Dr. Murai Éva szerk.: Parasitologia Hungarica 14. (Budapest, 1982)
Studies on the protease inhibitor of a rumen fluke, Paramphistomum daubneyi Dr. Sándor JUHÁSZ Veterinary Medical Research Institute, Hungarian Academy of Sciences, Budapest, Hungary "Studies on the protease inhibitor of a rumen fluke, Paramphistomum daubneyi" - Juhász, S. - Parasit, hung., 14: 67-72. 1981-82. ABSTRACT, Trypsin and chymotrypsin inhibiting factor was obtained by affinity chromatography in a partially purified form from the extracts of P. daubneyi Dinnik, 1962, a fluke of the rumen of cattle. The same molecule can inhibit both trypsin and chymotrypsin by the same (or closely adjacent) active sites. The molecular weight of the inhibitor as estimated by gel chromatography is 6 000. The inhibitor is heat stable, resistant to pH changes, treatment by urea, but sensitive to 2-mercaptoethanol. The complex-formation between enzyme and inhibitor is time-dependent. ) Protease inhibitors were originally detected in nematodes and cestodes (BRAND, 1979). The first observation on trematodes was made by HALTON and DERMOTT (1967) who supposed a protective role of the secretion of acidophilic secretory cells surrounding the oral sucker and the pharynx of Opisthio- glyphe ranae against the enzymes of host. KLTMENKO and KENINA (1971) have not found any inhibitory effect in the extracts of Fasciola hepatica, F . gigantica and Caliphoron sp. against trypsin and chymotrypsin. HAJDU et al. (1979) demonstrated proteolytic inhibitory activity in somatic extracts of F . he patica and in the maintenance fluid, against trypsin arid chymotrypsin of mammalian origin. JUHASZ (1980) reported that somatic extracts of Paramphistomum daubneyi inhibited the activity of trypsin and chymotrypsin A and B, but had no influence on elástase, subtilisin. pepsin, rennin, papain and collagenase activities. Since inhibitory effects of trematodes were observed only jn crude worm extracts, it was necessary to study the real inhibitor(s), in purified form. This paper^escribes the purification and some properties of the inhibitor of P. daubneyi . MATERIALS AND METHODS P. daubneyi Dinnik, 1962 worms were collected in the abbatoire from the rumen of freshly slaughtered cattle, washed in five changes of tapwater, homogenized with distilled water (1 g worm + 4 ml distilled water) in Sorvall Omni-Mixer under constant cooling. The homogenate was centrifuged at 20 000 g for 40 min at 5°C. The clear supernatant was collected and kept in refrigerator at 4°C until used. The supernatant had no measurable trypsin and chymotrypsin activities. Enzyme activity measurements were conducted spectrophotometrically (Unicam SP 1800 Ultraviolet Spectrophotometer, AR-25 Linear Recorder, thermoregulated cuvettes). Trypsin activity was measured on N- cc-benzoyl-L-arginine ethyl ester (BAEE) substrate as suggested by SCHWERT and TAKENAKA (1955), the chymotrypsin activity on N-benzoyl-L-tyrosine ethyl ester (BTEE) by the method of HUMMEL (1959). For detailed description of the methods, see JUHÁSZ (1979a).
