title: Side-specific effects of sodium on (Na,K)-ATPase. Studies with inside-out red cell membrane vesicles.
abstract: Using inside-out vesicles of human red cell membranes, the side-specific effects of Na+ on phosphorylation of (Na,K)-ATPase have been studied using low concentrations of [gamma-32P]ATP (less than or equal to 0.1 microM). Phosphorylation is stimulated by Na+ at the cytoplasmic membrane surface (extravesicular Na+) alone and not by Na+ at the external surface (intravesicular Na+). At 37 degrees C, external Na+ (less than or equal to 10 mM) does, however, increase the steady state level (approximately 2 1/2-fold) of phosphoenzyme above that observed with cytoplasmic Na+ alone; hydrolysis is increased to only a small extent. Little stimulation by external Na+ is observed at 0 degrees C. As Na+ at the cytoplasmic side is decreased to very low levels (less than or equal to 0.2 mM) several kinetic changes are observed: (i) the apparent turnover of phosphoenzyme (ratio Na+-ATP-ase/phosphoenzyme level) is markedly increased (approximately 3-fold, (ii) Rbext sensitivity (inhibition of (Na)-ATPase at low ATP levels) is reduced, and (iii) the ratio of Na+ ions transported per molecule of ATP hydrolyzed is decreased. These results are compatible with a reaction pathway involving a transition from one form of phosphoenzyme, E1-P, to another, E2-P of which the hydrolysis is decreased by moderate levels of external Na+. It is suggested also that an alternate reaction pathway for Na+-ATPase occurs at very low cytoplasmic Na+, one via hydrolysis of E1-P and not associated with Na+ translocation.
mesh_terms: D001693:Biological Transport, Active; D004910:Erythrocyte Membrane / Q000201:enzymology*; D004912:Erythrocytes / Q000201:enzymology*; D006096:Gramicidin / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D010766:Phosphorylation; D012964:Sodium / Q000494:pharmacology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000097:blood*
chemical_list: D006096:Gramicidin; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase