Affinity labeling and localization of the ATP binding site in the (Na+,K+)-ATPase.

The (Na+,K+)-ATPase has been covalently labeled with oxidized ATP. The extent of oxidized ATP incorporation which inactivates the enzyme corresponds to one nucleotide site per equivalent of ouabain binding site. ATP and ADP protect against the covalent labeling by oxidized ATP; the dissociation constants for the protective effects of ATP and ADP are 0.6 mM and 2.5 mM, respectively. The covalent incorporation is accelerated by K+ and by ouabain in the presence of Mg2+. The site of incorporation of oxidized ATP is located on a Mr = 58,000 fragment situated at the COOH-terminal end of the Mr = 94,000 alpha chain, while the phosphorylation site and the ouabain site are in the other (NH2-terminal) part of the chain.