Studies on ouabain-complexed (Na+ +K+)-ATPase carried out with vanadate.
Vanadate is able to promote the binding of ouabain to (Na+ +K+)-ATPase and it is shown that vanadate is trapped in the enzyme-ouabain complex. Also ouabain-bound enzyme, the formation of which was facilitated by (Mg2+ +Na+ +ATP) or (Mg2+ +Pi), is accessible to vanadate when washed free of competing ligands used for the promotion of ouabain binding. For vanadate binding to (Na+ +K+)-ATPase and to enzyme-ouabain complexes a divalent cation (Mg2+ or Mn2+) is indispensable, indicating that the cation does not remain attached to the ouabain-bound enzyme. K+ further increases vanadate binding in the absence of ouabain, but seems to have no additional role in case of vanadate binding to enzyme-ouabain complexes. Mn2+ is more efficient than Mg2+ in promoting binding of vanadate and ouabain to (Na+ +K+)-ATPase. That K+ in combination with Mn2+, in analogy with the effect in combination with Mg2+, increases the equilibrium binding level of vanadate and decreases that of ouabain does not seem to favour the hypothesis of selection of a special E2-subconformation by Mn2+. The vanadate-trapped enzyme-ouabain complex was examined for simultaneous nucleotide binding which could demonstrate a two-substrate mechanism per functional unit of the enzyme. The acceleration by (Na+ +ATP) of ouabain release from the (Mg2+ +Pi)-facilitated enzyme-ouabain complex does not, as anticipated, support such a mechanism. On the other hand, the deceleration of vanadate release as well as of ouabain release from a (Mg2+ +vanadate)-promoted complex could be consistent with a two-substrate mechanism working out-of-phase.
D000818:Animals; D007679:Kidney Medulla / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D010042:Ouabain / Q000378:metabolism*; D011485:Protein Binding; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013552:Swine; D014638:Vanadates; D014639:Vanadium / Q000494:pharmacology*
D014639:Vanadium; D014638:Vanadates; D008345:Manganese; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium