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. 1986 Jan 1;87(1):47–72. doi: 10.1085/jgp.87.1.47

Effects of altering the ATP/ADP ratio on pump-mediated Na/K and Na/Na exchanges in resealed human red blood cell ghosts

PMCID: PMC2217126  PMID: 3950576

Abstract

Resealed human red blood cell ghosts were prepared to contain a range of ADP concentrations at fixed ATP concentrations and vice versa. ATP/ADP ratios ranging from approximately 0.2 to 50 were set and maintained (for up to 45 min) in this system. ATP and ADP concentrations were controlled by the addition of either a phosphoarginine- or phosphocreatine-based regenerating system. Ouabain- sensitive unidirectional Na efflux was determined in the presence and absence of 15 mM external K as a function of the nucleotide composition. Na/K exchange was found to increase to saturation with ATP (K 1/2 approximately equal to 250 microM), whereas Na/Na exchange (measured in K-free solutions) was a saturating function of ADP (K 1/2 approximately equal to 350 microM). The elevation of ATP from approximately 100 to 1,800 microM did not appreciably affect Na/Na exchange. In the presence of external Na and a saturating concentration of external K, increasing the ADP concentration at constant ATP was found to decrease ouabain-sensitive Na/K exchange. The decreased Na/K exchange that still remained when the ADP/ATP ratio was high was stimulated by removal of external Na. Assuming that under normal substrate conditions the reaction cycle of the Na/K pump is rate- limited by the conformational change associated with the release of occluded K [E2 X (K) X ATP----E1 X ATP + K], increasing ADP inhibits the rate of these transformations by competition with ATP for the E2(K) form. A less likely alternative is that inhibition is due to competition with ATP at the high-affinity site (E1). The acceleration of the Na/K pump that occurs upon removing external Na at high levels of ADP evidently results from a shift in the forward direction of the transformation of the intermediates involved with the release of occluded Na from E1P X (Na). Thus, the nucleotide composition and the Na gradient can modulate the rate at which the Na/K pump operates.

