Phosphatase activity of Na +/K +-ATPase. Enzyme conformation from ligands interactions and Rb occlusion experiments

Abstract

The present work compares the effects of several ligands (phosphatase substrates, MgCl 2, RbCl and inorganic phosphate) and temperature on the phosphatase activity and the E 2(Rb) occuluded conformation of Na +/K +-ATPase. Cooling from 37°C to 20°C and 0°C (hydrolysis experiments) or from 20°C to 0°C (occlusion experiments) had the following consequences: (i) dramatically reduced the V max for p_ nitrophenyl phosphate and acetyl phosphate hydrolysis but it produced little or no changes in the K m for the subtrates; (ii) led to a 5-fold drop in the K m for the inorganic phosphate-induced di-occlusion of E 2(Rb); (iii) reduced the K 0.5 and curve sigmoidicity of the Rb-stimulated hydrolysis of p- nitrophenyl phosphate and acetyl phosphate and the Rb-promoted E 2(Rb) formation. At 20 °C, in the presence of 1 mM RbCl and no Mg 2+, acetyl phosphate did not affect E 2(Rb); with 3 mM MgCl 2, acetyl phosphate stimulated a release of Rb from E 2(Rb) both in the presence and absence of RbCl in the incubation mixture. As a function of acetyl phosphate concentration the K m for Rb release was indistinguishable from the K m found for stimulation of hydrolysis and enzyme phosphorylation under identical experimental conditions; in addition, the extrapolated di-occluded fraction corresponding to maximal dydrolysis was not different from 100%. These results indicate that although E 2(K) might be an intermediary in the phosphatase reaction, the most abundant enzyme conformation during phosphatase turnover is E 2 which has no K + occluded in it. The ligand interactions associated to phosphatase activity do not support an equivalence of this reaction with the dephosphorylation step in the Na ++ K + -dependent AT Phydrolysis; on the other hand, there are similarities with the reversible binding of inorgnic phosphate in the presence of Mg 2+ and K + ions.