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The equilibrium between different conformations of the unphosphorylated sodium pump: effects of ATP and of potassium ions, and their relevance to potassium transport
Author(s) -
Beaugé L. A.,
Glynn I. M.
Publication year - 1980
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.1980.sp013130
Subject(s) - potassium , chemistry , sodium , enzyme , nucleotide , stereochemistry , biochemistry , organic chemistry , gene
1. Changes in the intrinsic fluorescence of Na, K‐ATPase protein have been used to monitor the interconversion of E 1 (low fluorescence) and E 2 (high fluorescence) forms of the unphosphorylated enzyme. 2. In media lacking sodium and nucleotides, 1 m M ‐potassium was sufficient to convert practically all of the enzyme into the E 2 form. In media containing 1 m M ‐potassium, 1 m M ‐EDTA, and no sodium or magnesium, the addition of ATP, or its β, γ‐imido or methylene analogues, converted the enzyme back into the E 1 form. The relation between nucleotide concentration and the fraction of the enzyme that was in the E 1 form could be described by a rectangular hyperbola, with a K ½ of about 15 μ M for ATP, 65 μ M for adenylyl‐imidodiphosphate (AMP‐PNP) and 180 μ M for adenylyl (β, γ‐methylene)‐diphosphonate (AMP‐PCP). ADP also converted the enzyme back into the E 1 form, with a K ½ of about 25 μ M , but the relation between concentration and fraction converted was not well described by a rectangular hyperbola. 3. In similar media containing 50 m M ‐potassium, much higher concentrations of ATP were required to convert the enzyme back into the E 1 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 E 1 and E 2 forms of the enzyme, we are able to conclude (i) that potassium ions bind to the E 1 form with a moderately low affinity, (ii) that, in the absence of nucleotides, the equilibrium between E 1 K and E 2 K is poised strongly in favour of E 2 K, (iii) that the binding of ATP to a low‐affinity site alters the equilibrium constant for the interconversion of E 1 K and E 2 K by two to three orders of magnitude, so that, at saturating levels of ATP, the equilibrium is probably slightly in favour of E 1 K, 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 E 2 K. ATP complex. 5. The pattern of the equilibrium constants for the various reactions between E 1 , E 2 , ATP and potassium is compatible with the hypothesis that the ATP‐accelerated conversion of E 2 K into E 1 K, 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.