Epidermal Proteases of American Eel

Fish skin proteases are believed to serve as nonspecific defense factors but their roles remain to be elucidated. A fluorescent-sensitive assay demonstrated protease activities in the dorsal skin of American eel Anguilla rostrata. Extracts were prepared from skin mucus and epidermal cell layers, but only the epidermal extract was reactive to a variety of substrates. The optimum hydrolysis pHs of the epidermal extract activities were found in acidic and neutral ranges, and their optimum hydrolysis temperatures were between 35°C and 50°C. These activities were differentially affected by potential protease inhibitors, metals, and sulfhydryls. Among these reagents, p-tosyl-l-phenylalanyl-chloromethylketone, chymostatin, CdCl2, CuCl2, HgCl2, and ZnCl2 significantly inhibited all tested activities. All cysteine protease inhibitors markedly inactivated hydrolyses of five substrates, and serine protease inhibitors and aminopeptidase inhibitors specifically inactivated other hydrolyses of a single substrate and two substrates, respectively. These findings indicate that epidermal cell layers of American eel reserve at least four skin proteases, which are likely assigned to cathepsins L and B, a serine protease, and an aminopeptidase.