Irreversible inhibition of ³H-ouabain binding to Na⁺+K⁺-ATPase by digoxigenin-3,12-dibromoacetate, an alkylating derivative of digoxigenin

Digoxigenin-3,12-dibromoacetate (DDB), an alkylating derivative of digoxigenin, was synthesized and tested as a cardiotonic steroid (CS) site directed affinity label for Na⁺+K⁺-ATPase (ATP phosphohydrolase EC 3.6.1.3). DDB inhibited rat brain Na⁺+K⁺-ATPase with an I₅₀ of 5 × 10^{-6 M} and readily displaced specifically bound ³H-ouabain from its binding sites on Na⁺+K⁺-ATPase. If the enzyme was exposed to DDB prior to the addition of ³H-ouabain its ability to bind ³H-ouabain was decreased, consistent with the concept that DDB interacted irreversibly with the cardiotonic steroid binding sites of Na⁺+K⁺-ATPase. However, DDB proved to be an even more effective inhibitor of ³H-ouabain binding under conditions where it was unlikely that it could interact with the CS binding sites of this enzyme, suggesting that DDB inhibited ³H-ouabain binding by non-cardiotonic site directed actions. Similarly, the presence of excess strophanthidin did not protect this enzyme against irreversible inhibition by DDB. The data suggest that the presence of a bromoacetate group at the 12 position on cardiotonic steroids does not confer CS binding site directed alkylating properties on these drugs.