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Selected References

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  1. Alberty R. A. Standard Gibbs free energy, enthalpy, and entropy changes as a function of pH and pMg for several reactions involving adenosine phosphates. J Biol Chem. 1969 Jun 25;244(12):3290–3302. [PubMed] [Google Scholar]
  2. Baker P. F., Blaustein M. P., Keynes R. D., Manil J., Shaw T. I., Steinhardt R. A. The ouabain-sensitive fluxes of sodium and potassium in squid giant axons. J Physiol. 1969 Feb;200(2):459–496. doi: 10.1113/jphysiol.1969.sp008703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beaugé L. A., Glynn I. M. Occlusion of K ions in the unphosphorylated sodium pump. Nature. 1979 Aug 9;280(5722):510–512. doi: 10.1038/280510a0. [DOI] [PubMed] [Google Scholar]
  4. Beaugé L. A., Glynn I. M. The equilibrium between different conformations of the unphosphorylated sodium pump: effects of ATP and of potassium ions, and their relevance to potassium transport. J Physiol. 1980 Feb;299:367–383. doi: 10.1113/jphysiol.1980.sp013130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Beaugé L., Berberian G. The effects of several ligands on the potassium-vanadate interaction in the inhibition of the (Na+ + K+)-ATPase and the Na+, K+ pump. Biochim Biophys Acta. 1983 Jan 19;727(2):336–350. doi: 10.1016/0005-2736(83)90419-4. [DOI] [PubMed] [Google Scholar]
  6. Beaugé L., Di Polo R. The effects of ATP on the interactions between monovalent cations and the sodium pump in dialysed squid axons. J Physiol. 1981 May;314:457–480. doi: 10.1113/jphysiol.1981.sp013719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bodemann H. H., Hoffman J. F. Effects of Mg and Ca on the side dependencies of Na and K on ouabain binding to red blood cell ghosts and the control of Na transport by internal Mg. J Gen Physiol. 1976 May;67(5):547–561. doi: 10.1085/jgp.67.5.547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bodemann H., Passow H. Factors controlling the resealing of the membrane of human erythrocyte ghosts after hypotonic hemolysis. J Membr Biol. 1972;8(1):1–26. doi: 10.1007/BF01868092. [DOI] [PubMed] [Google Scholar]
  9. Brinley F. J., Jr, Mullins L. J. Sodium fluxes in internally dialyzed squid axons. J Gen Physiol. 1968 Aug;52(2):181–211. doi: 10.1085/jgp.52.2.181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cavieres J. D., Ellory J. C. Allosteric inhibition of the sodium pump by external sodium. Nature. 1975 May 22;255(5506):338–340. doi: 10.1038/255338a0. [DOI] [PubMed] [Google Scholar]
  11. Cavieres J. D., Glynn I. M. Sodium-sodium exchange through the sodium pump: the roles of ATP and ADP. J Physiol. 1979 Dec;297(0):637–645. doi: 10.1113/jphysiol.1979.sp013061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. De Weer P. Axoplasmic free magnesium levels and magnesium extrusion from squid giant axons. J Gen Physiol. 1976 Aug;68(2):159–178. doi: 10.1085/jgp.68.2.159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. De Weer P. Effects of intracellular adenosine-5'-diphosphate and orthophosphate on the sensitivity of sodium efflux from squid axon to external sodium and potassium. J Gen Physiol. 1970 Nov;56(5):583–620. doi: 10.1085/jgp.56.5.583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Eisner D. A., Richards D. E. The interaction of potassium ions and ATP on the sodium pump of resealed red cell ghosts. J Physiol. 1981;319:403–418. doi: 10.1113/jphysiol.1981.sp013917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Flatman P. W., Lew V. L. The magnesium dependence of sodium-pump-mediated sodium-potassium and sodium-sodium exchange in intact human red cells. J Physiol. 1981 Jun;315:421–446. doi: 10.1113/jphysiol.1981.sp013756. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Garrahan P. J., Glynn I. M. Facftors affecting the relative magnitudes of the sodium:potassium and sodium:sodium exchanges catalysed by the sodium pump. J Physiol. 1967 Sep;192(1):189–216. doi: 10.1113/jphysiol.1967.sp008296. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Garrahan P. J., Glynn I. M. The behaviour of the sodium pump in red cells in the absence of external potassium. J Physiol. 1967 Sep;192(1):159–174. doi: 10.1113/jphysiol.1967.sp008294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Garrahan P. J., Glynn I. M. The stoicheiometry of the sodium pump. J Physiol. 