Isolation and characterization of complex I, rotenone-sensitive NADH: ubiquinone oxidoreductase, from the procyclic forms of Trypanosoma brucei.
Additional characterization of complex I, rotenone-sensitive NADH:ubiquinone oxidoreductase, in the mitochondria of Trypanosoma brucei brucei has been obtained. Both proline:cytochrome c reductase and NADH:ubiquinone oxidoreductase of procyclic T. brucei were inhibited by the specific inhibitors of complex I rotenone, piericidin A, and capsaicin. These inhibitors had no effect on succinate: cytochrome c reductase activity. Antimycin A, a specific inhibitor of the cytochrome bc1 complex (ubiquinol:cytochrome c oxidoreductase), blocked almost completely cytochrome c reductase activity with either proline or succinate as electron donor, but had no inhibitory effect on NADH:ubiquinone oxidoreductase activity. The rotenone-sensitive NADH:ubiquinone oxidoreductase of procyclic T. brucei was partially purified by sucrose density centrifugation of mitochondria solubilized with dodecyl-beta-D-maltoside, with an approximately eightfold increase in specific activity compared to that of the mitochondrial membranes. Four polypeptides of the partially purified enzyme were identified as the homologous subunits of complex I (51 kDa, PSST, TYKY, and ND4) by immunoblotting with antibodies raised against subunits of Paracoccus denitrificans and against synthetic peptides predicted from putative complex I subunit genes encoded by mitochondrial and nuclear T. brucei DNA. Blue Native polyacrylamide gel electrophoresis of T. brucei mitochondrial membrane proteins followed by immunoblotting revealed the presence of a putative complex I with a molecular mass of 600 kDa, which contains a minimum of 11 polypeptides determined by second-dimensional Tricine-SDS/PAGE including the 51 kDa, PSST and TYKY subunits.
Fang
J
J
Department of Biochemistry, West Virginia University School of Medicine, Morgantown, WV 26506-9142, USA.
Wang
Y
Y
Beattie
D S
DS
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000968:Antimycin A / Q000494:pharmacology; D002211:Capsaicin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D042967:Electron Transport Complex I; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015151:Immunoblotting; D008928:Mitochondria / Q000201:enzymology; D009245:NADH Dehydrogenase / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry*; D011725:Pyridines / Q000494:pharmacology; D012402:Rotenone / Q000737:chemistry*; D013386:Succinates / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000201:enzymology*; D014475:Uncoupling Agents / Q000494:pharmacology*
D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D011725:Pyridines; D013386:Succinates; D014475:Uncoupling Agents; D012402:Rotenone; D000968:Antimycin A; C100213:piericidin A; D009247:NADH, NADPH Oxidoreductases; D009245:NADH Dehydrogenase; D042967:Electron Transport Complex I; D002211:Capsaicin