title: Phosphatase activity of (Na+ + K+)-ATPase. Ligand interactions and related enzyme forms.
abstract: The prevailing conformations of partially purified pig kidney (Na+ + K+)-ATPase interacting with ligands related to its phosphatase activity were determined following time-dependent trypsin digestion and inactivation as well as the amounts of Rb+ or Ca2+ bound to the enzyme after passage through cation-exchange resin columns. In the presence of 150 mM choline chloride, alone or with 3 mM MgCl2, 3 mM MnCl2 or 1 mM CaCl2, the major enzyme conformation was E1. Similar forms were seen with 5 mM p-nitrophenyl phosphate with and without 3 mM MgCl2. KCl, at 0.5 mM or 150 mM, produced an E2 enzyme state; the effects of 0.5 mM KCl were completely counteracted by 5 mM p-nitrophenyl phosphate. Under optimal conditions for phosphatase activity (3 mM MgCL2/5 mM p-nitrophenyl phosphate/10 mM KCl) the (Na+ + K+)-ATPase was in the E2 state. At low ionic strength and 20 degrees C and under 85% of maximal RbCl-stimulated phosphatase turnover (1 mM RbCl/3 mM MgCl2/5 mM p-nitrophenyl phosphate) no Rb+ occlusion could be detected. Ca2+, at low ionic strength and in the presence of 3 mM MgCl2, stimulated an ouabain-sensitive phosphatase activity. The rates of hydrolysis obtained wit 1 mM CaCl2 were similar to those seen with 0.5 mM KCl; under both conditions, similar patterns of trypsin digestion and inactivation of the enzyme were obtained. On the other hand, Ca2+ could not mimic Rb+ in its ability to induce an E2-occluding state. These results suggest that during phosphatase activity of (Na+ + K+)-ATPase, the most abundant form is a non-occluding E2 and that at least one of the mechanisms of potassium stimulation of that activity it to take the enzyme into the E2 state.
mesh_terms: D000818:Animals; D002118:Calcium / Q000494:pharmacology; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000032:analysis; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000302:isolation & purification / Q000378:metabolism*; D013552:Swine; D014357:Trypsin
chemical_list: D010446:Peptide Fragments; D010744:Phosphoric Monoester Hydrolases; D014357:Trypsin; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium; D011188:Potassium; D002118:Calcium