The calmodulin-binding domain as an endogenous inhibitor of the p-nitrophenylphosphatase activity of the Ca2+ pump from human red cells.
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 Ca2+ 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. Ca2+ inhibited the chymotrypsin-induced p-nitrophenylphosphatase (Ki for Ca2+ = 2 microM). In the absence of ATP, C20W and Ca2+ did not interact in apparent affinity as inhibitors of this activity. On the other hand, in the presence of 2 mM ATP, Ca2+ antagonized the inhibition produced by C20W. The results are consistent with the idea that the calmodulin-binding site is an 'autoinhibitory domain' of the Ca2+ 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 Ca2+.
Caride
A J
AJ
IQUIFIB, Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Argentina.
Penniston
J T
JT
Rossi
J P
JP
D009597:4-Nitrophenylphosphatase / Q000037:antagonists & inhibitors* / Q000097:blood; D000328:Adult; D002118:Calcium / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000097:blood / Q000187:drug effects / Q000502:physiology*; D002148:Calmodulin-Binding Proteins / Q000097:blood / Q000494:pharmacology / Q000502:physiology*; D002918:Chymotrypsin; D004789:Enzyme Activation / Q000187:drug effects; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D006868:Hydrolysis; D010455:Peptides / Q000494:pharmacology
D002148:Calmodulin-Binding Proteins; D010455:Peptides; D009597:4-Nitrophenylphosphatase; D002918:Chymotrypsin; D000252:Calcium-Transporting ATPases; D002118:Calcium