar 25;250(6):2013–2021. [PubMed] [Google Scholar]
  10. Gietzen K., Tejcka M., Wolf H. U. Calmodulin affinity chromatography yields a functional purified erythrocyte (Ca+ + Mg2+)-dependent adenosine triphosphatase. Biochem J. 1980 Jul 1;189(1):81–88. doi: 10.1042/bj1890081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Glynn I. M., Chappell J. B. A simple method for the preparation of 32-P-labelled adenosine triphosphate of high specific activity. Biochem J. 1964 Jan;90(1):147–149. doi: 10.1042/bj0900147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Herscher C. J., Rega A. F., Garrahan P. J. The dephosphorylation reaction of the Ca(2+)-ATPase from plasma membranes. J Biol Chem. 1994 Apr 8;269(14):10400–10406. [PubMed] [Google Scholar]
  13. Ikemoto N., Nelson R. W. Oligomeric regulation of the later reaction steps of the sarcoplasmic reticulum calcium ATPase. J Biol Chem. 1984 Oct 10;259(19):11790–11797. [PubMed] [Google Scholar]
  14. Kosk-Kosicka D., Bzdega T. Activation of the erythrocyte Ca2+-ATPase by either self-association or interaction with calmodulin. J Biol Chem. 1988 Dec 5;263(34):18184–18189. [PubMed] [Google Scholar]
  15. Kosk-Kosicka D., Bzdega T. Effects of calmodulin on erythrocyte Ca2(+)-ATPase activation and oligomerization. Biochemistry. 1990 Apr 17;29(15):3772–3777. doi: 10.1021/bi00467a025. [DOI] [PubMed] [Google Scholar]
  16. Kosk-Kosicka D., Inesi G. Cooperative calcium binding and calmodulin regulation in the calcium-dependent adenosine triphosphatase purified from the erythrocyte membrane. FEBS Lett. 1985 Sep 9;189(1):67–71. doi: 10.1016/0014-5793(85)80843-7. [DOI] [PubMed] [Google Scholar]
  17. Kosk-Kosicka D., Scaillet S., Inesi G. The partial reactions in the catalytic cycle of the calcium-dependent adenosine triphosphatase purified from erythrocyte membranes. J Biol Chem. 1986 Mar 5;261(7):3333–3338. [PubMed] [Google Scholar]
  18. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  19. Lehotsky J., Raeymaekers L., Missiaen L., Wuytack F., De Smedt H., Casteels R. Stimulation of the catalytic cycle of the Ca2+ pump of porcine plasma-membranes by negatively charged phospholipids. Biochim Biophys Acta. 1992 Mar 23;1105(1):118–124. doi: 10.1016/0005-2736(92)90169-m. [DOI] [PubMed] [Google Scholar]
  20. Levi V., Rossi J. P., Castello P. R., González Flecha F. L. Oligomerization of the plasma membrane calcium pump involves two regions with different thermal stability. FEBS Lett. 2000 Oct 20;483(2-3):99–103. doi: 10.1016/s0014-5793(00)02093-7. [DOI] [PubMed] [Google Scholar]
  21. Mahaney J. E., Froehlich J. P., Thomas D. D. Conformational transitions of the sarcoplasmic reticulum Ca-ATPase studied by time-resolved EPR and quenched-flow kinetics. Biochemistry. 1995 Apr 11;34(14):4864–4879. doi: 10.1021/bi00014a044. [DOI] [PubMed] [Google Scholar]
  22. Mårdh S., Zetterqvist O. Phosphorylation and dephosphorylation reactions of bovine brain (Na+-K+)-stimulated ATP phosphohydrolase studied by a rapid mixing technique. Biochim Biophys Acta. 1974 Jun 18;350(2):473–483. doi: 10.1016/0005-2744(74)90523-3. [DOI] [PubMed] [Google Scholar]
  23. Niggli V., Adunyah E. S., Penniston J. T., Carafoli E. Purified (Ca2+-Mg2+)-ATPase of the erythrocyte membrane. Reconstitution and effect of calmodulin and phospholipids. J Biol Chem. 1981 Jan 10;256(1):395–401. [PubMed] [Google Scholar]
  24. Penniston J. T., Filoteo A. G., McDonough C. S., Carafoli E. Purification, reconstitution, and regulation of plasma membrane Ca2+-pumps. Methods Enzymol. 1988;157:340–351. doi: 10.1016/0076-6879(88)57089-1. [DOI] [PubMed] [Google Scholar]
  25. Peterson G. L. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem. 1977 Dec;83(2):346–356. doi: 10.1016/0003-2697(77)90043-4. [DOI] [PubMed] [Google Scholar]
  26. Ronner P., Gazzotti P., Carafoli E. A lipid requirement for the (Ca2+ + Mg2+)-activated ATPase of erythrocyte membranes. Arch Biochem Biophys. 1977 Mar;179(2):578–583. doi: 10.1016/0003-9861(77)90146-1. [DOI] [PubMed] [Google Scholar]
  27. Sackett D. L., Kosk-Kosicka D. The active species of plasma membrane Ca2+-ATPase are a dimer and a monomer-calmodulin complex. J Biol Chem. 1996 Apr 26;271(17):9987–9991. doi: 10.1074/jbc.271.17.9987. [DOI] [PubMed] [Google Scholar]
  28. Strehler E. E., James P., Fischer R., Heim R., Vorherr T., Filoteo A. G., Penniston J. T., Carafoli E. Peptide sequence analysis and molecular cloning reveal two calcium pump isoforms in the human erythrocyte membrane. J Biol Chem. 1990 Feb 15;265(5):2835–2842. [PubMed] [Google Scholar]
  29. Sumida M., Wang T., Schwartz A., Younkin C., Froehlich J. P. The Ca2+-ATPase partial reactions in cardiac and skeletal sarcoplasmic reticulum. A comparison of transient state kinetic data. J Biol Chem. 1980 Feb 25;255(4):1497–1503. [PubMed] [Google Scholar]
  30. Takisawa H., Tonomura Y. Factors affecting the transient phase of the Ca2+, Mg2+-dependent ATPase reaction of sarcoplasmic reticulum from skeletal muscle. J Biochem. 1978 May;83(5):1275–1284. doi: 10.1093/oxfordjournals.jbchem.a132034. [DOI] [PubMed] [Google Scholar]
  31. Vorherr T., Kessler T., Hofmann F., Carafoli E. The calmodulin-binding domain mediates the self-association of the plasma membrane Ca2+ pump. J Biol Chem. 1991 Jan 5;266(1):22–27. [PubMed] [Google Scholar]

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