Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1997 Feb 1;321(Pt 3):671–676. doi: 10.1042/bj3210671

Effects of pH on phosphorylation of the Ca2+-ATPase of sarcoplasmic reticulum by inorganic phosphate.

Y M Khan 1, J M East 1, A G Lee 1
PMCID: PMC1218121  PMID: 9032452

Abstract

The fluorescence intensity of the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum (SR) labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin has been shown to decrease on phosphorylation of the ATPase with P(i), this providing a convenient measure of the level of phosphorylation. Comparison of the fluorescence decrease observed with ATP and with high concentrations of P(i) fix the value of the equilibrium constant for the phosphorylation reaction E2PMg<==>E2P(i)Mg at pH 6.0 at about 2. Studies of the pH-dependence of phosphorylation show that H2PO4- and HPO4(2)- bind to the ATPase with equal affinity, but that only binding of H2PO4- leads to phosphorylation, described by an equilibrium constant of 2.3. Luminal Ca2+ can bind to a pair of sites on the ATPase, with affinities of 1.3 x 10(3) and 1.7 x 10(3) M(-1) for the unphosphorylated and phosphorylated forms of the ATPase respectively, with stronger binding of Ca2+ to the phosphorylated form resulting in an increase in the effective equilibrium constant for phosphorylation.

