Reaction of (Na,K)-ATPase with fluorescent maleimide derivatives. Probes for studying ATP site(s) function

The fluorescent maleimide derivatives, 2-(4'-maleimidylanilino)naphthalene 6-sulfonic acid (Mal-ANS) and N-(1-pyrene)-maleimide (Mal-pyrene), both alkylate sulfhydryl groups on the alpha subunit of the (Na,K)-ATPase to inhibit (Na,K)-ATPase and p-nitrophenyl phosphatase activities and phosphoenzyme formation. Reaction of the enzyme with Mal-pyrene, but not with Mal-ANS, also inhibits MgPi- and Mg.ATP.Na-supported [3H]ouabain-binding to the enzyme. Mal-pyrene and Mal-ANS react, in part, with different sulfhydryl groups on the enzyme protein. On the average, the sulfhydryl groups which react with Mal-pyrene are located in a more shielded or hydrophobic environment than are those which react with Mal-ANS. It is the reaction of Mal-pyrene with sulfhydryl groups, which are not accessible to Mal-ANS, that results in the decreased [3H]ouabain-binding capacity of the (Na,K)-ATPase. The results indicate that phosphorylation of (Na,K)-ATPase is not required for Mg.ATP.Na-stimulated ouabain binding, and suggest that the ATP and sodium sites which modulate the interaction of ouabain with the (Na,K)-ATPase may be different from those which promote phosphorylation.