1967 Sep;192(1):217–235. doi: 10.1113/jphysiol.1967.sp008297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Glynn I. M., Hoffman J. F. Nucleotide requirements for sodium-sodium exchange catalysed by the sodium pump in human red cells. J Physiol. 1971 Oct;218(1):239–256. doi: 10.1113/jphysiol.1971.sp009612. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Glynn I. M., Karlish S. J. ATP hydrolysis associated with an uncoupled sodium flux through the sodium pump: evidence for allosteric effects of intracellular ATP and extracellular sodium. J Physiol. 1976 Apr;256(2):465–496. doi: 10.1113/jphysiol.1976.sp011333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Glynn I. M., Karlish S. J. The sodium pump. Annu Rev Physiol. 1975;37:13–55. doi: 10.1146/annurev.ph.37.030175.000305. [DOI] [PubMed] [Google Scholar]
  22. Glynn I. M., Richards D. E. Occlusion of rubidium ions by the sodium-potassium pump: its implications for the mechanism of potassium transport. J Physiol. 1982 Sep;330:17–43. doi: 10.1113/jphysiol.1982.sp014326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. HOFFMAN J. F. The active transport of sodium by ghosts of human red blood cells. J Gen Physiol. 1962 May;45:837–859. doi: 10.1085/jgp.45.5.837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hara Y., Nakao M. Sodium ion discharge from pig kidney Na+, K+-ATPase Na+-dependency of the E1P-E2P equilibrium in the absence of KCl. J Biochem. 1981 Oct;90(4):923–931. doi: 10.1093/oxfordjournals.jbchem.a133580. [DOI] [PubMed] [Google Scholar]
  25. Hegyvary C., Post R. L. Binding of adenosine triphosphate to sodium and potassium ion-stimulated adenosine triphosphatase. J Biol Chem. 1971 Sep 10;246(17):5234–5240. [PubMed] [Google Scholar]
  26. Hexum T., Samson F. E., Jr, Himes R. H. Kinetic studies of membrane (Na+-K+-Mg2+)-ATPase. Biochim Biophys Acta. 1970 Aug 15;212(2):322–331. doi: 10.1016/0005-2744(70)90213-5. [DOI] [PubMed] [Google Scholar]
  27. Hobbs A. S., Dunham P. B. Interaction of external alkali metal ions with the Na-K pump of human erythrocytes: a comparison of their effects on activation of the pump and on the rate of ouabain binding. J Gen Physiol. 1978 Sep;72(3):381–402. doi: 10.1085/jgp.72.3.381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Hobbs A. S., Dunham P. B. Letter: Evidence for two sodium sites on the external aspect of Na-K pump in human erythrocytes. Nature. 1976 Apr 15;260(5552):651–652. doi: 10.1038/260651a0. [DOI] [PubMed] [Google Scholar]
  29. Hoffman J. F. The link between metabolism and active transport of sodium in human red cell ghosts. J Membr Biol. 1980 Dec 15;57(2):143–161. doi: 10.1007/BF01869000. [DOI] [PubMed] [Google Scholar]
  30. Kanazawa T., Saito M., Tonomura Y. Formation and decomposition of a phosphorylated intermediate in the reaction of Na plus-K plus dependent ATPase. J Biochem. 1970 May;67(5):693–711. doi: 10.1093/oxfordjournals.jbchem.a129297. [DOI] [PubMed] [Google Scholar]
  31. Kaplan J. H., Hollis R. J. External Na dependence of ouabain-sensitive ATP:ADP exchange initiated by photolysis of intracellular caged-ATP in human red cell ghosts. Nature. 1980 Dec 11;288(5791):587–589. doi: 10.1038/288587a0. [DOI] [PubMed] [Google Scholar]
  32. Kaplan J. H., Kenney L. J. ADP supports ouabain-sensitive K-K exchange in human red blood cells. Ann N Y Acad Sci. 1982;402:292–295. doi: 10.1111/j.1749-6632.1982.tb25750.x. [DOI] [PubMed] [Google Scholar]
  33. Kaplan J. H. Sodium pump-mediated ATP:ADP exchange. The sided effects of sodium and potassium ions. J Gen Physiol. 1982 Dec;80(6):915–937. doi: 10.1085/jgp.80.6.915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Karlish S. J., Yates D. W., Glynn I. M. Conformational transitions between Na+-bound and K+-bound forms of (Na+ + K+)-ATPase, studied with formycin nucleotides. Biochim Biophys Acta. 1978 Jul 7;525(1):252–264. doi: 10.1016/0005-2744(78)90219-x. [DOI] [PubMed] [Google Scholar]
  35. Kennedy B. G., De Weer P. Relationship between Na:K and Na:Na exchange by the sodium pump of skeletal muscle. Nature. 1977 Jul 14;268(5616):165–167. doi: 10.1038/268165a0. [DOI] [PubMed] [Google Scholar]
