The equilibrium between different conformations of the unphosphorylated sodium pump: effects of ATP and of potassium ions, and their relevance to potassium transport.
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 mM-potassium was sufficient to convert practically all of the enzyme into the E(2) form. In media containing 1 mM-potassium, 1 mM-EDTA, and no sodium or magnesium, the addition of ATP, or its beta, gamma-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((1/2)) of about 15 muM for ATP, 65 muM for adenylyl-imidodiphosphate (AMP-PNP) and 180 muM for adenylyl (beta, gamma-methylene)-diphosphonate (AMP-PCP). ADP also converted the enzyme back into the E(1) form, with a K((1/2)) of about 25 muM, 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 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.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology*; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D001693:Biological Transport, Active / Q000187:drug effects; D005470:Fluorometry; D007679:Kidney Medulla / Q000201:enzymology; D011188:Potassium / Q000378:metabolism / Q000494:pharmacology*; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013552:Swine
D000266:Adenylyl Imidodiphosphate; C005147:5'-adenylyl (beta,gamma-methylene)diphosphonate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; C002630:alpha,beta-methyleneadenosine 5'-triphosphate; D011188:Potassium