title: Sodium ions, acting at high-affinity extracellular sites, inhibit sodium-ATPase activity of the sodium pump by slowing dephosphorylation.
abstract: 1. It is known that extracellular Na+ ions, in low concentrations, inhibit Na+-ATPase activity in resealed red cell ghosts and that this inhibition is reversed by high concentrations of extracellular Na+. We have attempted to elucidate these actions of extracellular Na+ by investigating the dependence on Na+ concentration of (a) ATP-ADP exchange and Na+-ATPase activity both in native and in N-ethylmaleimide (NEM)-treated (Na+ + K+)-ATPase from pig kidney, and (b) the rate of hydrolysis of the phosphorylated kidney enzyme in the absence of K+ ions. 2. With the native enzyme, ATP-ADP exchange and Na+-ATPase activity showed similar responses to changes in Na+ concentration: a steep but S-shaped rise between 0 and 2.5 mM, a slight fall (exchange) or a plateau (ATPase) between 2.5 and 10 mM, and a roughly linear rise between 10 and 150 mM. With NEM-treated enzyme, the ATP-ADP exchange, which was greatly accelerated, showed no sign either of inhibition at intermediate Na+ concentrations or of the reversal of that inhibition at higher concentrations. The exchange rate increased with Na+ concentration in a smooth curve and was half-maximal at about 7 mM. 3. The effects, on ATP-ADP exchange, of changing the concentrations of ATP, ADP and Mg have also been investigated. With both native and NEM-treated enzyme, the interactions of ATP, ADP and Mg are complicated; they show that, for the reaction leading to ATP formation, either free ADP rather than MgADP is the substrate, or Mg2+ ions are inhibitory (or both). 4. Since NEM, in the conditions in which we have used it, is believed to act by inhibiting the conversion of an ADP-sensitive form of the phosphoenzyme (E1P) to an ADP-insensitive form (E2P), the absence of Na+ inhibition of ATP-ADP exchange in NEM-treated enzyme, together with the parallel effects of Na+ ions on the ATP-ADP exchange activity and on the Na+-ATPase activity of native enzyme, suggests that the inhibitory effect of external Na+ occurs after the conversion of E1P into E2P. 5. To test whether this inhibitory effect of Na+ reflected inhibition of the hydrolysis of E2P, we measured the rate of loss of incorporated 32P when enzyme, newly phosphorylated by [gamma32P]ATP, was squirted into a large volume of ice-cold solution containing 1,2-cyclohexylenedinitrilotetraacetic aicd (CDTA), unlabelled ATP and 0, 5 or 150 mM-Na+. The rate of loss of radioactivity from the membranes was least at 5 mM-Na+, about twice as great at 150 mM-Na+, and about 5 times as great at 20 microM (final) Na+. 6. An unexpected feature of the results was that the pattern of stimulation of ATP-ADP exchange in intact cells. If Na+ ions are absent externally, a different could be fitted better on the assumption that activation by internal Na+ occurs at two sites with equal affinities, than on the assumptions that activation occurs at a single site or at three sites with equal affinities.
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D066298:In Vitro Techniques; D007679:Kidney Medulla / Q000201:enzymology; D007700:Kinetics; D010766:Phosphorylation; D012964:Sodium / Q000378:metabolism* / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013552:Swine
chemical_list: D000244:Adenosine Diphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase