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

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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