Modulation of erythrocyte Ca2+-ATPase by selective calpain cleavage of the calmodulin-binding domain.
The activity of the membrane-bound and the purified erythrocyte Ca2+-ATPase in the absence of calmodulin was stimulated by calpain digestion but could be further increased to maximal levels by calmodulin (CaM). Thus, CaM sensitivity was retained by the digested ATPase, at least at short times of incubation. In membranes digested at higher temperatures and in the purified ATPase digested at higher calpain/ATPase ratios, the ATPase became fully activated. The membrane-bound and the purified 138-kDa ATPase were converted by calpain to a fragment of approximately 124 kDa which still bound CaM and could be isolated on CaM columns when proteolysis occurred slowly but not when it occurred rapidly. Carboxypeptidase digestion of the purified enzyme and of its fragment of about 124 kDa has shown that calpain attacked the CaM-binding domain near the C terminus of the ATPase. This has also been supported by digestion of the purified enzyme and of its fragment of about 124 kDa. A first cut occurred in the middle of the domain producing a fragment of about 14 kDa and a (CaM-binding) fragment of about 124 kDa. A second cut closer to the N terminus of the domain also produced a fragment of about 124 kDa and accounted for the loss of CaM binding at prolonged times of incubation of the ATPase with calpain.
James
P
P
Laboratory of Biochemistry Swiss Federal Institute of Technology (ETH), Switzerland.
Vorherr
T
T
Krebs
J
J
Morelli
A
A
Castello
G
G
McCormick
D J
DJ
Penniston
J T
JT
De Flora
A
A
Carafoli
E
E
D000595:Amino Acid Sequence; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000097:blood*; D002147:Calmodulin / Q000378:metabolism* / Q000494:pharmacology; D002154:Calpain / Q000378:metabolism* / Q000494:pharmacology; D002268:Carboxypeptidases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004910:Erythrocyte Membrane / Q000201:enzymology*; D005615:Freezing; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism
D002147:Calmodulin; D010446:Peptide Fragments; D002268:Carboxypeptidases; D002154:Calpain; D000252:Calcium-Transporting ATPases