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. 1980 Feb;299:367–383. doi: 10.1113/jphysiol.1980.sp013130

The equilibrium between different conformations of the unphosphorylated sodium pump: effects of ATP and of potassium ions, and their relevance to potassium transport

L A Beaugé 1, I M Glynn 1
PMCID: PMC1279230  PMID: 6247481

Abstract

1. Changes in the intrinsic fluorescence of Na, K-ATPase protein have been used to monitor the interconversion of E1 (low fluorescence) and E2 (high fluorescence) forms of the unphosphorylated enzyme.

2. In media lacking sodium and nucleotides, 1 mM-potassium was sufficient to convert practically all of the enzyme into the E2 form. In media containing 1 mM-potassium, 1 mM-EDTA, and no sodium or magnesium, the addition of ATP, or its β, γ-imido or methylene analogues, converted the enzyme back into the E1 form. The relation between nucleotide concentration and the fraction of the enzyme that was in the E1 form could be described by a rectangular hyperbola, with a K½ of about 15 μM for ATP, 65 μM for adenylyl-imidodiphosphate (AMP-PNP) and 180 μM for adenylyl (β, γ-methylene)-diphosphonate (AMP-PCP). ADP also converted the enzyme back into the E1 form, with a K½ of about 25 μM, but the relation between concentration and fraction converted was not well described by a rectangular hyperbola.

3. In similar media containing 50 mM-potassium, much higher concentrations of ATP were required to convert the enzyme back into the E1 form, and the conversion was probably incomplete.

4. If we assume that ATP and potassium ions affect each other's binding solely by altering the equilibrium between E1 and E2 forms of the enzyme, we are able to conclude (i) that potassium ions bind to the E1 form with a moderately low affinity, (ii) that, in the absence of nucleotides, the equilibrium between E1K and E2K is poised strongly in favour of E2K, (iii) that the binding of ATP to a low-affinity site alters the equilibrium constant for the interconversion of E1K and E2K by two to three orders of magnitude, so that, at saturating levels of ATP, the equilibrium is probably slightly in favour of E1K, and (iv) that in sodium-free, potassium-containing media, ATP will appear to bind to the enzyme more tightly than would be expected from the dissociation constant of the E2K. ATP complex.

5. The pattern of the equilibrium constants for the various reactions between E1, E2, ATP and potassium is compatible with the hypothesis that the ATP-accelerated conversion of E2K into E1K, and the subsequent release of potassium ions from low-affinity inward-facing sites, are part of the normal sequence of events during potassium influx in physiological conditions.

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Selected References

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

  1. 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]
  2. Joiner C. H., Lauf P. K. The correlation between ouabain binding and potassium pump inhibition in human and sheep erythrocytes. J Physiol. 1978 Oct;283:155–175. doi: 10.1113/jphysiol.1978.sp012494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Jorgensen P. L. Purification and characterization of (Na+ + K+)-ATPase. VI. Differential tryptic modification of catalytic functions of the purified enzyme in presence of NaCl and KCl. Biochim Biophys Acta. 1977 Apr 1;466(1):97–108. doi: 10.1016/0005-2736(77)90211-5. [DOI] [PubMed] [Google Scholar]
  4. Jorgensen P. L. Purification and characterization of (Na+, K+)-ATPase. V. Conformational changes in the enzyme Transitions between the Na-form and the K-form studied with tryptic digestion as a tool. Biochim Biophys Acta. 1975 Sep 2;401(3):399–415. doi: 10.1016/0005-2736(75)90239-4. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Karlish S. J., Yates D. W., Glynn I. M. Elementary steps of the (Na+ + K+)-ATPase mechanism, studied with formycin nucleotides. Biochim Biophys Acta. 1978 Jul 7;525(1):230–251. doi: 10.1016/0005-2744(78)90218-8. [DOI] [PubMed] [Google Scholar]
  7. Karlish S. J., Yates D. W. Tryptophan fluorescence of (Na+ + K+)-ATPase as a tool for study of the enzyme mechanism. Biochim Biophys Acta. 1978 Nov 10;527(1):115–130. doi: 10.1016/0005-2744(78)90261-9. [DOI] [PubMed] [Google Scholar]
  8. Lamola A. A., Eisinger J., Blumberg W. E., Kometani T., Burnham B. F. Quantitative determination of erythrocyte zinc protoporphyrin. J Lab Clin Med. 1977 Apr;89(4):881–890. [PubMed] [Google Scholar]
  9. Norby J. G., Jensen J. Binding of ATP to Na+, K+-ATPase. Ann N Y Acad Sci. 1974;242(0):158–167. doi: 10.1111/j.1749-6632.1974.tb19088.x. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. 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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