Identification and partial characterization of the proteases from different developmental stages of Schistocephalus solidus (Cestoda: Pseudophyllidae).

The proteolytic activities of extracts prepared from procercoids and plerocercoids as well as adults of the pseudophyllidean cestode Schistocephalus solidus were examined using several proteins and synthetic peptides as substrates. Whole bodies of procercoids and the isolated syncytial tegument of plerocercoids and adults prepared by freeze-thaw were studied. Extracts of procercoids contained a chymotrypsin-like proteinase exhibiting a molecular weight of 23,500 determined by gel filtration chromatography. The proteinase showed collagenolytic activity and had a pH optimum at 8. Such a proteinase was absent in plerocercoids and adults. In these developmental stages leucine aminopeptidases were detected in the isolated syncytial tegument having molecular weights of 93,500 (plerocercoids) and 89,000 (adults), respectively. The aminopeptidases in both stages displayed optimal activity at pH 8.5. The chymotrypsin-like proteinase of the procercoid is possibly necessary for the penetration of the host's intestinal wall, whereas the aminopeptidases of the plerocercoid and the adult of S. solidus may aid in parasite nutrition by degrading oligopeptides at the tegumental surface.

Proteinase activity in the plerocercoid of Proteocephalus ambloplitis (Cestoda).

Host invasion and tissue migration of several helminths have been linked to expression and release of parasite-derived proteinases. The plerocercoid of the cestode Proteocephalus ambloplitis can migrate into the visceral organs or, in the case of bass, from them into the intestinal tract of the same individual fish. It does this within a few hours, aided by secretion of a substance from its apical gland. Proteinase activity in this plerocercoid, obtained from the host liver, was defined by pH optimum, by substrate and inhibitor specificity, and by electrophoretic and chromatographic techniques. Homogenates of plerocercoid contained a metalloproteinase exhibiting a molecular weight of 30,000 determined by gelatin substrate gel electrophoresis. Peak activity of this proteolytic enzyme in gel filtration fractions when azocoll was used as substrate then corresponded to a molecular weight of 31,500. The proteinase showed collagenolytic, haemoglobinolytic and slight elastinolytic activity, and it had a pH optimum at 9.0. Enzyme activity could be inhibited by various chelating agents. The metalloproteinase identified in this study constitutes the only enzyme class present in this larval stage of P. ambloplitis. We suggest that the plerocercoid's metalloproteinase is the substance secreted from the apical organ, necessary for the previously recognized tissue migration phase. This enzyme might also have a nutritional function.

Proteolytic enzymes of Pomphorhynchus laevis and in three other acanthocephalan species.

Cystacanths and adults of the acanthocephalan Pomphorhynchus laevis were found to release proteolytic enzymes during in vitro culture. Culture media in which cystacanths had been kept contained a trypsinlike collagenolytic proteinase exhibiting a molecular mass of 29 kDa on gelatin substrate gel electrophoresis and a pH optimum at pH 9.0. Samples of adults possessed a trypsinlike collagenolytic proteinase with a molecular mass of 26 kDa and showed a pH optimum at 9.0. Leucine aminopeptidase activity was also present in culture media of adults. An apparent molecular mass of 92 kDa and a pH optimum at pH 8.5 were determined for this proteolytic enzyme. It was concluded that the trypsinlike proteinases of both stages were necessary for the complete and quick perforation of the fishes' intestinal wall, whereas the leucine aminopeptidase seemed to have a nutritional function in the terminal stages of protein hydrolysis at the surface of the worm's body wall. Cystacanths of 3 other species of fish-parasitizing Acanthocephala (Acanthocephalus anguillae, Acanthocephalus lucii, Paratenuisentis ambiguus) did not show any histolytic activity.

Leucine aminopeptidase activity in adults of Pomphorhynchus laevis (Acanthocephala): a histochemical study.

In frozen sections of the acanthocephalan Pomphorhynchus laevis, which is a frequent intestinal parasite of cyprinid and salmonid fishes, leucine aminopeptidase (APase) was localized histochemically in outer parts of the presomal bulbus as well as in all layers and most nuclei of the metasomal body wall. Enzyme activity visualized at pH 6.5 using L-leucyl-4-methoxy-2-naphtylamide as the substrate was also associated with ovarian balls, immature larvae, and the testes. The results are discussed with respect to the possible function of APases and the proposed sites of amino acid uptake in tissues of P. laevis.

Identification and characterization of the proteolytic enzymes in the developmental stages of the eel-pathogenic nematode Anguillicola crassus.

The proteolytic activities of homogenates prepared from the second larva (L2) and the third larva (L3) as well as the adult stage of the eel-pathogenic nematode Anguillicola crassus were examined using hemoglobin, azocoll, elastin-orcein, and keratin azure as substrates. Whole bodies of L2 larvae, the anterior third of the bodies of L3 larvae, and the anterior fifth of the bodies of adults were studied. Extracts of L2 contained a trypsin-like proteinase exhibiting a molecular weight of 38,000 Da on gelatin-substrate gel electrophoresis. The proteinase showed a pH optimum at 8 and activity against azocoll and keratin. An apparent molecular weight of 25,000 Da was determined for the trypsin-like proteinase of the L3. This enzyme possessed collagenolytic, keratinolytic and slight elastinolytic activity at an optimal pH of 8. Samples of adults contained an aspartyl proteinase with a molecular weight of 90,000 Da. When hemoglobin was used as the substrate, the enzyme displayed optimal activity at pH 5. It was concluded that the proteinases of the larval stages are penetration enzymes, whereas that of the adult stage is a digestive enzyme.