CHARACTERIZATION OF A MULTICATALYTIC PROTEINASE COMPLEX (20S-PROTEASOME) FROM TRYPANOSOMA-BRUCEI-BRUCEI

African trypanosomes are tsetse-transmitted protozoan parasites that c ause sleeping sickness in humans and 'Nagana' in animals. A high relat ive molecular mass multicatalytic proteinase complex (MCP) was purifie d and biochemically characterized from the cytosolic fraction of Trypa nosoma brucei brucei. The isolation procedure consisted of fractionati on of the lysate by high speed centrifugation, chromatography on Q-sep harose, molecular sieve filtration on Sephacryl S-300, chromatography on HA-Ultrogel and glycerol density gradient centrifugation (10-40%). The final enzyme preparation yielded a single protein band correspondi ng to a relative molecular mass of 630 kDa on a non-denaturing polyacr ylamide gel, The enzyme hydrolyses a wide range of peptide substrates characteristic of chymotrypsin-like, trypsin-like, peptidylglutamylpep tide-hydrolysing activities determined by fluorogenic peptides, Z-Gly- Gly-Leu-NHMec, Z-Arg-Arg-NHMec and Z-Leu-Leu-Glu-beta NA, respectively . The enzyme was found to have a wide variation in pH optimal activity profile, with optimum activity against Z-Gly-Gly-Leu-NHMec at 7.8, Z- Arg-Arg-NHMec at pH 10.5 and Z-Leu-Leu-Glu-beta NA at pH 8.0, showing that the different activities are distinct. The enzyme hydrolysed oxid ized proteins. In addition, the chymotryptic and trypsin-like activiti es were susceptible to inhibition by peptide aldehyde inhibitors with variable inhibition effects. The study demonstrates the presence of a non-lysosomal proteasome pathway of intracellular protein degradation in the bloodstream form of T. b. brucei. Further, the ability of the e nzyme to hydrolyse most oxidized proteins, and the high immunogenicity exhibited suggests a possible involvement of the enzyme in pathogenes is of the disease.