Quantitation of tissue calpain activity after isolation by hydrophobic chromatography.
A rapid and reliable method for quantitating tissue calpains (Ca2+-activated, neutral, thiol proteases) was developed using hydrophobic chromatography with phenyl-Sepharose. Calpains I and II isolated by this method are free of endogenous inhibitor(s) (calpastatin), activator(s), and nonspecific proteases. These calpains expose hydrophobic regions in the presence of Ca2+ and bind tightly to phenyl-Sepharose. Inactivation of bound calpain is prevented by the addition of leupeptin (20 microM). Calpains I and II bound initially by phenyl-Sepharose in a Ca2+-dependent manner are then eluted successively on the basis of their Ca2+-independent binding to phenyl-Sepharose. Because calpastatin may prevent binding of calpain to phenyl-Sepharose by forming a protease-inhibitor complex in the presence of Ca2+, preadsorbing the protease to a suspension of phenyl-Sepharose beads initially in the absence of Ca2+ separates most of the calpain present in tissue extracts from calpastatin. The isolated calpains obtained are assayed by casein digestion. This quantitation procedure is suitable for measuring calpain activity in various tissues and cells including erythrocytes.
D000818:Animals; D001921:Brain / Q000201:enzymology; D002118:Calcium; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D002154:Calpain / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002845:Chromatography / Q000379:methods; D005260:Female; D051381:Rats; D011919:Rats, Inbred Strains; D012685:Sepharose / Q000031:analogs & derivatives
D002135:Calcium-Binding Proteins; D002147:Calmodulin; C033452:Phenyl-Sepharose CL-4B; C033668:calpastatin; D012685:Sepharose; D002154:Calpain; D002118:Calcium