Identification of an essential sulfhydryl group in the ouabain binding site of (Na,K)-ATPase.

Ellman's reagent 5,5'-dithiobis-(2-nitrobenzoic acid) inhibits sodium- and potassium-stimulated ATPase, p-nitrophenyl phosphatase activity, and [3H]ouabain binding to lamb kidney (Na,K)-ATPase. The inactivation of [3H]ouabain binding follows pseudo-first order reaction kinetics at pH values less than or equal to 8.2. The inactivation of [3H]ouabain binding, but not of enzymatic activity, can be blocked by preincubation with ouabagenin, a rapidly reversible aglycone derivative of ouabain. The reduction in [3H]ouabain binding is due to a decrease in the number of binding sites rather than an alteration of the affinity of the enzyme for ouabain. Differential labeling at pH 8.2 with 1.0 mM 5,5'-dithiobis-(2-nitrobenzoic acid), preincubated with or without 5 microM ouabagenin, followed by tryptic digestion and reverse-phase high performance liquid chromatography of the generated soluble peptides reveals a single peptide labeled by the sulfhydryl probe that is protected by ouabagenin. From these results it is concluded that there is a single sulfhydryl group, essential for ouabain binding, presumably located in the ouabain binding site of lamb kidney (Na,K)-ATPase.