Purification and characterization of calpains from pig epidermis and their action on epidermal keratin.
Two forms of Ca++-dependent cysteine proteinases, calpain I, requiring low Ca++ (microM concentration), and calpain II, requiring high Ca++ (mM concentration), were purified from the cytosolic fraction of pig epidermis. Calpains I and II were separated on DEAE-cellulose chromatography, and thereafter they were purified by separate but almost identical procedures, which included chromatographies on Sephacryl S-300, Blue Sepharose CL-6B, and DEAE Bio-Gel A. Purified calpains I and II required 10 and 450 microM Ca++ for half-maximal activation, respectively, and had an optimal pH of 7.0 to 8.0. Both enzymes were heterodimers and composed of one heavy subunit (83 kDa for calpain I and 80 kDa for calpain II) and one light subunit (29 kDa for both enzymes). The action of calpains I and II on keratin extracted from the same tissue was studied. Both enzymes rapidly cleaved keratin into small fragments. The cleavage depends on Ca++ and could be blocked by leupeptin and calpastation, an endogenous calpain-specific inhibitor, which was also found in the cytosolic fraction of pig epidermis and partially purified.
D000818:Animals; D002135:Calcium-Binding Proteins / Q000302:isolation & purification / Q000494:pharmacology; D002154:Calpain / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D002845:Chromatography; D004817:Epidermis / Q000378:metabolism*; D066298:In Vitro Techniques; D007633:Keratins / Q000378:metabolism*; D013552:Swine
D002135:Calcium-Binding Proteins; D007633:Keratins; C033668:calpastatin; D002154:Calpain