The order of addition of sodium and release of potassium at the inside of the sodium pump of the human red cell.

Abstract

SUMMARY1. Inhibition of Na,K-adenosine 5'-triphosphatase (Na,K-ATPase) activity of human red cell membranes by vanadate rapidly reaches a steady-state level and is rapidly reversible.2. In K-free cells vanadate inhibits the Na-K exchange at low concentrations, the uncoupled Na efflux at higher concentrations, and has little effect on the Na-Na exchange even at high concentrations. Increasing intracellular K concentration increases the sensitivity of the Na-Na exchange to vanadate.3. Vanadate inhibition of the Na-K exchange is uncompetitive with extracellular K and inhibition of the K-K exchange is partially uncompetitive with intracellular K. Na-Li exchange is less sensitive to vanadate inhibition than Na-K exchange. The results indicate that vanadate inhibits by combining with an enzyme form which occurs between the addition of K at the outside and its release to the inside.4. Inhibition by vanadate is non-competitive with Na at low adenosine 5'-triphosphate (ATP) concentrations and high concentrations of K. At high ATP and low K, vanadate inhibition becomes partially uncompetitive with Na. At high or at low ATP and in the absence of cell K, inhibition is strictly uncompetitive with cell Na.5. These results are consistent with a ping-pong model for the reaction mechanism of the Na pump, but they are not consistent with a sequential mechanism or with a branched-chain mechanism in which Na adds after the release of K in one branch and before in the other.

Changes in Steady-state Conformational Equilibrium Resulting from Cytoplasmic Mutations of the Na,K-ATPase α-Subunit

“…The reaction mechanism is probably consecutive (Cleland's ping-pong mechanism), whereby Na ϩ is released before K ϩ binds (5,6). Most reaction schemes are based on the original Albers-Post mechanism involving phosphorylated and dephosphorylated forms of the enzyme, both of which undergo conformational transitions (E 1 P 3 E 2 P and E 2 3 E 1 ), which are coupled to ion translocation steps.…”

Inhibition of the Na,K pump by vanadate in high-Na solutions. Modification of the reaction mechanism by external Na acting at a high-affinity site.

“…Because the Na pump 292 can carry out an efflux of Na into Na-and K-free solutions that is not coupled to the influx of a cation (uncoupled Na efflux), it is not possible to use the standard methods of steady state kinetics to distinguish between the ping-pong model and alternative models in which Na and K are bound to the pump simultaneously at some point in the transport cycle (Sachs, 1979(Sachs, , 1986a . However, by examining the effects of external and internal Na and K on the characteristics of pump inhibition by oligomycin (Sachs, 1980) and by vanadate (Sachs, 1986b), it is possible to conclude that, at least in Na-free solutions, Na is released to the outside before K adds and, at least in K-free cells, K is released to the inside before Na adds . Moreover, the presence of two cation exchanges THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 90 -1987 Resealed ghosts were prepared by a gel filtration method similar to that described by Kaplan (1982) ; the method has been described in detail (Sachs, 1986b).…”

“…However, by examining the effects of external and internal Na and K on the characteristics of pump inhibition by oligomycin (Sachs, 1980) and by vanadate (Sachs, 1986b), it is possible to conclude that, at least in Na-free solutions, Na is released to the outside before K adds and, at least in K-free cells, K is released to the inside before Na adds . Moreover, the presence of two cation exchanges THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 90 -1987 Resealed ghosts were prepared by a gel filtration method similar to that described by Kaplan (1982) ; the method has been described in detail (Sachs, 1986b). Briefly, cells were washed with an isosmotic choline chloride solution buffered to pH 5.5 until the pH of the cell suspension was -6.0.…”

“…ATPase activity was measured, with and without 2.5 x 10' M ouabain, by a coupled enzyme assay in which rephosphorylation of ADP by pyruvate kinase and phosphoenolpyruvate (PEP) is coupled to the oxidation of NADH by lactic dehydrogenase . A complete description of the method has recently been published (Sachs, 1986b). p-Nitrophenolphosphatase (pNPPase) activity was measured by incubating membranes or ghosts in the presence of 5 mM p-nitrophenolphosphate (pNPP), 5.5 mM MgC12, 0.5 mM EDTA, and other substances indicated in the figure legends .…”

“…The biphasic relation between 29 6 THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 90 " 1987 the pump rate and the Ko concentration occurred only when the external solution contained Na . When the pump rate was measured as a function of the Ko concentration in Na-free solutions, vanadate reduced both the apparent VM and the apparent Ky, for K., but the curve increased monotonically; i.e ., vanadate was a noncompetitive inhibitor with respect to Ko (Beauge and Berberian, 1983 ;Sachs, 1986b). Fig.…”