  36. Kennedy B. G., De Weer P. Strophanthidin-sensitive sodium fluxes in metabolically poisoned frog skeletal muscle. J Gen Physiol. 1976 Oct;68(4):405–420. doi: 10.1085/jgp.68.4.405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Keynes R. D., Steinhardt R. A. The components of the sodium efflux in frog muscle. J Physiol. 1968 Oct;198(3):581–599. doi: 10.1113/jphysiol.1968.sp008627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Levin M. L., Rector F. C., Jr, Seldin D. W. Effects of potassium and ouabain on sodium transport in human red cells. Am J Physiol. 1968 Jun;214(6):1328–1332. doi: 10.1152/ajplegacy.1968.214.6.1328. [DOI] [PubMed] [Google Scholar]
  39. Lienhard G. E., Secemski I. I. P 1 ,P 5 -Di(adenosine-5')pentaphosphate, a potent multisubstrate inhibitor of adenylate kinase. J Biol Chem. 1973 Feb 10;248(3):1121–1123. [PubMed] [Google Scholar]
  40. Mercer R. W., Dunham P. B. Membrane-bound ATP fuels the Na/K pump. Studies on membrane-bound glycolytic enzymes on inside-out vesicles from human red cell membranes. J Gen Physiol. 1981 Nov;78(5):547–568. doi: 10.1085/jgp.78.5.547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Mullins L. J., Brinley F. J., Jr Some factors influencing sodium extrusion by internally dialyzed squid axons. J Gen Physiol. 1967 Nov;50(10):2333–2355. doi: 10.1085/jgp.50.10.2333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Neufeld A. H., Levy H. M. A second ouabain-sensitive sodium-dependent adenosine triphosphate in brain microsomes. J Biol Chem. 1969 Dec 10;244(23):6493–6497. [PubMed] [Google Scholar]
  43. Norby J. G., Jensen J. Binding of ATP to brain microsomal ATPase. Determination of the ATP-binding capacity and the dissociation constant of the enzyme-ATP complex as a function of K+ concentration. Biochim Biophys Acta. 1971 Mar 9;233(1):104–116. doi: 10.1016/0005-2736(71)90362-2. [DOI] [PubMed] [Google Scholar]
  44. Parker J. C., Hoffman J. F. The role of membrane phosphoglycerate kinase in the control of glycolytic rate by active cation transport in human red blood cells. J Gen Physiol. 1967 Mar;50(4):893–916. doi: 10.1085/jgp.50.4.893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Post R. L., Hegyvary C., Kume S. Activation by adenosine triphosphate in the phosphorylation kinetics of sodium and potassium ion transport adenosine triphosphatase. J Biol Chem. 1972 Oct 25;247(20):6530–6540. [PubMed] [Google Scholar]
  46. Proverbio F., Hoffman J. F. Membrane compartmentalized ATP and its preferential use by the Na,K-ATPase of human red cell ghosts. J Gen Physiol. 1977 May;69(5):605–632. doi: 10.1085/jgp.69.5.605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Robinson J. D., Flashner M. S. The (Na+ + K+)-activated ATPase. Enzymatic and transport properties. Biochim Biophys Acta. 1979 Aug 17;549(2):145–176. doi: 10.1016/0304-4173(79)90013-2. [DOI] [PubMed] [Google Scholar]
  48. Robinson J. D. Substrate sites for the (Na+ + K+)-dependent ATPase. Biochim Biophys Acta. 1976 May 13;429(3):1006–1019. doi: 10.1016/0005-2744(76)90345-4. [DOI] [PubMed] [Google Scholar]
  49. Robinson J. D. The (Na + K+)-dependent ATPase. Mode of inhibition of ADP/ATP exchange activity by MgC12. Biochim Biophys Acta. 1976 Sep 13;440(3):711–722. doi: 10.1016/0005-2728(76)90053-0. [DOI] [PubMed] [Google Scholar]
  50. Sachs J. R. Interaction of external K, Na, and cardioactive steroids with the Na-K pump of the human red blood cell. J Gen Physiol. 1974 Feb;63(2):123–143. doi: 10.1085/jgp.63.2.123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Sachs J. R. Kinetics of the inhibition of the Na-K pump by external sodium. J Physiol. 1977 Jan;264(2):449–470. doi: 10.1113/jphysiol.1977.sp011677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Sachs J. R. Sodium movements in the human red blood cell. J Gen Physiol. 1970 Sep;56(3):322–341. doi: 10.1085/jgp.56.3.322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Sakamoto J., Tonomura Y. Order of release of ADP and Pi from phosphoenzyme with bound ADP of Ca2+-dependent ATPase from sarcoplasmic reticulum and of Na+, K+-dependent ATPase studied by ADP-inhibition patterns. J Biochem. 1980 Jun;87(6):1721–1727. doi: 10.1093/oxfordjournals.jbchem.a132916. [DOI] [PubMed] [Google Scholar]
  54. Segel G. B., Feig S. A., Glader B. E., Muller A., Dutcher P., Nathan D. G. Energy metabolism in human erythrocytes: the role of phosphoglycerate kinase in cation transport. Blood. 1975 Aug;46(2):271–278. [PubMed] [Google Scholar]
  55. Simons T. J. Potassium: potassium exchange catalysed by the sodium pump in human red cells. J Physiol. 1974 Feb;237(1):123–155. doi: 10.1113/jphysiol.1974.sp010474. [DOI] [PMC free article] [PubMed] [Google Scholar]

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