title: Phosphatase activity of Na+/K+-ATPase. Enzyme conformations from ligands interactions and Rb occlusion experiments.
abstract: The present work compares the effects of several ligands (phosphatase substrates, MgCl2, RbCl and inorganic phosphate) and temperature on the phosphatase activity and the E2(Rb) occluded conformation of Na+/K+-ATPase. Cooling from 37 degrees C to 20 degrees C and 0 degrees C (hydrolysis experiments) or from 20 degrees C to 0 degrees C (occlusion experiments) had the following consequences: (i) dramatically reduced the Vmax for p-nitrophenyl phosphate and acetyl phosphate hydrolysis but it produced little or no changes in the Km for the substrates; (ii) led to a 5-fold drop in the Km for the inorganic phosphate-induced di-occlusion of E2(Rb); (iii) reduced the K0.5 and curve sigmoidicity of the Rb-stimulated hydrolysis of p-nitrophenyl phosphate and acetyl phosphate and the Rb-promoted E2(Rb) formation. At 20 degrees C, in the presence of 1 mM RbCl and no Mg2+, acetyl phosphate did not affect E2(Rb); with 3 mM MgCl2, acetyl phosphate stimulated a release of Rb from E2(Rb) both in the presence and absence of RbCl in the incubation mixture. As a function of acetyl phosphate concentration the Km for iRb release was indistinguishable from the Km found for stimulation of hydrolysis and enzyme phosphorylation under identical experimental conditions; in addition, the extrapolated di-occluded fraction corresponding to maximal hydrolysis was not different from 100%. These results indicate that although E2(K) might be an intermediary in the phosphatase reaction, the most abundant enzyme conformation during phosphatase turnover is E2 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 ATP hydrolysis; on the other hand, there are similarities with the reversible binding of inorganic phosphate in the presence of Mg2+ and K+ ions.
authors.affiliation: Divisíon de Biofisica, Instituto M. y M. Ferreyra, Córdoba, Argentina.
authors.forename: M
authors.initials: M
authors.lastname: Campos
authors.forename: G
authors.initials: G
authors.lastname: Berberián
authors.forename: L
authors.initials: L
authors.lastname: Beaugé
mesh_terms: D000818:Animals; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008024:Ligands; D008274:Magnesium / Q000494:pharmacology; D010042:Ouabain / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases; D011188:Potassium / Q000494:pharmacology; D011487:Protein Conformation; D012413:Rubidium / Q000378:metabolism* / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013552:Swine
chemical_list: D008024:Ligands; D010042:Ouabain; D010744:Phosphoric Monoester Hydrolases; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium; D012413:Rubidium; D011188:Potassium