PROTEASES FROM TRYPANOSOMA-BRUCEI-BRUCEI - PURIFICATION, CHARACTERIZATION AND INTERACTIONS WITH HOST REGULATORY MOLECULES

African trypanosomes contain proteases that may be released into the b loodstream of their infected hosts. This paper describes a novel, comb ined isolation of a cysteine proteinase (called trypanopain-Tb) and a serine oligopeptidase (which we call oligopeptidase-Tb) from Trypanoso ma brucei brucei, as well as a comparison of the activities of these t wo enzymes against several host regulatory molecules. The enzymes diff ered in various respects. Firstly, purified trypanopain-Tb was shown t o readily cleave proteins such as gelatin maximally at acidic pH. In c ontrast, oligopeptidase-Tb, which is optimally active at alkaline pH, did not hydrolyse proteins larger than 4 kDa. However, it readily hydr olysed various polypeptides, including neurotensin and atrial natriure tic factor. The interaction of the two enzymes with mammalian inhibito rs also differed. Cystatins and alpha(2)-macroglobulin effectively inh ibited trypanopain-Tb, with the K-i values for cystatin C and low-mole cular-mass kininogen (approximate to 10(-11) M) predicting that trypan opain-Tb is likely to be effectively controlled by these inhibitors if released into the host bloodstream. In contrast, oligopeptidase-Tb wa s not inhibited by serpins or alpha(2)-macroglobulin, suggesting that it may remain active if released into the host bloodstream. In support of these in vitro results, the blood of trypanosome-infected rats dis played no trypanopain-Tb-like activity, but exhibited high oligopeptid ase-Tb-like activity. Thus, while trypanopain-Tb seems likely to be co nfined to an intracellular role within the parasite, oligopeptidase-Tb has the potential to remain active in the host bloodstream and so con tribute directly to pathogenesis.