Activity of lysosomal cysteine proteinase during differentiation of rat skeletal muscle.
Cysteine-proteinase activities were measured in extracts of pre- and post-fusion populations of rat myogenic line L6 cells and in extracts of whole rat muscle. Activities of cathepsins B, L and H were compared. The substrates used included Z-Phe-Arg-NMec (cathepsins B and L), Z-Arg-Arg-NMec (cathepsin B), and Arg-NMec (cathepsin H) (where Z = benzyloxycarbonyl, and NMec = 4-methyl-7-coumarylamide); the enzyme activities were more specifically differentiated by appropriate concentrations of the inhibitors Z-Phe-Phe-CHN2 (CHN2 = diazomethane), bestatin and E-64 [L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane]. These experiments have demonstrated the feasibility of determining the cysteine-proteinase activities of myoblasts from a single (60 mm-diameter) Petri dish, with enzyme concentrations in the range of 5-20 ng/ml. Specific activities of the enzymes in L6 cells increased 2-20-fold after fusion. Concentrations of cysteine proteinases in extracts from cultured myoblasts were two orders of magnitude greater than those in muscle-tissue extracts. Cultured-cell extracts contained endogenous inhibitor(s) to purified rat cathepsins B, L and H.
D000818:Animals; D002403:Cathepsins / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D003546:Cysteine Endopeptidases; D010450:Endopeptidases / Q000378:metabolism*; D008099:Liver / Q000201:enzymology; D008247:Lysosomes / Q000201:enzymology; D008297:Male; D009132:Muscles / Q000166:cytology / Q000201:enzymology*; D011480:Protease Inhibitors; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity
D011480:Protease Inhibitors; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases