Isolation and characterization of complex I, rotenone-sensitive NADH:ubiquinone oxidoreductase, from the procyclic forms of Trypanosoma brucei
Isolation and characterization of complex I, rotenone-sensitive NADH:ubiquinone oxidoreductase, from the procyclic forms of Trypanosoma brucei
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DOI：
10.1046/j.1432-1327.2001.02205.x
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发表时间：
2001-05-01
期刊：
影响因子：
--
通讯作者：
Beattie, DS
中科院分区：
文献类型：
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作者：
Fang, J;Wang, YD;Beattie, DS
复合物I，鱼藤酮敏感的NADH：泛醌氧化还原酶，在布氏锥虫布氏线粒体的其他特征已获得。脯氨酸：细胞色素c还原酶和NADH：泛醌氧化还原酶的原环T。布氏杆菌被复合物I鱼藤酮、杀粉蝶菌素A和辣椒素的特异性抑制剂抑制。这些抑制剂对琥珀酸：细胞色素c还原酶活性没有影响。抗霉素A是细胞色素bc（1）复合物（泛醌醇：细胞色素c氧化还原酶）的特异性抑制剂，几乎完全阻断了脯氨酸或琥珀酸作为电子供体的细胞色素c还原酶活性，但对NADH：泛醌氧化还原酶活性没有抑制作用。对鱼藤酮敏感的NADH：泛醌氧化还原酶进行了研究。通过蔗糖密度离心用十二烷基-β-D-麦芽糖苷溶解的线粒体，部分纯化了布氏杆菌，与线粒体膜相比，其比活性增加了约8倍。通过免疫印迹，用抗副球菌亚基和抗合成肽的抗体鉴定了部分纯化的酶的四种多肽为复合物I的同源亚基（51 kDa，PSST，TYKY和ND 4），所述合成肽是从线粒体和核T编码的推定复合物I亚基基因预测的。布鲁氏菌DNA。T.蓝色非变性聚丙烯酰胺凝胶电泳。随后通过免疫印迹分析的布氏杆菌线粒体膜蛋白揭示了分子量为600 kDa的推定复合物I的存在，其含有通过二维Tricine-SDS/PAGE确定的至少11个多肽，包括51 kDa、PSST和TYKY亚基。
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 bc(1) 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.