Sodium ions, acting at high‐affinity extracellular sites, inhibit sodium‐ATPase activity of the sodium pump by slowing dephosphorylation.

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Abstract
Extracellular Na+ ions, in low concentrations, inhibit Na+-ATPase activity in resealed red cell ghosts and this inhibition is reversed by high concentrations of extracellular Na+. These actions of extracellular Na+ were demonstrated by investigating the dependence on Na+ concentration of ATP-ADP exchange and Na+-ATPase activity both in native and in N-ethylmaleimide(NEM)-treated (Na+ + K+)-ATPase from pig kidney, and the rate of hydrolysis of the phosphorylated kidney enzyme in the absence of K+ ions. 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. The effects, on ATP-ADP exchange, of changing the concentrations of ATP, ADP and Mg were also 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 Mg-ADP is the substrate and Mg2+ ions are inhibitory. Since NEM, under these conditions, 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. To test whether this inhibitory effect of Na+ reflected inhibition of the hydrolysis of E2P, the rate of loss of incorporated 32P was measured when enzyme, newly phosphorylated by [.gamma.32P]ATP, was added to a large volume of ice-cold solution containing 1,2-cyclohexylenedinitrilotetraacetic acid (CDTA), unlabeled 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 .mu.M (final) Na+. An unexpected feature was the pattern of stimulation of ATP-ADP exchange in intact cells. If Na+ ions are absent externally, the difference could be fitted better on the assumption that activation by internal Na+ occurs at 2 sites with equal afinities, than on the assumption that activation occurs at a single site or at 3 sites with equal affinities.