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Physiological Laboratory, University of Cambridge, Cambridge CB2 3EG

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 , -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 K of about 15 µM for ATP, 65 µM for adenylyl-imidodiphosphate (AMP-PNP) and 180 µM for adenylyl (, -methylene)-diphosphonate (AMP-PCP). ADP also converted the enzyme back into the E₁ form, with a K 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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