Analyzes of physiological properties in defensive mechanisms against oxidative stress in yeasts

对两种酵母（Hansenula mrakii）和酿酒酵母（Saccharomyces cerevisiae）的氧化应激防御机制进行了生化和分子生物学分析。所得结果可总结如下：H。mrakii IFO 0895对脂质过氧化氢引起的氧化应激表现出较高的抗性，这是由于在脂质过氧化氢存在下特异性诱导谷胱甘肽过氧化物酶（GPx）。发现GPx与细胞质膜以及线粒体内外膜结合。线粒体是呼吸过程中产生大量活性氧的细胞器，因此发现GPx清除活性氧以保护细胞免受氧化损伤。还原型谷胱甘肽（GSH）通过GPx反应氧化为谷胱甘肽二硫（GSSG）， GSSG在NADPH存在下通过谷胱甘肽还原酶（GR）还原为谷胱甘肽。由GR反应生成的NADP^+被葡萄糖-6-磷酸脱氢酶（G6PDH）还原为NADPHA。我们发现氧化应激也会诱导hmraki的G6PDH。对G6PDH进行了纯化和表征。从酿酒酵母中克隆出抗过氧化脂质基因OSR （oxidative stress resistance）。我们测定了OSR基因的DNA序列，发现它包含一个1278 bp的开放阅读框。基因产物的分子量计算为47,075。构建了OSR基因的敲除突变体，并分析了其表型特征。基因敲除突变体仍然存活，因此该基因产物不是酿酒酵母生长所必需的；然而，敲除突变体对脂质过氧化氢敏感。与野生型相比，敲除突变体的细胞内GSH水平下降了约40%，而osr过表达细胞的细胞内GSH水平是对照的3倍。因此，OSR基因产物的功能被认为是调节细胞内GSH含量以适应氧化应激。少

Biochemical and molecular biological analyzes in the defensive mechanisms against oxidative stress in two kinds of yeasts, Hansenula mrakii and Saccharomyces cerevisiae, were done in the course of this study. The results obtained can be summarized as follows :H.mrakii IFO 0895 showed high resistance against oxidative stress caused by lipid hydroperoxide, which was due to the specific induction of glutathione peroxidase (GPx) in the presence of lipid hydroperoxide. The GPx was found to bind the cytoplasmic membrane as well as inner and outer membrane of mitochondria. Mitochondria are the organelles in which a large amount of reactive oxygen species are generated during the respiration, thus the GPx was found to scavenge the reactive oxygen species to protect the cells against the oxidative damage. Reduced form of glutatione (GSH) is oxidized to glutahione disulfide (GSSG) by the GPx reaction, and GSSG is reduced to GSH by glutathione reductase (GR) in the presence of NADPH.NADP^+ formed … More by the GR reaction is reduced to NADPHA by glucose-6-phosphate dehydrogenase (G6PDH). We found that the G6PDH was also induced by oxidative stress in H.mrakii. G6PDH was purified and characterized.The OSR (oxidative stress resistance) gene was cloned as a gene that enhanced the resistance against lipid hydroperoxide from S.cerevisiae. We determined the DNA sequence of the OSR gene, and it was found to contain an open reading frame with 1,278 bp. The molecular weight of the gene product was calculated to be 47,075. The knockout mutant of the OSR gene was constructed and its phenotypic characters were analyzed. The knockout mutant was still viable, therefore, the gene product was not necessary for the growth of S.cerevisiae ; however, the knockout mutant was hypersensitive to lipid hydroperoxide. Intracellular GSH level of the knockout mutant decreased approximately 40% compared with that of wild type, whereas that of the OSR-overexpressing cell was 3-times higher than control. Therefore, the function of the OSR gene product was thought to regulate the intracellular GSH content to apapt the oxidative stress. Less