Full Text

The Full Text of this article is available as a PDF (470.5 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barrabin H., Scofano H. M., Inesi G. Adenosinetriphosphatase site stoichiometry in sarcoplasmic reticulum vesicles and purified enzyme. Biochemistry. 1984 Mar 27;23(7):1542–1548. doi: 10.1021/bi00302a031. [DOI] [PubMed] [Google Scholar]
  2. Barrabin H., de Meis L. Vanadate inhibition of the Ca-ATPase activity of sarcoplasmic reticulum vesicles. An Acad Bras Cienc. 1982 Dec;54(4):743–751. [PubMed] [Google Scholar]
  3. Beil F. U., von Chak D., Hasselbach W. Phosphorylation from inorganic phosphate and ATP synthesis of sarcoplasmic membranes. Eur J Biochem. 1977 Nov 15;81(1):151–164. doi: 10.1111/j.1432-1033.1977.tb11936.x. [DOI] [PubMed] [Google Scholar]
  4. Caldeira M. T., de Meis L. Effects of a Ca2+ gradient and water activity on the phosphorylation of Ca(2+)-ATPase by Pi. FEBS Lett. 1991 Aug 19;288(1-2):10–12. doi: 10.1016/0014-5793(91)80990-k. [DOI] [PubMed] [Google Scholar]
  5. Coll R. J., Murphy A. J. Purification of the CaATPase of sarcoplasmic reticulum by affinity chromatography. J Biol Chem. 1984 Nov 25;259(22):14249–14254. [PubMed] [Google Scholar]
  6. East J. M., Lee A. G. Lipid selectivity of the calcium and magnesium ion dependent adenosinetriphosphatase, studied with fluorescence quenching by a brominated phospholipid. Biochemistry. 1982 Aug 17;21(17):4144–4151. doi: 10.1021/bi00260a035. [DOI] [PubMed] [Google Scholar]
  7. Fabiato A., Fabiato F. Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J Physiol (Paris) 1979;75(5):463–505. [PubMed] [Google Scholar]
  8. Froud R. J., Lee A. G. A model for the phosphorylation of the Ca2+ + Mg2+-activated ATPase by phosphate. Biochem J. 1986 Jul 1;237(1):207–215. doi: 10.1042/bj2370207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gould G. W., East J. M., Froud R. J., McWhirter J. M., Stefanova H. I., Lee A. G. A kinetic model for the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum. Biochem J. 1986 Jul 1;237(1):217–227. doi: 10.1042/bj2370217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Henderson I. M., Khan Y. M., East J. M., Lee A. G. Binding of Ca2+ to the (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum: equilibrium studies. Biochem J. 1994 Feb 1;297(Pt 3):615–624. doi: 10.1042/bj2970615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hughes G., Starling A. P., East J. M., Lee A. G. Mechanism of inhibition of the Ca(2+)-ATPase by spermine and other polycationic compounds. Biochemistry. 1994 Apr 26;33(16):4745–4754. doi: 10.1021/bi00182a001. [DOI] [PubMed] [Google Scholar]
  12. Inesi G., Lewis D., Murphy A. J. Interdependence of H+, Ca2+, and Pi (or vanadate) sites in sarcoplasmic reticulum ATPase. J Biol Chem. 1984 Jan 25;259(2):996–1003. [PubMed] [Google Scholar]
  13. Jencks W. P., Yang T., Peisach D., Myung J. Calcium ATPase of sarcoplasmic reticulum has four binding sites for calcium. Biochemistry. 1993 Jul 13;32(27):7030–7034. doi: 10.1021/bi00078a031. [DOI] [PubMed] [Google Scholar]
  14. Kanazawa T., Boyer P. D. Occurrence and characteristics of a rapid exchange of phosphate oxygens catalyzed by sarcoplasmic reticulum vesicles. J Biol Chem. 1973 May 10;248(9):3163–3172. doi: 10.2172/4473783. [DOI] [PubMed] [Google Scholar]
  15. Lacapère J. J., Gingold M. P., Champeil P., Guillain F. Sarcoplasmic reticulum ATPase phosphorylation from inorganic phosphate in the absence of a calcium gradient. Steady state and kinetic fluorescence studies. J Biol Chem. 1981 Mar 10;256(5):2302–2306. [PubMed] [Google Scholar]
  16. Lee A. G., Baker K., Khan Y. M., East J. M. Effects of K+ on the binding of Ca2+ to the Ca(2+)-ATPase of sarcoplasmic reticulum. Biochem J. 1995 Jan 1;305(Pt 1):225–231. doi: 10.1042/bj3050225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Martin D. W., Tanford C. Phosphorylation of calcium adenosinetriphosphatase by inorganic phosphate: van't Hoff analysis of enthalpy changes. Biochemistry. 1981 Aug 4;20(16):4597–4602. doi: 10.1021/bi00519a013. [DOI] [PubMed] [Google Scholar]
  18. Myung J., Jencks W. P. Lumenal and cytoplasmic binding sites for calcium on the calcium ATPase of sarcoplasmic reticulum are different and independent. Biochemistry. 1994 Jul 26;33(29):8775–8785. doi: 10.1021/bi00195a020. [DOI] [PubMed] [Google Scholar]
