Studies on Cyanide-Resistant Respiration in yeast, Hansenula anomala

(1)在抗霉素A或特定硫化合物的作用下，从汉菜中分离出抗氰呼吸的线粒体。凝胶电泳分析表明，线粒体蛋白诱导了一个36 kDa的蛋白。当细胞在抗霉素A和CCCP存在的情况下被标记为^<35> s -蛋氨酸时，放射性被纳入一个39 kDa的线粒体蛋白。在去除CCCP后，该蛋白被加工成36 kDa的形式，这表明39 kDa蛋白是36 kDa蛋白的前体。(2) Fe^<2+>的特异性螯合剂完全抑制了抗霉素a诱导的抗氰化物呼吸。在细胞中加入Fe^<2+>后，细胞抗氰化物呼吸活性迅速增加，提示Fe^<2+>参与了细胞抗氰化物呼吸的反应机制。(3)由于一种植物样替代氧化酶被报道为锥虫的唯一终末氧化酶，因此研究抗氰呼吸的特异性抑制剂对锥虫感染的化疗具有重要意义。我们发现一些具有非共轭双键的脂肪酸、自由基清除剂（如CoQ衍生物和类黄酮）和阳离子离子载体对抗氰化物呼吸有特定的抑制作用。(4) Q_i位点抑制剂（如抗霉素A）可诱导呼吸氰化物敏感线粒体生成O_2。自由基清除剂抑制O_2的生成，抑制抗氰呼吸的诱导和线粒体36kda蛋白的出现。这些结果表明，O_2参与了抗氰化物呼吸的诱导。(5)克隆了抗霉素A诱导的mRNA cDNA。核苷酸序列显示了一个由342个密码子编码的长长的开放阅读框。免疫印迹分析显示，线粒体中的36kda蛋白是该cDNA编码蛋白的成熟形式。少

(1) Mitochondria exihibiting cyanide-resistant respiration were isolated from Hansenula anomala incubated in the presence of antimycin A or a certain sulfur-compounds. Analysis of mitochondrial proteins by gel electrophoresis indicates that a 36 kDa protein was induced. When the cells were labeled with ^<35>S-methionine in the presence of both antimycin A and CCCP, the radioactivity was incorporated into a 39 kDa mitochondrial protein. Upon removal of CCCP, this protein was processed into a 36 kDa form, suggesting that the 39 kDa protein is the precursor of 36 kDa protein.(2) The antimycin A-dependent induction of cyanide-resistant respiration was completely repressed by specific chelators for Fe^<2+>. The addition of Fe^<2+> to the cells resulted in a rapid increase in cyanide-resistant respiration activity, suggesting that Fe^<2+> is involved in the reaction mechanism of this respiration.(3) Since a plant-like alternative oxidase was reported to serve as a sole terminal oxidase of Tr … More ypanosoma brucei brucei, the studies on the specific inhibitors of cyanide-resistant respiration have taken on a great sibnificance for the chemotherapy of trypanosome infections. We found specific inhibitory effects on cyanide-resistant respiration of some fatty acids with nonconjugated duoble bonds, radical scavengers such as CoQ derivatives and flavonoids, and cation ionophores.(4) Q_i site inhibitors such as antimycin A induce O_2 generation in respiring cyanide-sensitive mitochondria of H. anomala. Radical scavengers suppressed O_2 generation, and inhibited both the induction of cyanide-resistant respiration and the appearance of the mitochondrial 36 kDa protein. These results suggest that O_2 is involved in the induction of cyanide-resistant respiration.(5) A cDNA for mRNA induced by antimycin A was cloned. The nucleotide sequence revealed a long open reading frame of 342 codons encoding a protein. Immunoblotting showed that the 36 kDa protein localized in mitochondria was a mature form of the protein encoded by the cDNA. Less