Abstract

Oxidized [³H]ouabain was prepared and its binding to renal plasma membranes was studied. More than 90% of the oxidized ouabain was bound by the (Na⁺ + K⁺)-ATPase (EC 3.6.1.3) component of the membranes. This binding required the simultaneous presence of Na⁺, Mg⁺⁺, and ATP. K⁺ inhibited binding. No binding took place in the absence of Mg⁺⁺. Oxidized [³H]ouabain was covalently attached to the membranes by reducing the ouabain-membrane complex with NaBH₄ at pH 9.0. The covalent nature of this binding was evident from its stability against dissociation by acid, 90% methanol, or heating. In the presence of Na⁺, ATP, and Mg⁺⁺ oxidized [³H]ouabain was bound preferentially by an electrophoretically single membrane protein. This protein had an apparent molecular weight of 89,000 as estimated by polyacrylamide gel electrophoresis, and it is thought to be part of (Na⁺ + K⁺)-ATPase because (a) it bound ouabain in the presence of Na⁺, Mg⁺⁺, and ATP but not in the presence of Tris-EDTA, and (b) it could be phosphorylated by [³²P]ATP in the presence of Na⁺ and Mg⁺⁺ but not in the presence of K⁺ and Mg⁺⁺.