Subcellular fractionation (together with immunocytochemical localisation studies) showed that the parasite Trypanosoma brucei brucei possesses a multicatalytic protease complex (MCPTb). This complex is predominantly cytosolic but some activity is also present in the nuclear fraction. MCP-Tb was isolated from T. b. brucei and compared to the properties of other proteasomes reported in the literature and to the 20S MCP isolated from bovine red blood cells (MCP-rbc). The isolation procedure employed four-steps: anion exchange chromatography on Q-Sepharose, adsorption chromatography on HA-Ultrogel, molecular exclusion chromatography on Sephacryl S-300 and glycerol density gradient sedimentation. The molecular mass of intact MCP-Tb was shown to be smaller than that of MCP-rbc. Separation of the different proteasome subunits by 2D-PAGE showed that MCP-Tb has 12 different polypeptide components compared to the 28 different polypeptide components of MCP-rbc. The N-terminal sequence of an MCP-Tb subunit showed that this subunit did not have any obvious sequence homology with the subunits of proteasomes from other cells. Furthermore, anti-MCP-Tb antibodies (which exhibited the in vitro inhibitory activity of MCP-Tb) did not cross-react with MCP-rbc showing that MCP-Tb and MCP-rbc are antigenically distinct. The basic enzymatic properties of MCP-Tb were fairly typical of other 20S proteasomes. MCP-Tb had multiple peptidase activities (identified as chymotrypsin-like, trypsin-like and peptidyl glutamylpeptide hydrolase activities) that are characteristic of proteasomes. Furthermore, the characteristics of inhibition by a variety of inhibitors were similar to those of other proteasomes, including MCP-rbc. The activities of 20S proteasomes from most cell types are activated by endogenous high molecular mass complexes such as the bovine 19S complex called PA700. These complexes form end-on associations with the 20S proteasome. However, no endogenous MCP-activator was found in T. b. brucei. Nevertheless, MCP-Tb was activated in an ATP-dependent manner by bovine PA700. Inhibition of the intrinsic phosphatase activity of PA700 inhibited the protease enhancing effect of PA700. Electron microscopic examination of negatively stained MCP-Tb and MCP-rbc showed particles that were morphologically indistinguishable. However, the MCP-Tb also exhibited unique end-on associations between individual units forming long (up to 200 nm) ribbon-like chains. Since access to the active sites of proteasomes occurs through the pores at the end of the complexes, this end-on association, when coupled to our observation of an apparent lack of an endogenous activator, suggests that T. b. brucei may have evolved an alternate mechanism for controlling their proteasome activity. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1999.

The protozoan parasite Trypanosoma brucei brucei causes nagana in cattle and is a widely used model for human sleeping sickness. The major lysosomal cysteine proteinases (trypanopains) of African trypanosomes may contribute to pathogenesis by degrading proteins in the mammalian bloodstream and also appear to be essential for the viability of T. cruzi and T. congolense. This study describes the first purification to electrophoretic homogeneity of trypanopain-Tb from T. b. brucei and the first reported characterisation of its enzymatic properties. Trypanopain-Tb was purified from bloodstream forms of T. b. brucei by a combination of three phase partitioning (between ammonium sulfate and tertiary butanol), and chromatography on quaternary amine or pepstatin A-Sepharose resins. Trypanopain-Tb was found to be a typical cysteine proteinase, in that it is inhibited by typical cysteine proteinase inhibitors and requires reducing agents for full activity. Trypanopain has cathepsin L-like specificity for synthetic substrates and readily degrades various proteins. In vitro analysis of the kinetics of trypanopain interaction with cystatins suggested that these are likely to inhibit any trypanopain released into the mammalian bloodstream. Furthermore, no trypanopain-like activity was detectable in the blood of infected hosts, so it appears that trypanopain is unlikely to contribute directly to pathogenesis by degrading bloodstream host proteins. Antibodies against a peptide corresponding to a region of the trypanopain active site were produced in rabbits and chickens. Both enzyme activity-enhancing and enzyme activity inhibiting antibodies were produced and these effects varied with the substrate tested. Thus, the in vivo effects of anti-trypanopain antibodies will only become clearly understood once the physiological substrates of trypanopain have been identified. Various cysteine proteinase inhibitors, including peptidyl diazomethylketones, killed cultured bloodstream forms of T. b. brucei. Use of biotinylated derivatives of peptidyl diazomethylketone and fluoromethylketone inhibitors suggested that trypanopain is the likely intracellular target of these inhibitors, indicating that the enzyme is essential for parasite viability. Furthermore, chalcones (a class of reversible cysteine proteinase inhibitors) killed in vitro cultured parasites and also prolonged the life of T. b. brucei-infected mice. Thus, trypanopain-Tb seems to be a possible target for new anti-trypanosomal drugs. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1997.