  19. Orlowski S., Lund S., Møller J., Champeil P. Phosphoenzymes formed from Mg.ATP and Ca.ATP during pre-steady state kinetics of sarcoplasmic reticulum ATPase. J Biol Chem. 1988 Nov 25;263(33):17576–17583. [PubMed] [Google Scholar]
  20. Prager R., Punzengruber C., Kolassa N., Winkler F., Suko J. Ionized and bound calcium inside isolated sarcoplasmic reticulum of skeletal muscle and its significance in phosphorylation of adenosine triphosphatase by orthophosphate. Eur J Biochem. 1979 Jun;97(1):239–250. doi: 10.1111/j.1432-1033.1979.tb13108.x. [DOI] [PubMed] [Google Scholar]
  21. Punzengruber C., Prager R., Kolassa N., Winkler F., Suko J. Calcium gradient-dependent and calcium gradient-independent phosphorylation of sarcoplasmic reticulum by orthophosphate. The role of magnesium. Eur J Biochem. 1978 Dec;92(2):349–359. doi: 10.1111/j.1432-1033.1978.tb12754.x. [DOI] [PubMed] [Google Scholar]
  22. Sagara Y., Fernandez-Belda F., de Meis L., Inesi G. Characterization of the inhibition of intracellular Ca2+ transport ATPases by thapsigargin. J Biol Chem. 1992 Jun 25;267(18):12606–12613. [PubMed] [Google Scholar]
  23. Shigekawa M., Wakabayashi S., Nakamura H. Effect of divalent cation bound to the ATPase of sarcoplasmic reticulum. Activation of phosphoenzyme hydrolysis by Mg2+. J Biol Chem. 1983 Dec 10;258(23):14157–14161. [PubMed] [Google Scholar]
  24. Starling A. P., East J. M., Lee A. G. Effects of phosphatidylcholine fatty acyl chain length on calcium binding and other functions of the (Ca(2+)-Mg2+)-ATPase. Biochemistry. 1993 Feb 16;32(6):1593–1600. doi: 10.1021/bi00057a025. [DOI] [PubMed] [Google Scholar]
  25. Starling A. P., East J. M., Lee A. G. Effects of phospholipid fatty acyl chain length on phosphorylation and dephosphorylation of the Ca(2+)-ATPase. Biochem J. 1995 Sep 15;310(Pt 3):875–879. doi: 10.1042/bj3100875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Stefanova H. I., East J. M., Gore M. G., Lee A. G. Labeling the (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum with 4-(bromomethyl)-6,7-dimethoxycoumarin: detection of conformational changes. Biochemistry. 1992 Jul 7;31(26):6023–6031. doi: 10.1021/bi00141a010. [DOI] [PubMed] [Google Scholar]
  27. Suko J., Plank B., Preis P., Kolassa N., Hellmann G., Conca W. Formation of magnesium-phosphoenzyme and magnesium-calcium-phosphoenzyme in the phosphorylation of adenosine triphosphatase by orthophosphate in sarcoplasmic reticulum. Models of a reaction sequence. Eur J Biochem. 1981 Oct;119(2):225–236. doi: 10.1111/j.1432-1033.1981.tb05598.x. [DOI] [PubMed] [Google Scholar]
  28. Tanford C., Martin D. W. Equilibrium constants for some steps of the reaction cycle of the sarcoplasmic reticulum calcium pump. Z Naturforsch C. 1982 May-Jun;37(5-6):522–526. doi: 10.1515/znc-1982-5-626. [DOI] [PubMed] [Google Scholar]
  29. Wictome M., Khan Y. M., East J. M., Lee A. G. Binding of sesquiterpene lactone inhibitors to the Ca(2+)-ATPase. Biochem J. 1995 Sep 15;310(Pt 3):859–868. doi: 10.1042/bj3100859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wictome M., Michelangeli F., Lee A. G., East J. M. The inhibitors thapsigargin and 2,5-di(tert-butyl)-1,4-benzohydroquinone favour the E2 form of the Ca2+,Mg(2+)-ATPase. FEBS Lett. 1992 Jun 15;304(2-3):109–113. doi: 10.1016/0014-5793(92)80599-c. [DOI] [PubMed] [Google Scholar]
  31. de Meis L., Martins O. B., Alves E. W. Role of water, hydrogen ion, and temperature on the synthesis of adenosine triphosphate by the sarcoplasmic reticulum adenosine triphosphatase in the absence of a calcium ion gradient. Biochemistry. 1980 Sep 2;19(18):4252–4261. doi: 10.1021/bi00559a017. [DOI] [PubMed] [Google Scholar]
  32. de Meis L., Vianna A. L. Energy interconversion by the Ca2+-dependent ATPase of the sarcoplasmic reticulum. Annu Rev Biochem. 1979;48:275–292. doi: 10.1146/annurev.bi.48.070179.001423. [DOI] [PubMed] [Google Scholar]
  33. de Meis L., de Souza Otero A., Martins O. B., Alves E. W., Inesi G., Nakamoto R. Phosphorylation of sarcoplasmic reticulum ATPase by orthophosphate in the absence of Ca2+ gradient. Contribution of water activity to the enthalpy and the entropy changes. J Biol Chem. 1982 May 10;257(9):4993–4998. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES