Ouabain binding and the conformational change in Na⁺, K⁺-ATPase

Abstract

The addition of ouabain to the Na⁺, K⁺-ATPase [EC. 3. 6. 1. 3] of pig kidney modified with N-[p-(2-benzimidazolyl) phenyl] maleimide gradually increased the fluorescence and the amount of phosphoenzyme from Pi with the same time course in the presence of 0.43mM Mg²⁺, 16mM Na⁺, 27μM ADP and 27μM Pi. The extent of the increment of the fluorescence intensity was dependent on the concentration of ouabain. A Hill plot of the data showed that n (Hill coefficient) and K_1/2 (apparent affinity) were equal to 0.27 and 0.84μM, respectively. Addition of ouabain to give 93μM increased the intensity to the highest level, similar to that of K⁺-sensitive phosphoenzyme (E₂P), and increased the extent of phosphorylation to half the amount of E₂P formed with Mg²⁺, Na⁺ and ATP. ADP inhibited the phosphorylation from Pi without affecting the binding of ouabain. The extent of the fluorescence intensity induced by ouabain in the presence of 0.43mM Mg²⁺, 16mM Na⁺ and 27μM ADP was the same irrespective of the presence of 27μM Pi. Addition of inorganic phosphate to give 2.6mM accelerated the rate of fluorescence increase and 27μM ADP retarded it without affecting the extent of the increment. The addition of ouabain to the Na⁺-bound enzyme increased the fluorescence with time to a level similar to that of E₂P. These results and those of others indicate that ouabain can bind to nonphosphorylated Na⁺, K⁺-ATPase, and the relative fluorescence intensity of ouabain bound Na⁺, K⁺-ATPase was similar to that of E₂P irrespective of the phosphorylation.