Abstract

The interaction of strophanthidin bromoacetate (SBA) with rat brain (Na⁺ + K⁺)-ATPase ATP phosphohydrolase, EC 3.6.1.3) was investigated. SBA displaced [³H]ouabain from the Na⁺-, Mg⁺⁺-, and ATP-dependent binding site on the enzyme. The apparent affinity of SBA for the cardiotonic steroid binding site of the enzyme was at least one-third of that of ouabain for this site. When SBA was allowed to bind to the cardiotonic steroid binding site in the presence of Na⁺, Mg⁺⁺, and ATP or of Mg⁺⁺ and P_i, [³H]ouabain added at various intervals after SBA equilibrated with all the cardiotonic steroid binding sites. If SBA was allowed to bind to the enzyme in the presence of Mg⁺⁺ and P_i or of Na⁺, Mg⁺⁺, and UTP, its subsequent phosphorylation form [γ-³²P]ATP was reduced by 60-80%. Exposure of this inhibited enzyme to high concentrations of EDTA resulted in recovery of phosphorylation capacity in less than 5 min at 37°. Since SBA did not irreversibly occupy the cardiotonic steroid binding sites or give rise to irreversible inhibition of the enzyme, SBA does not appear to be an effective affinity label for Na⁺ + K⁺)-ATPase. Because a challenge to the concept that (Na⁺ + K⁺)-ATPase may be the cardiotonic receptor for cardiotonic steroids [B. F. Roth-Schechter, G. T. Okita, R. E. Thomas, and F. F. Richardson, J. Pharmacol. Exp. Ther. 171, 13-19 (1970)] is based on the supposed irreversibility of the SBA-(Na⁺ + K⁺)-ATPase interaction, our results leave such an interpretation open to question.