The order of release of sodium and addition of potassium in the sodium-potassium pump reaction mechanism.

1. The characteristics of oligomycin inhibition of the Na--K pump of human red cell membranes was investigated. Oligomycin inhibition of the pump was found to be reversible. 2. Inhibition of Na--K ATPase activity was uncompetitive with respect to ATP in broken membrane preparations. In intact cells inhibition was uncompetitive with respect to both intracellular and extracellular Na. 3. Oligomycin did not significantly inhibit the K--K exchange if the cells were Na-free, but if the cells contained a small amount of Na, oligomycin inhibition of the K--K exchange became significant. Taken together with the findings described above, this indicates that oligomycin combines with E1P-Na conformation of the pump and not with any E2 conformation. 4. When measurements are made in solutions high in Na, oligomycin is a non-competitive inhibitor with respect to external K, but in Na-free solutions, oligomycin inhibition is uncompetitive with respect to external K. 5. These findings indicate that, in the normal operation of the pump, Na is released to the outside before K adds.