Binding of monovalent cations to Na+,K+-dependent ATPase purified from porcine kidney. II. Acceleration of transition from a K+-bound form to a Na+-bound form by binding of ATP to a regulatory site of the enzyme.
Two kinds of ATP binding sites were found to exist on the ATPase molecule. One was the catalytic site (1 mol/mol phosphorylation site) and its apparent dissociation constant for ATP was about 1 microM. The other was the regulatory site(s) and its apparent dissociation constant for ATP was equal to or higher than about 0.2 mM. The affinities of both sites for AMPPNP were three times lower than those for ATP. The affinity of the ATPase for ATP was reduced by the addition of KCl, but unaffected by the addition of NaCl. As thermodynamically expected, the affinity of the Na+-binding sites for Na+ ions was almost completely unaffected by the addition of ATP, which markedly decreased that of the K+-binding sites for K+ and Rb+ ions. In the absence of KCl, Na+ ions were bound very rapidly to the Na+-binding sites [(1979) J. Biochem. 86, 509--523]. However, Na+ ions were bound very slowly to the enzyme preincubated with 50 microM KCl, and the Na+ binding was markedly accelerated by the addition of ATP or AMPPNP at concentrations much higher than several microM. On the other hand, in the presence of 50 microM KCl, 1 mol of ATP was bound to the catalytic site with the same dissociation constant as that in the absence of KCl, and another 1 mol of ATP bound with a dissociation constant of about 0.1 mM. Therefore, we concluded that the Na+ binding to the enzyme in a K+ form is markedly accelerated by the binding at ATP to the regulatory site.
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D011188:Potassium / Q000378:metabolism*; D011485:Protein Binding; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013552:Swine
D000266:Adenylyl Imidodiphosphate; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium