AN ELASTASE-LIKE ENDOPEPTIDASE IN THE PENETRATION GLANDS OF THE CERCARIAE OF NEOGLYPHE SOBOLEVI (DIGENEA, PLAGIORCHIIDAE)

An elastase-like proteinase was localized histochemically in the penet ration glands of the cercariae of Neoglyphe sobolevi. The enzyme extra cted from the larvae hydrolyzed azocoll, gelatin, azoalbumin, azocasei n, and elastin-orcein at optimal pH of 8.4, 8.4, 8.0, 7.6, and 8.4, re spectively. The nonionic detergent Triton X-100 slightly enhanced its activity toward azocoll, whereas the anionic detergent SDS, and the ca tionic detergent cetyltrimethylammonium bromide acted as strong inhibi tors. Magnesium ions stabilized the proteinase activity. Strong calciu m and magnesium chelators (EGTA, EDTA) and the serine proteinase inhib itor DFP (0.1 mM) inhibited it. 2 mM 1,10-phenanthroline, a relatively specific chelator of zinc, produced a weak inhibition. The results in dicate, therefore, that the active proteinase represents a metal-enzym e complex rather than a metalloenzyme. Being capable of hydrolyzing N- blocked L-alanine-1-naphthylester, N-blocked L-methionine-1-naphthyles ter, and naphthyl AS-D chloroacetate at pH 6.8, the proteinase activit y was insensitive to 1 mM p-nitrophenyl phosphate, an inhibitor of som e mammalian esterproteinases. The enzyme did not split N-blocked-DL-ph enylalanine-2-naphthylester and also N-blocked L-aminoacyl- and N-bloc ked L-peptidyl-naphthylamides bearing L-arginine, L-alanine, L-phenyla lanine, L-leucine, or L-proline at the P-1 subsite. At operative pH va lues of 4.8 and 3.5 generated during electrophoresis in a stacking and a resolving gel, respectively, the cercarial proteinase migrated towa rd the cathode. The separated enzyme produced four bands of proteolysi s in a gelatin-containing polyacrylamide gel, at the optimal pH of 8.4 .