Interaction of sodium and potassium ions with Na+,K(+)-ATPase. IV. Affinity change for K+ and Na+ of Na+,K(+)-ATPase in the cycle of the ATP hydrolysis reaction.
The Kd 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), Pi, and Mg2+. From the results of the K+ binding experiments, Kd for Na+ was estimated by using an equation describing the competitive inhibition between the K+ and Na+ binding. 1) The Kd for K+ binding was 1.9 microM when no ligand was present. Addition of 2 mM Mg2+ increased the Kd to 15-17 microM. In the presence of 2 mM Mg2+, addition of 3 mM AMPPCP with or without 3 mM Na+ increased the Kd to 1,000 or 26 microM, respectively. These Kds 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 Kd from 15-17 microM to 5 or 0.8 microM, 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 Kd 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 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Homareda
H
H
Department of Biochemistry, Kyorin University School of Medicine, Tokyo.
Nagano
Y
Y
Matsui
H
H
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D004285:Dogs; D006868:Hydrolysis; D066298:In Vitro Techniques; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D010042:Ouabain / Q000494:pharmacology; D010710:Phosphates / Q000494:pharmacology; D010766:Phosphorylation; D011188:Potassium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D010710:Phosphates; D010042:Ouabain; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium