|dc.contributor.advisor||Coetzer, Theresa Helen Taillefer.||
|dc.contributor.advisor||Lonsdale-Eccles, John David.||
|dc.creator||Lomo, Peter Onyimbo.||
|dc.description||Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1999.||en
|dc.description.abstract||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.||en
|dc.subject||Trypanosoma brucei brucei--Molecular aspects.||en
|dc.title||Studies on a multicatalytic, protease complex from Trypanosoma brucei brucei.||en