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. 1996 Apr 15;315(Pt 2):673–677. doi: 10.1042/bj3150673

Pre-steady-state kinetic study of the effects of K+ on the partial reactions of the catalytic cycle of the plasma membrane Ca(2+)-ATPase.

C J Herscher 1, A F Rega 1, H P Adamo 1
PMCID: PMC1217249  PMID: 8615846

Abstract

The effects of 100 mM K+ on the partial reactions that take place during ATP hydrolysis on the calcium ion-dependent ATPase from plasma membrane (PM-Ca(2+)-ATPase) were studied at 37 degrees C on fragmented intact membranes from pig red cells by means of a rapid chemical quenching technique. At 10 microM [gamma-32P]ATP plus non-limiting concentrations of Ca2+ and Mg2+, K+ increased the k(app) of formation by 140% to 84 11 s-1 and the steady-state level of phosphoenzyme (EP) by 25% to 3.4 0.17 pmol/mg of protein. If added together with [gamma-32P]ATP at the beginning of phosphorylation, K+ was much less effective than if added earlier, indicating that it did not act on the phosphorylation reaction. Measurements of the E2 --> E1 transition by phosphorylation showed that in medium with Ca2+ and Mg2+, K+ increased the k(app) of the transition by 55% to 14 3 s-1 and the apparent concentration of E1 by 45%, suggesting that this may be the cause of the increased rate of phosphorylation observed in enzyme preincubated with K+. The presence of K+ did not change the slow decay of EP without Mg2+ but activated the decay of EP made with Mg2+, increasing its k(app) by 60% to 91 12 s-1. In contrast with observations made during phosphorylation, if added at the beginning of dephosphorylation K+ was fully effective in favouring decomposition of EP made in medium containing no K+. In the presence of either 3mM ATP or 3 mM ATP plus calmodulin, which activate hydrolysis of CaE2P, the effect of K+ on dephosphorylation was conserved. Because the sites for K+ are intracellular and the concentration of K+ in normal red cells is above 100 mM, the effects described here must be taken into account to describe the catalytic cycle of the PM-Ca(2+)-ATPase under physiological conditions.

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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. Briggs F. N., Wise R. M., Hearn J. A. The effect of lithium and potassium on the transient state kinetics of the (Ca + Mg)-ATPase of cardiac sarcoplasmic reticulum. J Biol Chem. 1978 Sep 10;253(17):5884–5885. [PubMed] [Google Scholar]
  4. 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]
  5. Chaloub R. M., de Meis L. Effect of K+ on phosphorylation of the sarcoplasmic reticulum ATPase by either Pi or ATP. J Biol Chem. 1980 Jul 10;255(13):6168–6172. [PubMed] [Google Scholar]
  6. Champeil P., Guillain F., Vénien C., Gingold M. P. Interaction of magnesium and inorganic phosphate with calcium-deprived sarcoplasmic reticulum adenosinetriphosphatase as reflected by organic solvent induced perturbation. Biochemistry. 1985 Jan 1;24(1):69–81. doi: 10.1021/bi00322a012. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Jones L. R., Besch H. R., Jr, Watanabe A. M. Regulation of the calcium pump of cardiac sarcoplasmic reticulum. Interactive roles of potassium and ATP on the phosphoprotein intermediate of the (K+,Ca2+)-ATPase. J Biol Chem. 1978 Mar 10;253(5):1643–1653. [PubMed] [Google Scholar]
  10. 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]
  11. Kratje R. B., Garrahan P. J., Rega A. F. The effects of alkali metal ions on active Ca2+ transport in reconstituted ghosts from human red cells. Biochim Biophys Acta. 1983 May 26;731(1):40–46. doi: 10.1016/0005-2736(83)90395-4. [DOI] [PubMed] [Google Scholar]
  12. Larocca J. N., Rega A. F., Garrahan P. J. Phosphorylation and dephosphorylation of the Ca2+ pump of human red cells in the presence of monovalent cations. Biochim Biophys Acta. 1981 Jul 6;645(1):10–16. doi: 10.1016/0005-2736(81)90505-8. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Medda P., Fassold E., Hasselbach W. The effect of monovalent and divalent cations on the ATP-dependent Ca2+-binding and phosphorylation during the reaction cycle of the sarcoplasmic reticulum Ca2+-transport ATPase. Eur J Biochem. 1987 Jun 1;165(2):251–259. doi: 10.1111/j.1432-1033.1987.tb11435.x. [DOI] [PubMed] [Google Scholar]
  16. Moutin M. J., Dupont Y. Interaction of potassium and magnesium with the high affinity calcium-binding sites of the sarcoplasmic reticulum calcium-ATPase. J Biol Chem. 1991 Mar 25;266(9):5580–5586. [PubMed] [Google Scholar]
  17. Shigekawa M., Akowitz A. A. On the mechanism of Ca2+-dependent adenosine triphosphatase of sarcoplasmic reticulum. Occurrence of two types of phosphoenzyme intermediates in the presence of KCl. J Biol Chem. 1979 Jun 10;254(11):4726–4730. [PubMed] [Google Scholar]
  18. Shigekawa M., Dougherty J. P. Reaction mechanism of Ca2+-dependent ATP hydrolysis by skeletal muscle sarcoplasmic reticulum in the absence of added alkali metal salts. II. Kinetic properties of the phosphoenzyme formed at the steady state in high Mg2+ and low Ca2+ concentrations. J Biol Chem. 1978 Mar 10;253(5):1451–1457. [PubMed] [Google Scholar]
  19. Shigekawa M., Pearl L. J. Activation of calcium transport in skeletal muscle sarcoplasmic reticulum by monovalent cations. J Biol Chem. 1976 Nov 25;251(22):6947–6952. [PubMed] [Google Scholar]

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