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.
Highlights
The exchangereaction was stimulated by oligomycin, but the degree of stimulation relative to the degree of inhibition of ATP hydrolysis was low at 37“ comparedto 0”
Preliminary experiments were carried out to test the effects of oligomycin and ouabain on the Na+- plus K+-activated hydrolysis of ATP at saturating concentration (2 mu)
Addition of Naf and K+ increased the hydrolysis of T-~~P-ATP from 3.3 to 10.5 mpmoles of a2Pi released per min per mg of protein
Summary
Erythrocyte membranes were prepared from fresh, human erythrocytes as described previously [11]. Membranes were washed once more with 2 mM Tris-HCI containing 4 PM Tris-EDTA, pH 7.4, and the volume was adjusted with the same solution to one-half the original packed cell volume. This extra wash eliminated residual adenylate kinase activity. Membranes were initially incubated for 20 min at 37”, with or without inhibitors, prior to addition to reaction medium. Procedures for labeling membranes with T-~~P-ATP, terminating reactions with trichloracetic acid, measuring ATP hydrolysis (azPi released), (14C)ADPATP exchange and protein were carried out as described [11]. When the (14C)ADPATP exchange activity was high relative to ATP hydrolysis (O”), the amount of isotope incorporated into product was corrected for approach to isotopic equilibrium [22]; otherwise, results were expressed on the basis of the initial specific radioactivity of 14CADP
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