[Ouabain binding and the conformational change in Na+, K+ -ATPase].
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 0.43 mM Mg2+, 16 mM Na+, 27 microM ADP and 27 microM 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 K1/2 (apparent affinity) were equal to 0.27 and 0.84 microM, respectively. Addition of ouabain to give 93 microM 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 0.43 mM Mg2+, 16 mM Na+ and 27 microM ADP was the same irrespective of the presence of 27 microM Pi. Addition of inorganic phosphate to give 2.6 mM accelerated the rate of fluorescence increase and 27 microM 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.
D000244:Adenosine Diphosphate; D055598:Chemical Phenomena; D002621:Chemistry; D008274:Magnesium; D008301:Maleimides; D008968:Molecular Conformation; D010042:Ouabain; D010710:Phosphates; D010766:Phosphorylation; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase
D008301:Maleimides; D010710:Phosphates; C000245:N-(4-(2-benzimidazolyl)phenyl)maleimide; D010042:Ouabain; D000244:Adenosine Diphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium