Dr. Murai Éva szerk.: Parasitologia Hungarica 16. (Budapest, 1983)
Parasit, hung. 16. 1983. Studies on a protease inhibitor secreted by Paramphistomum daubneyi Dr. Sándor JUHÁSZ Veterinary Medical Research Institute, Hungarian Academy of Sciences, Budapest, Hungary "Studies on a protease inhibitor secreted by Paramphistomum daubneyi" - Juhász, S. - Parasit, hung., 16: 59-62. 1983. ABSTRACT. In vitro maintained Paramphistomum daubneyi worms released trypsin-chymotrypsin inhibitor into the maintenance medium. The properties of this metabolic inhibitor are identical to those of the somatic inhibitor of P. daubneyi. KEY WORDS: Paramphistomum daubneyi, enzyme inhibition, trypsin, chymotrypsin. In an earlier paper (JUHÁSZ, 1982) it was demonstrated that the somatic extracts of a rumen fluke, Paramphistomum d aubneyi contained a protease inhibitor capable of inhibiting both trypsin and chymotrypsin. The inhibitor was partially purified by affinity chromatography. The inhibition of both enzymes depends on the same (or closely adjacent) sites of the molecule. The molecular weight of the inhibitor is 6 000. The complex formation between the enzyme and the inhibitor requires about five minutes. The inhibitor is unaffected by heating, pH-changes, urea, but is sensitive to 2-mercaptoethanol. Parasitic worms containing somatic inhibitors are able to release inhibitors into the maintenance medium. Ascaris suum under in vitro conditions released trypsin and chymotrypsin inhibitors into the maintenance medium even if the anterior and posterior ends of the worm were sealed (JUHÁSZ, 1979). Protease inhibitors could be demonstrable in culture media of in vitro maintained Ligula intestinalis (MATSKÁSI and JUHÁSZ, 1977). Fasciola hepatica, maintained in vitro, was able to decrease the activity of bovine proteases added to the medium (HAJDÚ et al. , 1979). In the maintenance fluid of the metacestodes of Taenia pisi formis , trypsin and chymotrypsin inhibiting activity was demonstrated (NÉMETH and JUHÁSZ, 1981). Studies on the metabolic protease inhibitor of P. daubneyi Dinnik, 1962 are reported in this paper. MATERIAL AND METHODS P. daubneyi worms were collected in the abattoir from the rumen of freshly slaughtered cattle, washed in five changes of tapwater and placed in a maintaining medium consisting of 14 g TRIS and 4 g NaCl in 1 000 ml distilled water (pH 7.8). Penicillin, streptomycin and sterogenol were added to the medium in order to prevent bacterial and fungal contamination (JUHÁSZ and BABOS, 1969). Ten parasites (0.28 g) were placed into each Erlenmeyer flask, the volume of the medium corresponded to 10 times the weight of the worms. In another flask 5 g worms were maintained in 50 ml medium. The incubation lasted 6 h and 16 h at 37°C. Incubation beyond 16 h was no longer well tolerated by the parasites. After incubation the maintenance medium was centrifuged (16 000 g, 20 min) before inhibitor assays. All the other methods used are detailed elsewhere (JUHÁSZ, 1982).
