✓ In previous studies of topical application of calphostin C, a specific inhibitor of the regulatory domain of protein kinase C (PKC), and calpeptin, a selective inhibitor of calpain, to spastic canine basilar artery (BA) researchers have suggested that the catalytic fragment of PKC (known as PKM) is probably formed by a limited proteolysis of continuously activated µ-calpain, but there has been no direct evidence for PKM formation in vasospasm. The present immunoblot study with anti-PKCα antibody shows a significant decrease in cytosolic 80-kD PKCα and a concomitantly significant increase in membrane PKCα in the spastic canine BA. In addition, an immunoblot study in which cleavage site—directed antibodies were used demonstrated a significant increase in immunoreactive 45-kD PKM. The changes in membrane PKCα and PKM were enhanced with the lapse of time after subarachnoid hemorrhage. The cleavage site—directed antibodies distinguish the proteolyzed from the unproteolyzed forms of PKC for in situ analyses of enzyme regulation mediated by proteolysis. The data indicate that PKCα in spastic canine BA is translocated to the cell membrane, where PKCα is rapidly cleaved into PKM as a result of proteolysis of the isozyme by µ-calpain but not by µ-calpain. The authors hypothesize that µ-calpain is continuously activated in spastic canine BA and produces PKM by limited proteolysis of PKCα.
Effect of phospholipid on substrate phosphorylation by a catalytic fragment of protein kinase C.