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

异常汉逊酵母中抗氰呼吸作用的研究

基本信息

批准号：
05680535
负责人：
YOSHIMOTO Akio
金额：
$ 1.28万
依托单位：
Nigata University of Phermacy and Applied Life Sciences
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

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.

据报道，包括高等植物和微生物在内的多种生物都具有抗氰化物呼吸作用。该途径在斑状虫、布氏锥虫中也有发现。该途径由CoQ位点传统电子传递系统的分支组成，由一种不同于细胞色素氧化酶的特异性“替代氧化酶”催化。然而，其生理作用尚不清楚，酶的表征仍不完整。在酵母（Hansenula anomala）中，我们发现一些呼吸抑制剂能快速诱导抗氰呼吸，克隆了替代氧化酶的cDNA，并从cDNA的核苷酸序列推断出氨基酸序列。本课题研究结果如下：(1)获得了一种抗植物中替代氧化酶的单克隆抗体，该单克隆抗体可与异芽草中替代氧化酶发生交叉反应。这些酶在氨基酸序列上有相似之处。(2)苯并- (h) -喹啉等几种氮碱基不具有螯合能力，可有效抑制金丝桃线粒体的抗氰化物呼吸。(3)在Q循环的Q位点阻断H.anomala线粒体的电子传递，O_2生成增加。自由基清除剂抑制了O_2的生成和抗氰呼吸的诱导，表明O_2可能在向细胞核表达替代氧化酶基因的信号形成中起作用。(4)一种抗生素ascofuranone特异性抑制了引起昏睡病的原虫——布氏锥虫线粒体电子传递系统中的甘油-3-磷酸脱氢酶位点，与甘油ascofuranone联合使用对布氏锥虫体外生长有明显的抑制作用。