Study of thiol proteases of normal human skin fibroblasts.
The protease activity of cultured normal human skin fibroblasts was studied using the synthetic fluorigenic peptides, the modified protein 4-methylumbelliferyl-casein, the thiol inhibitors and the affinity for concanavalin A-Sepharose. The majority of the activity to N-benzyloxycarbonyl-L-phenylalanyl-L-arginyl-7-amido-4-methyl-coumarin and N-a-benzyloxycarbonyl-L-arginyl-arginyl-7-amido-4-methylcoumarin had a pH optimum of 6.0, and was thiol-dependent and inhibited by leupeptin and antipain. The activity toward N-benzyloxycarbonyl-L-phenylalanyl-L-arginyl-7-amido-4-methylcoumarin represents both cathepsin B and cathepsin L, whereas the activity towards 4-methylumbelliferyl-casein represent only cathepsin L. Cathepsin H could not be detected when assayed with L-arginine-7-amido-4-methylcoumarin substrate. Cathepsin D was present in comparatively small amounts when assayed with 4-methylumbelliferyl-casein. Activity towards 4-methylumbelliferyl-casein had pH optima at 3 and 6 and was stimulated by dithiothreitol. A proportion of the activity at pH 6.0 was not dependent on thiols and not inhibited by leupeptin, and had the general characteristics of a carboxyl proteinase. Over 70 per cent of the activity was in the lysosomal fraction and showed structure-linked latency. All the detectable protein emerged from the immobilized concanavalin A column and the fractions eluted by alpha-methyl-D-mannoside were significantly hydrolysed the synthetic peptides. Only that fraction which bound to concanavalin A was active towards 4-methylumbelliferyl-casein. Cathepsin B had no affinity for concanavalin A-Sepharose due to the absence of glycoprotein content, unlike cathepsin L which showed a strong affinity for concanavalin A-Sepharose.
Khalfan
H A
HA
Discipline of Biochemistry, College of Medicine and Medical Science, Arabian Gulf University, State of Bahrain.
D002364:Caseins / Q000378:metabolism; D002401:Cathepsin B / Q000378:metabolism; D056655:Cathepsin H; D056668:Cathepsin L; D002403:Cathepsins / Q000378:metabolism; D002458:Cell Fractionation; D002478:Cells, Cultured; D003208:Concanavalin A / Q000378:metabolism; D003374:Coumarins / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004151:Dipeptides / Q000378:metabolism; D010450:Endopeptidases; D005347:Fibroblasts / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology; D008247:Lysosomes / Q000201:enzymology* / Q000378:metabolism; D008861:Microsomes / Q000201:enzymology* / Q000378:metabolism; D012867:Skin / Q000166:cytology
C037949:4-methylumbelliferyl-casein; D002364:Caseins; D003374:Coumarins; D004151:Dipeptides; D007976:Leupeptins; D003208:Concanavalin A; D006923:Hymecromone; C021159:benzyloxycarbonyl-phenylalanylarginine-4-methylcoumaryl-7-amide; C069012:benzyloxycarbonylarginyl-arginine 4-methylcoumarin-7-ylamide; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; C534281:CTSL protein, human; D056668:Cathepsin L; C534126:CTSH protein, human; D056655:Cathepsin H; C032854:leupeptin