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 derivation 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 times 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.