Abstract

[12α-³H]Digoxigenin was prepared by the reduction of 12-dehydrodigoxigenin with NaB³H₄. Binding to the sodium- and potassium-dependent adenosine triphosphatase was studied at room temperature. Digoxigenin bound to the enzyme with high affinity. This binding was eliminated by prior ouabain treatment and was dependent on the presence of ligands. As in cardiac glycoside binding, mixtures of Mg²⁺ and P_i or of Na⁺, Mg²⁺, and ATP were both effective as ligands. Scatchard plots of binding were linear and showed that the dissociation constant did not change at different concentrations of each ligand except in the very low concentration range, but that the number of binding sites on the enzyme was reduced on descreasing the ligand concentration. The present results show that the binding of digoxigenin to the (Na⁺ + K⁺)-ATPase does not follow the usual equation representing a reversible reaction. At saturating concentrations of ligands, i.e., 2 mM Mg²⁺ and 2 mM P_i, or 50 mM Na⁺, 2 mM Mg²⁺, and 2 mM ATP, the number of binding sites was close to the number of ouabain binding sites for each set of ligands. The dissociation constant was 0.041 µM in the Mg²⁺-P_i system and 0.078 µM in the Na⁺-Mg²⁺-ATP system. These dissociation constants are lower than the I₅₀ value of digoxigenin (0.4 µM) under the conditions used for assay.