Ouabain binding to (Na⁺ + K⁺)-ATPase effects of nucleotide analogues and ethacrynic acid

To determine the mechanism of Na⁺, M²⁺ and ATP stimulated [³H]ouabain binding to (Na⁺ + K⁺)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) the effects on this reaction of ethacrynic acid and analogues of ATP were investigated. β,7-Methylene-ATP inhibited [³H]ouabain binding to this enzyme under all conditions tested. This inhibition was synergistic with that of Na⁺, and Na⁺ acted to increase the apparent affinity of the enzyme for β,γ-methylene-ATP. β,γ-Methylene ADP also inhibited [⁺H]ouabain binding to this enzyme. Similarly, the overall effect of β,γ-imido-ATP on [³H]ouabain binding to guinea pig kidney (Na^- - K⁺)-ATPase was inhibitory. Ethacrynic acid prolonged transient pulses of [³H]ouabain binding supported by ATP or ADP. Ethacynic acid produced this effect by stabilizing the form of the enzyme which interacted with ouabain rather than by affecting the rates at which ouabain was bound to or dissociated from the enzyme. The data suggest that nucleotides with a stable terminal phosphate bond cannot support [³H]ouabain binding. These observations are consistent with the hypothesis that Na⁺-stimulated phosphorylation of the (Na⁺ + K⁺)-ATPase is a primary event in the Na⁺-stimulated binding of [³H]-ouabain to this enzyme.