The activation of phosphatase activity of the Ca 2+-ATPase from human red cell membranes by calmodulin, ATP and partial proteolysis

Abstract

(1) Depending on the assay conditions, the ability of the Ca 2+-ATPase from intact human red cell membranes to catalyze the hydrolysis of p-nitrophenylphosphate is elicited by either calmodulin or ATP. The response of the phosphatase activity to p-nitrophenylphosphate, ATP, Mg 2+ and K + is the same for the activities elicited by ATP or by calmodulin, suggesting that a single process is responsible for both activities. (2) In media with calmodulin, high-affinity activation is followed by high-affinity inhibition of the phosphatase by Ca 2+ so that the activity becomes negligible above 30 μM Ca 2+. Under these conditions, addition of ATP leads to a large decrease in the apparent affinity for inhibition by Ca 2+. (3) In membranes submitted to partial proteolysis with trypsin, neither calmodulin nor Ca 2+ are needed and phosphatase activity is maximal in media without Ca 2+. This is the first report of an activity sustained by the Ca 2+-ATPase of red cell membranes in the absence of Ca 2+. Under these conditions, however, ATP still protects against high-affinity inhibition by Ca 2+. These results strongly suggest that during activation by calmodulin, Ca 2+ is needed only to form the calmodulin-Ca 2+ complex which is the effective cofactor. (4) Protection by ATP of the inhibitory effects of Ca 2+ and the induction of phosphatase activity by ATP + Ca 2+ suggests that activation of the phosphatase by Ca 2+ in media with ATP requires the combination of the cation at sites in the ATPase. (5) Results can be rationalized assuming that E 2, the conformer of the Ca 2+-ATPase, is endowed with phosphatase activity. Under this assumption, either the calmodulin-Ca 2+ complex or partial proteolysis would elicit phosphatase activity by displacing the equilibrium between E 1 and E 2 towards E 2. On the other hand, ATP + Ca 2+ would elicit the activity by establishing through a phosphorylation-dephosphorylation cycle a steady-state in which E 2 predominates over other conformers of the ATPase.