Studies of Cyanide-Resistat Respiratrion in the Yeast, Hansenula anomala

抗氰呼吸作用在包括高等植物和微生物在内的多种生物中均有报道。该途径在动物寄生虫、布氏锥虫中也有发现。该途径由一个分支，从传统的电子传递系统在辅酶Q网站催化的一个特定的“交替氧化酶”不同于细胞色素氧化酶。然而，生理作用仍然未知，酶的表征仍然不完整。本研究在异常汉逊酵母（Hansenulaanomala）中发现了呼吸抑制剂能快速诱导其抗氰呼吸，克隆了交替氧化酶的cDNA，并根据其核苷酸序列推导出了相应的氨基酸序列，结果如下：（1）抗植物交替氧化酶的单克隆抗体与来自汉逊酵母的酶发生了交叉反应。这些酶之间的氨基酸序列具有相似性。(2)几个氮碱，如苯并-（h）-喹啉，没有螯合能力，是有效的抑制剂的抗氰呼吸的线粒体分离自H. dichroma。(3)在Q-循环的Q位点阻断线粒体的电子传递后，O_2的产生增加。自由基清除剂抑制O_2的产生和抗氰呼吸的诱导，提示O_2可能在向细胞核传递信号以表达交替氧化酶基因中起作用。(4)一种抗生素，ascofuranone，特异性地抑制甘油-3-磷酸脱氢酶位点的线粒体电子传递系统从布氏锥虫布氏锥虫，一种原生动物寄生虫负责睡眠病，并在与甘油ascofuranone组合显示出有效的抑制体外生长的寄生虫。

The cyanide-resistant respiration was reported in a variety of organisms, including higher plants, and microorganisms. This pathway was also found zoomatigophoria, Trypanosoma brucei brucei. The pathway consists of a branching from the conventional electron transport system at CoQ site catalyzed by a specific "alternative oxidase" distinct from cytochrome oxidase. However, the physiological role remains unknown, and the characterization of the enzyme is still incomplete. In the yeast, Hansenula anomala, we found the rapid induction of the cyanide-resistant respiration with some respiratory inhibitors, cloned cDNA for the alternative oxidase, and deduced the amino acid sequence from the nucleotide sequence of the cDNA.Our finding in this research project are as follows :(1) A monoclonal antibody against the alternative oxidase in the plant cross-reacted with the enzyme from H.anomala. There was a similarity in the amino acid sequence between these enzymes.(2) Several nitrogen bases such as benzo- (h) -quinoline, with no chelating ability, were potent inbitors of cyanide resistant respiration of mitochondria isolated from H.anomala.(3) When the electron transport of the mitochondria from H.anomala was blocked at Q site of Q-cycle, O_2 generation was increased. Both O_2 generation and the induction of cyanide-resistant respiration were suppressed by radical scavengers, suggesting that O_2 might play a role in the signal formation toward nuclei to express the alternative oxidase gene.(4) An antibiotic, ascofuranone, specifically inhibits glycerol-3-phosphate dehydrogenase site in the mitochondria electron transport system from Trypanosoma brucei brucei, a protozoan parasite responsible for sleeping sickeness, and in combination with glycerol ascofuranone showed a potent inhibition on the in vitro growth of the parasite.