Sodium-activated Adenosine Triphosphatase Activity of the Erythrocyte Membrane

Abstract

Abstract Evidence for a phosphorylated intermediate of Na+-activated ATPase of the erythrocyte membrane has been presented previously in studies of a Na+-stimulated component of membrane phosphorylation at low ATP concentrations. In this investigation, the effects of ouabain and oligomycin on Na+-activated membrane phosphorylation and (14C)ADP-ATP exchange were studied in relation to effects on Na+-activated ATP hydrolysis. In agreement with findings with other particulate preparations, ouabain interaction with membranes resulted in inhibition of (a) Na+-activated ATP hydrolysis, (b) Na+-stimulated increase and K+-stimulated decrease in the steady state level of phosphorylated intermediate, and (c) Na+-activated (14C)ADP-ATP exchange. Ouabain inhibition of hydrolysis was diminished by addition of potassium ions. The ratio of Na+-stimulated (14C)ADP-ATP exchange activity to ATPase activity was low at 37° compared to 0°. The exchange reaction was stimulated by oligomycin, but the degree of stimulation relative to the degree of inhibition of ATP hydrolysis was also low at 37° compared to 0°. Oligomycin inhibited Na+-activated ATP hydrolysis without affecting the level of 32P bound in the presence of sodium ions alone. K+-stimulated dephosphorylation was decreased by oligomycin. With increasing concentrations of ATP, the catalytic center activity of Na+, K+-activated ATPase increases, to 1980 per minute determined at 0.05 mm ATP and to approximately 6000 per min estimated at 2 mm ATP.