Autolytic transition of mu-calpain upon activation as resolved by antibodies distinguishing between the pre- and post-autolysis forms.
A novel method to observe the autolytic activation of a mammalian cytoplasmic calcium protease, mu-calpain, was developed using a set of antipeptidic antibodies capable of distinguishing between the pre- and post-autolysis forms of the enzyme. Antibodies raised against synthetic peptides designed to match the N-terminal sequences of the pre- and post-autolysis forms of the mu-calpain large subunit reacted specifically with the corresponding form of calpain and not with the other. The antibodies were specific and sensitive enough to detect the antigens in crude cell lysates. The relevance of the immunochemical detection of calpain activation was confirmed by the observation that proteolysis of a substrate protein by purified mu-calpain paralleled autolysis at various pCa as probed by these antibodies and that autolysis preceded substrate proteolysis. We also observed calcium-dependent autolysis of calpain accompanying subsequent proteolysis of substrate in intact cells using the antibodies. The method will provide a novel approach to assess the physiological targets of the enzyme by determining the local intracellular sites of calpain activation.
Saido
T C
TC
Department of Molecular Biology, Tokyo Metropolitan Institute of Medical Science.
Nagao
S
S
Shiramine
M
M
Tsukaguchi
M
M
Sorimachi
H
H
Murofushi
H
H
Tsuchiya
T
T
Ito
H
H
Suzuki
K
K
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D002154:Calpain / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D002364:Caseins / Q000378:metabolism; D003593:Cytoplasm / Q000201:enzymology; D004789:Enzyme Activation; D006801:Humans; D007624:KB Cells; D008969:Molecular Sequence Data; D014407:Tumor Cells, Cultured
D000906:Antibodies; D002364:Caseins; D002154:Calpain; D002118:Calcium