Interaction of Sodium and Potassium Ions with Na+,K+-ATPase. HI. Cooperative Effect of ATP and Na+ on Complete Release of K+ from E2K1

The effects of Na+ and ATP on the K+ binding to Na+,K+-ATPase were investigated by the centrifugation method with radioactive K+ in the absence of Mg!+. In the presence of 10 fiM "KC1, 0.6 and 10 mM Na+ decreased the amount of bound K+ to one-half and zero, respectively. On the other hand, 10 /M and 10 mM ATP decreased the amount of K+ to 60 and 25-40%, respectively. When the combined effect of ATP and Na+ was tested, 10 /*M ATP decreased the Na+ concentration giving half-maximal inhibition of the K+ binding to one-third, showing synergistic inhibition by both ligands, though increase in ATP concentration seemed to depress the inhibitory effect of Na+. The synergistic inhibition by ATP and Na+ suggests that the release of K+ from E,K is not completed by the binding of ATP alone but is completed by the binding of Na+ in addition to ATP during the cycle of Na+,K+-dependent ATP-hydrolysis as well as ion-transport. Na+,K+-ATPase [ATP phosphohydrolase, EC 3.6. 1.3] is believed to have two different conformations, i.e., E, form with high-affinity sites for Na+ and ATP, and E, form with high-affinity sites for K+ (7, 2). According to the classical Post-Albers scheme (7), KMgE,-P is converted to E,K, which is an E, conformer with occluded K+, by its dephosphorylation, releasing Pi and Mg1+. E,