Inhibition of the sodium pump by inorganic phosphate in resealed red cell ghosts

Ouabain-sensitive Rb influx was measured into K-free resealed red cell ghosts. The effects of inorganic phosphate (P(i)) were examined. Phosphate decreased the magnitude of the influx. Increasing P(i) lowered the apparent affinity for both ATP and external rubidium ions. The effects of P(i) on the affinity for external Rb were greatest at low ATP concentrations. In the nominal absence of phosphate, increasing ATP from 1 to 100 μM had little effect on the Rb influx from solutions of low (1 μM) rubidium concentrations. In the presence of P(i) ATP increased Rb uptake markedly, even from solutions of low Rb concentration. The above interactions between P(i), ATP and external Rb are consistent with a consecutive scheme for the Na pump in which phosphate is released after potassium binds at the external surface and before ATP binds to release potassium ions to the internal solution. Previous failures to find an effect of phosphate on either the affinity for ATP or that for external potassium (rubidium) ions are shown to be equally consistent with the model. The lack of change of apparent affinity is shown to result from the restricted range of concentrations used in these previous experiments.