SIDE-SPECIFIC EFFECTS OF SODIUM ON (NA,K)-ATPASE - STUDIES WITH INSIDE-OUT RED-CELL MEMBRANE-VESICLES

Using inside-out vesicles of human red cell membranes, the side-specific effects of Na+ on phosphorylation of (Na,K)-ATPase were studied using low concentrations of [.gamma.-32P]ATP (.ltoreq. 0.1 .mu.M). 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.degree. C, external Na+ (.ltoreq. 10 mM) increases the steady state level (.apprx. 2 1/2-fold) of phosphoenzyme above that with cytoplasmic Na+ alone; hydrolysis is increased to only a small extent. Little stimulation by external Na+ is observed at 0.degree. C. As Na+ at the cytoplasmic side is decreased to very low levels (.ltoreq. 0.2 mM) kinetic changes are the apparent turnover of phosphoenzyme (ratio Na+-ATPase/phosphoenzyme level) is markedly increased (.apprx. 3-fold), RBext [external Rb] sensitivity (inhibition of (Na)-ATPase at low ATP levels) is reduced and the ratio of Na+ ions transported per molecule of ATP hydrolyzed is decreased. A reaction pathway involving a transition from 1 form of phosphoenzyme, E1-P, to another, E2-P of which the hydrolysis is decreased by moderate levels of external Na+ is suggested. An alternate reaction pathway for Na+-ATPase may occur at very low cytoplasmic Na+, 1 via hydrolysis of E1-P and not associated with Na+ translocation.