CHARACTERIZATION OF THE CYSTEINE PROTEINASES OF THE COMMON LIVER FLUKE FASCIOLA-HEPATICA USING NOVEL, ACTIVE-SITE-DIRECTED AFFINITY LABELS

The excreted/secreted proteinases of adult and juvenile Fasciola hepat ica maintained in vitro were found to hydrolyse the fluorogenic substr ates Cbz-Phe-Arg- and Cbz-Arg-Arg-NHMec. This activity was demonstrate d to have a classical cysteine proteinase inhibitor profile, with turn -over of both substrates being blocked by pre-incubation with E64 and peptidyl diazomethanes. The Cbz-Arg-Arg-NHMec hydrolysing activity of the mature fluke exhibited an alkaline stability not characteristic of its mammalian lysosomal counterparts. Further, the biotinylated affin ity reagents biotin-Phe-Ala CHN2 and biotin-Phe-Cys(SBzyl)-CHN2 were u sed to label and characterize these cysteine proteinases in terms of a pparent molecular weight and subsite specificity. Adult fluke media we re found to contain four species of molecular weights 66, 58, 50 and 2 5-26 kDa; juvenile media contained three species of molecular weights 66, 54 and 25-26 kDa. The major 25-26 kDa cysteine proteinase common t o both stages was shown to have a subsite specificity similar to that of mammalian cathepsin B.