Ouabain receptor interactions in (Na + K)-ATPase preparations. III. On the stability of the ouabain receptor against physical treatment, hydrolases and SH reagents

The action of ATP and its analogs as well as the effects of alkali ions were studied in their action on the ouabain receptor. One single ouabain receptor with a dissociation constant (K_D) of 13 nM was found in the presence of (Mg²⁺ + P_i) and (Na⁺ + Mg²⁺ + ATP). pH changes below pH 7.4 did not affect the ouabain receptor. Ouabain binding required Mg²⁺, where a curved line in the Scatchard plot appeared. The affinity of the receptor for ouabain was decreased by K⁺ and its congeners, by Na⁺ in the presence of (Mg²⁺ + P_i), and by ATP analogs (ADP-C-P, ATP-OCH₃). Ca²⁺ antagonized the action of K⁺ on ouabain binding. It was concluded that the ouabain receptor exists in a low affinity (R_α) and a high affinity conformational state (R_β). The equilibrium between both states is influenced by ligands of (Na⁺ + K⁺)-ATPase. With 3 mM Mg²⁺ a mixture between both conformational states is assumed to exist (curved line in the Scatchard plot).