Characterization of a multicatalytic proteinase complex (20S proteasome) from Trypanosoma brucei brucei.
African trypanosomes are tsetse-transmitted protozoan parasites that cause sleeping sickness in humans and 'Nagana' in animals. A high relative molecular mass multicatalytic proteinase complex (MCP) was purified and biochemically characterized from the cytosolic fraction of Trypanosoma brucei brucei. The isolation procedure consisted of fractionation of the lysate by high speed centrifugation, chromatography on Q-sepharose 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 corresponding to a relative molecular mass of 630 kDa on a non-denaturing polyacrylamide gel. The enzyme hydrolyses a wide range of peptide substrates characteristic of chymotrypsin-like, trypsin-like, peptidylglutamylpeptide-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 oxidized proteins. In addition, the chymotryptic and trypsin-like activities 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 enzyme to hydrolyse most oxidized proteins, and the high immunogenicity exhibited suggests a possible involvement of the enzyme in pathogenesis of the disease.
Lomo
P O
PO
Department of Biochemistry, University of Natal, Scottsville, South Africa.
Coetzer
T H
TH
Lonsdale-Eccles
J D
JD
D000818:Animals; D000913:Antibodies, Protozoan / Q000276:immunology; D002499:Centrifugation, Density Gradient; D002645:Chickens; D002850:Chromatography, Gel; D002918:Chymotrypsin / Q000737:chemistry / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006790:Host-Parasite Interactions; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008247:Lysosomes / Q000201:enzymology; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology; D010084:Oxidation-Reduction; D046988:Proteasome Endopeptidase Complex; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000276:immunology; D014353:Trypanosomiasis, African / Q000201:enzymology / Q000469:parasitology; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism
D000913:Antibodies, Protozoan; D009097:Multienzyme Complexes; D002918:Chymotrypsin; D014357:Trypsin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex