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

• Published in: Journal of biochemistry (Tokyo) Vol. 109; no. 1; pp. 70 - 77
• Main Authors: Homareda, Haruo, Nagano, Yoshine, Matsui, Hideo
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 01.01.1991
• Subjects: Adenosine Triphosphate - analogs & derivatives; Adenosine Triphosphate - metabolism; Adenosine Triphosphate - pharmacology; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Dogs; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Hydrolases; Hydrolysis; In Vitro Techniques; Kidney - enzymology; Kinetics; Ouabain - pharmacology; Phosphates - pharmacology; Phosphorylation; Potassium - metabolism; Sodium - metabolism; Sodium - pharmacology; Sodium-Potassium-Exchanging ATPase - metabolism; Hydrolysis; Active transport; Enzymatic activity; Radiolabelling; Sodium; Enzyme; Na,K-ATPase; Dissociation constant; ATP; Potassium; Conformational transition
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/oxfordjournals.jbchem.a123355

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-(β;,γ-methylene)diphos-phonate (AMPPCP), P1, 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 μM when no ligand was present. Addition of 2 mM Mg2+ increased the Kd to 15–17 μM. 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 μM, 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 μM to 5 or 0.8 μM, 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°C, it is suggested that K+ binds to E2-P-Mg under these ligand conditions. 3) The Kda 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 μM, respectively. The affinity ratio of K+ to Na+ of the enzyme (Kd for Na+/Kd 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 P1 in the presence of 2 mM Mg2+ increased the Kd for K+ from 17 μM to 40 μM. Addition of 3 mM Na+ in the presence of P1 and Mg2+ slightly increased the Kd from 40 to 49 μM, contrary to the result in the absence of Mg2+. The Kd for Na+ in the presence of P1 increased from 0.29 to 13 mM on addition of 2 mM Mg2+. Therefore, the K+-insensitive E2-P.Mg formed from Mg2+ and P1 [Post et al. (1975) J. Biol. Chem. 250, 691–701] was shown to have low affinity not only for K+ but also for Na+.