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

本研究从生物化学和分子生物学的角度分析了两种酵母菌（Hansenula mrakii和Saccharomyces cerevisiae）对氧化应激的防御机制。所得结果可概括如下：H.mrakii IFO 0895对脂质过氧化氢引起的氧化应激表现出高抗性，这是由于在脂质过氧化氢存在下谷胱甘肽过氧化物酶（GPx）的特异性诱导。发现GPx结合细胞质膜以及线粒体的内外膜。线粒体是呼吸过程中产生大量活性氧的细胞器，因此GPx被发现可以抑制活性氧，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化物（GSSG），GSSG在NADPH存在下被谷胱甘肽还原酶（GR）还原为GSH。 ...更多信息 通过GR反应被葡萄糖-6-磷酸脱氢酶（G6 PDH）还原为NADPHA。我们发现，G6 PDH也诱导的H. mrakii的氧化应激。对葡萄糖6磷酸脱氢酶（G6 PDH）进行了纯化和鉴定，并克隆了抗氧化应激（oxidative stress resistance，OSR）基因。我们测定了OSR基因的DNA序列，发现它含有一个1,278 bp的开放阅读框架。基因产物的分子量计算为47，075。构建了OSR基因敲除突变体，并对其表型特征进行了分析。基因敲除突变体仍然存活，因此，基因产物不是酿酒酵母生长所必需的;然而，基因敲除突变体对脂质过氧化氢高度敏感。与野生型相比，敲除突变体的细胞内GSH水平降低约40%，而OSR过表达细胞的细胞内GSH水平比对照高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