INTERACTION OF SODIUM AND POTASSIUM-IONS WITH NA+,K+-ATPASE .4. AFFINITY CHANGE FOR K+ AND NA+ OF NA+,K+-ATPASE IN THE CYCLE OF THE ATP HYDROLYSIS REACTION

The K(d) for ouabain-sensitive K+ or Rb+ binding to Na+,K+-ATPase was determined by the centrifugation method with radioactive K+ and Rb+ in the presence of various combinations of Na+, ATP, adenylylimidodiphosphate (AMPPNP), adenylyl-(beta,gamma-methylene)diphosphonate (AMPPCP), P(i), and Mg2+. From the results of the K+ binding experiments, K(d) for Na+ was estimated by using an equation describing the competitive inhibition between the K+ and Na+ binding. 1) The K(d) for K+ binding was 1.9-mu-M when no ligand was present. Addition of 2 mM Mg2+ increased the K(d) to 15-17-mu-M. In the presence of 2 mM Mg2+, addition of 3 mM AMPPCP with or without 3 mM Na+ increased the K(d) to 1,000 or 26-mu-M, respectively. These K(d)s correspond to those for K+ of Na.E1.AMPPCPMg or E1.AMPPCPMg, respectively. 2) Addition of 4 mM ATP with or without 3 mM Na+ decreased the K(d) from 15-17-mu-M to 5 or 0.8-mu-M, respectively. Because the phosphorylated intermediate was observed but ATPase activity was scarcely observed in the K+ binding medium containing 3 mM ATP and 2 mM Mg2+ in the absence of Na+ as well as in the presence of Na+ at 0-degrees-C, it is suggested that K+ binds to E2-P.Mg under these ligand conditions. 3) The K(d) for Na+ of the enzyme in the presence of 3 mM AMPPCP or 4 mM ATP with Mg2+ was estimated to be 80 or 570-mu-M, respectively. The affinity ratio of K+ to Na+ of the enzyme (K(d) for Na+/K(d) for K+) showed a large increase (240-fold) from 3 to 710 on changing the nucleotide used from AMPPCP to ATP. Consequently, it is presumed that the transition from E1.ATPMg to E2-P.Mg induces nearly 240-fold increase in the affinity ratio. 4) Addition of 4 mM P(i) in the presence of 2 mM Mg2+ increased the K(d) for K+ from 17-mu-M to 40-mu-M. Addition of 3 mM Na+ in the presence of P(i) and Mg2+ slightly increased the K(d) from 40 to 49-mu-M, contrary to the result in the absence of Mg2+. The K(d) for Na+ in the presence of P(i) increased from 0.29 to 13 mM on addition of 2 mM Mg2+. Therefore, the K+-insensitive E2-P.Mg formed from Mg2+ and P(i) [Post et al. (1975) J. Biol. Chem. 250, 691-701] was shown to have low affinity not only for K+ but also for Na+.