(Na+ + K+)-dependent adenosine triphosphatase. Regulation of inorganic phosphate, magnesium ion, and calcium ion interactions with the enzyme by ouabain.
1. (Na+ + K+)-dependent adenosine triphosphatase was phosphorylated on the alpha-subunit by Pi in the presence of Mg2+. Phosphorylation was stimulated by ouabain. The interactions of Pi, Mg2+, and ouabain with the enzyme could be explained by a random terreactant scheme in which the binding of each ligand to the enzyme increased the affinities for the other two. Dissociation constants of all steps of this scheme were estimated. 2. In the presence of Pi and ouabain and without added Mg2+, the phosphoenzyme was formed. Because this could be prevented by ethylenediaminetetraacetic acid, but not ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, phosphoenzyme formation under these conditions was probably dependent on traces of endogenous Mg2+. The ability of this Mg2+ to support phosphorylation could be explained by the large increase in the enzyme's affinity for Mg2+ by ouabain. 3. In the absence of ouabain, Ca2+ did not support phosphorylation and inhibited Mg2+-dependent phosphorylation. At lower concentrations, Ca2+ was competitive with Mg2+. With increasing Ca2+ concentration, negative cooperativity was observed, suggesting the existence of multiple divalent cation sites with equivalent affinities for Mg2+, but varying affinities for Ca2+. 4. In the presence of ouabain, the maximum inhibition of Mg2+-dependent phosphorylation by Ca2+ was 50%. With saturating Pi, Mg2+, and ouabain, the number of sites binding ouabain was equal to the number of sites phosphorylated. Although Ca2+ halved phosphorylation and reduced the affinity for ouabain about 100-fold, it did not affect the number of ouabain sites. 5. We suggest that the enzyme is an alpha-oligomer and that the half-of-the-sites reactivity for phosphorylation in the presence of Pi, Mg2+, ouabain, and optimal Ca2+ is caused by (a) ouabain-induced increase in the affinities of both protomers for Mg2+ and (b) the inability of Ca2+ to replace Mg2+ on one of the protomers.
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D004285:Dogs; D004492:Edetic Acid / Q000494:pharmacology; D007679:Kidney Medulla / Q000201:enzymology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D010042:Ouabain / Q000494:pharmacology*; D010710:Phosphates / Q000494:pharmacology*; D011485:Protein Binding; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D010710:Phosphates; D010042:Ouabain; D004492:Edetic Acid; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium; D002118:Calcium