Study of thiol proteases of normal human skin fibroblasts

✍ Scribed by Hussain A. Khalfan

• Publisher: John Wiley and Sons
• Year: 1991
• Tongue: English
• Weight: 637 KB
• Volume: 9
• Category: Article
• ISSN: 0263-6484
• DOI: 10.1002/cbf.290090109

✦ Synopsis

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-~-phenylalanyl-~-arginyl-7-amido-4-methy~-coumarin and N-abenzyloxycarbonyl-~-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-t-phenylalanyl-~-arginyl-7amido-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 t-arginine-7-amido-4methylcoumarin substrate. Cathepsin D was present in comparatively small amounts when assayed with 4methylumbelliferyl-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 a-methyl+-mannoside were significantly hydrolysed the synthetic peptides. Only that fraction which bound to concanavalin A was active towards 4-methylumbelliferyl-casein.