title: Purification and characterisation of an extracellularly released protease of Trypanosoma brucei.
abstract: Thrombocytopaenia, or platelet aggregation, is a serious complication of African trypanosomiasis. The biochemical basis is not clearly known. Proteases are known potent inducers of blood coagulation and platelet aggregation, and unknown factors released by Trypanosoma brucei have been shown to induce platelet aggregation. In attempts to define the biochemical mechanisms involved in thrombocytopaenia we purified and characterised a major proteolytic enzyme released extracellularly by T. brucei. Actively motile trypanosomes 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 polypeptides 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 ammonium sulfate precipitation followed by diethylaminoethyl (DEAE)-cellulose chromatography and Sephacryl S-300 gel filtration. The protease migrated as a single band of 63 kDa upon electrophoresis in both denaturing and non-denaturing gel co-polymerised with gelatin. The enzyme was strongly active against Z-ARR-AFC peptide substrate, with a pH optimum of 7.0. The proteolytic activity was enhanced by dithiothreitol and inhibited by E-64, leupeptin, TPCK and antipain. The released proteolytic enzyme is putatively identified as a cathepsin B-like cysteine protease.
authors.affiliation: Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, UK. d.okenu@lshtm.ac.uk
authors.forename: D M
authors.initials: DM
authors.lastname: Okenu
authors.forename: K N
authors.initials: KN
authors.lastname: Opara
authors.forename: R I
authors.initials: RI
authors.lastname: Nwuba
authors.forename: M
authors.initials: M
authors.lastname: Nwagwu
mesh_terms: D000818:Animals; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000254:growth & development; D014353:Trypanosomiasis, African / Q000469:parasitology
chemical_list: D010450:Endopeptidases