Purification and characterisation of an extracellularly released protease of Trypanosoma brucei

Thrombocytopaenia. or platelet aggregation, is a serious complication of Af rican trypanosomiasis. The biochemical basis is not clearly known. Protease s are known potent inducers of blood coagulation and platelet aggregation, and unknown factors released by Trypanosoma brucei have been shown to induc e platelet aggregation. In attempts to define the biochemical mechanisms in volved in thrombocytopaenia we purified and characterised a major proteolyt ic enzyme released extracellurally by T. brucei. Actively motile trypanosom es released proteins into the medium (phosphate/saline/glucose. pH 8.0) in which the organisms were incubated in vitro. The M-r of the released polype ptides ranged from 15 to > 200 kDa, amongst which are proteases, One of the major protein bands, a 250 kDa protease, was purified to homogeneity by am monium sulfate precipitation followed by diethylaminoethyl (DEAE)-cellulose chromatography and Sephacryl S-300 gel filtration. The protease migrated a s a single band of 63 kDa upon electrophoresis in both denaturing and non-d enaturing gel co-polymerised with gelatin. The enzyme was strongly active a gainst Z-ARR-AFC peptide substrate, with a pH optimum of 7.0. The proteolyt ic activity was enhanced by dithiothreitol and inhibited by E-64, leupeptin , TPCK and antipain. The released proteolytic enzyme is putatively identifi ed as a cathepsin B-like cysteine protease.