A cysteine proteinase in the penetration glands of the cercariae of Tylodelphys excavata (Trematoda, Diplostomidae).

A cysteine proteinase was detected in the penetration glands of the cercariae of Tylodelphys excavata and examined by means of biochemical and histochemical methods. The enzyme hydrolyzed azocoll, azocasein, azoalbumin, and N-benzoyl-l-arginine-4-nitroanilide at optimal pH 6.8, 6.4, 6.8, and 6.4, respectively, but was incapable of degrading elastin-orcein at the pH range of 6.0-10.0. Under histochemical conditions, the proteinase cleaved N-benzyloxycarbonyl-l-alanine-2-naphthyl ester and N-benzyloxycarbonyl-l-arginine-2-naphthylamide. A number of N-blocked synthetic substrates with l-methionine, l-leucine, glycine, dl- and l-phenylalanine at the P(1) position were not hydrolyzed. The metal cation complexane ethylenediamine tetraacetic acid (EDTA) and the reducing compound dithioerythritol stimulated the proteinase activity, whereas 0.1 mM zinc sulfate and the specific thiol-blocking reagent p-hydroxymercuribenzoate inhibited it. The proteinase activity was also sensitive to phenylmethylsulfonyl fluoride, leupeptin, tosyl-lysyl-chloromethylketone, and tosyl-phenylalanyl-chloromethylketone. An electrophoretic separation of extract proteins under non-denaturing conditions, at an operative pH of 3.5, in the presence of 5 mM EDTA and 5 mM 2-mercaptoethanol, revealed one main band of a relatively high gelatinolytic activity and two to three faint bands of low and very low activity, one of which was produced by a non-glandular intracellular cysteine proteinase related to cathepsin B. The other faint bands presumably were produced by partly autolysed molecules of the enzyme from the penetration glands.