Study of thiol proteases of normal human skin fibroblasts

AbstractThe 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