Dr. Murai Éva szerk.: Parasitologia Hungarica 12. (Budapest, 1979)

same sample of the fluke-protease from added bovine protease activity, the latter was determined by subtraction of the fluke enzyme activity measured in enzyme-free system from the total (bovine protease + proteases released by the worm) activity. The difference of the residual activity so established from the control was regarded as the measure of inactivation by the parasite-released protease inhibitors. c) Location of the sites of inhibitor release The foregoing experimental observations have unequivocally indicated that the liver flukes are able to inactivate host trypsin or chymotrypsin. Persistence of the inactivating effect in the maintenance medium after removal of the flukes suggested that the parasites synthesize protease inhibitors, and release these into their environment. No information was, however, emerging from these studies on the site(s) of inhibitor release. In order to obtain more information flukes with sealed oral sucker (group 4) were exposed to host enzymes of known activity (Table 10) in maintenance medium as previously described. The incubation media of the parasites showed lower BAEE- and BTEE-splitting activity than the parasite-free control systems, but both substrate-splitting activities were higher in the media of flukes with closed oral sucker than of not sealed flukes, indicating a lower inhibition by the former. The data of the supposed inhibition are shown in Table 11. The sealing of the oral part did not account for a notable depression of inactivation. Table 11 Protease inactivation in the maintenance medium of flukes with open and closed oral sucker Activity of bovine + released fluke enzym (see the Table 7) U/ml Inhibition (U/ml) oral sucker oral sucker open closed open closed BAEE-splitting activity 1.028 0.800 0.557 0.265 BTEE-splitting activity 4.041 3.477 1.999 1.649 Discussion Earlier studies on liver flukes were centered on the detection of acid - cathepsinlike - protease production by the parasite. We succeeded in demonstrating proteolytic activity in fluke extracts and maintenance media also at slightly alkaline pHs. On the basis of their substrate specificity the parasite enzyme(s) appeared to be trypsin- and chymotrypsinlike in nature. Experiments with protease inhibitors also indicated the presence of two - tryptic and chymotryptic - types of protease activity. Liver fluke extracts displayed a measurable BAEE- and BTEE-splitting activity also at low pHs. The BTEE-splitting activity showed two peaks in Na-phosphate buffer, an optimal one at pH 4.0, and a considerably lower peak at pH 8.0. BAEE-splitting also showed two maxima at pHs 4 and 8, respectively, in the same buffer system. This has suggested that the somatic extract of liver flukes probably contains several BAEE- or BTEE-splitting enzymes.