The calmodulin-binding domain as an endogenous inhibitor of the p-nitrophenylphosphatase activity of the Ca 2+ pump from human red cells

Abstract

Digestion of red cell membranes with chymotrypsin elicited p-nitrophenylphosphatase activity. During digestion, the p-nitrophenylphosphatase appeared in parallel with the activation of the Ca 2+-ATPase (in the absence of calmodulin). The chymotrypsin-activated p-nitrophenylphosphatase was inhibited by C20W, a 20 amino acid peptide modelled after the sequence of the calmodulin-binding site of the red cell Ca 2+ pump (Vorherr et al. (1990) Biochemistry 29, 355–365). On the contrary, the (ATP + Ca 2+)-dependent p-nitrophenylphosphatase activity of intact red cell membranes was not affected by C20W. Ca 2+ inhibited the chymotrypsin-induced p-nitrophenylphosphatase ( K ifor Ca 2+ = 2 μM). In the absence of ATP, C20W and Ca 2+ did not interact in apparent affinity as inhibitors of this activity. On the other hand, in the presence of 2 mM ATP, Ca 2+ antagonized the inhibition produced by C20W. The results are consistent with the idea that the calmodulin-binding site is an ‘autoinhibitory domain’ of the Ca 2+ pump, and that removal of this domain by proteolysis, or its modification by calmodulin binding is the reason for the activation of both the ATPase and the p-nitrophenylphosphatase activity of the pump. The results presented in this paper give new information about the mechanism of the two kinds of p-nitrophenylphosphatase and about the nature of the apparent competition between C20W and Ca 2+.