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

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RESUMO

1. 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.

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