Autolytic Transition of μCalpain upon Activation as Resolved by Antibodies Distinguishing between the Pre- and Post-Autolysis Forms1

Abstract

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.

Structural modifications associated with the change in Ca2+ sensitivity on activation of m‐calpain

“…Croall et al [18] have raised antibodies specific for unautolysed m-and pcalpain. Saido et al [19] have raised antibodies against unautolysed and autolysed forms ofp-calpain. The data in this paper should facilitate experiments using the antibody approach to investigate in vivo activation of m-calpain.…”

SH3 domain of spectrin participates in the activation of Rac in specialized calpain-induced integrin signaling complexes

“…Polyclonal antibodies were obtained against β3 integrin (Chemicon); against HA epitope (Santa Cruz Biotechnology); against bovine non-erythroid spectrin (P14), a gift from Aleksander Sikorski (University of Wroclaw, Poland); against calpain-induced β3 integrin fragment (Ab754), a gift from Xiaoping Du (College of Medicine, Chicago, Illinois). Polyclonal antibodies against autolysed form of µ-calpain and polyclonal antibodies against calpain-induced 150 kD fragment of spectrin were described previously (Saido et al, 1992;Saido et al, 1993), TRITC (tetramethyl rhodamine isothiocyanate)-conjugated phalloidin was from Sigma.…”

Calpain Cleavage of Focal Adhesion Proteins Regulates the Cytoskeletal Attachment of Integrin αIIbβ3 (Platelet Glycoprotein IIb/IIIa) and the Cellular Retraction of Fibrin Clots

“…Previous studies in human platelets have demonstrated calcium-dependent autoproteolytic conversion of the 80-kDa subunit of calpain to its 76-kDa active form (3). The antibodies used in these studies have been raised against synthetic peptides corresponding to the N-terminal sequence of the large subunit of both forms of calpain (28). Immunoblot analysis of whole cell lysates with these antibodies represents a sensitive, specific, and direct means of monitoring calpain activation within the cell (29).…”