3-O-Methylfluorescein phosphate as a fluorescent substrate for plasma membrane Ca-ATPase

3-O-Methylfluorescein phosphate hydrolysis, catalyzed by purified erythrocyte Ca²⁺-ATPase in the absence of Ca²⁺, was slow in the basal state, activated by phosphatidylserine and controlled proteolysis, but not by calmodulin. p-Nitrophenyl phosphate competitively inhibits hydrolysis in the absence of Ca²⁺, while ATP inhibits it with a complex kinetics showing a high and a low affinity site for ATP. Labeling with fluorescein isothiocyanate impairs the high affinity binding of ATP, but does not appreciably modify the binding of any of the pseudosubstrates. In the presence of calmodulin, an increase in the Ca²⁺ concentration produces a bell-shaped curve with a maximum at 50 μM Ca²⁺. At optimal Ca²⁺ concentration, hydrolysis of 3-O-methylfluorescein phosphate proceeds in the presence of fluorescein isothiocyanate, is competitively inhibited by p-nitrophenyl phosphate and, in contrast to the result observed in the absence of Ca²⁺, it is activated by calmodulin. In marked contrast with other pseudosubstrates, hydrolysis of 3-O-methylfluorescein phosphate supports Ca²⁺ transport. This highly specific activity can be used as a continuous fluorescent marker or as a tool to evaluate partial steps from the reaction cycle of plasma membrane Ca²⁺-ATPases.