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Detection of hydroperoxides of phospholipid and cholesterol by a novel glutathione peroxidase

用新型谷胱甘肽过氧化物酶检测磷脂和胆固醇的氢过氧化物

基本信息

批准号：
05556015
负责人：
KIMURA Akira
金额：
$ 3.46万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
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

A yeast Hansenula mrakii IFO 0895 is highly resistant against the oxidative stress caused by lipid hydroperoxides or reactive oxygen species such as superoxide anion radical and hydroxyl radical. It was due to the glutathione peroxidase (GPx) which was specifically induced if the cells were exposed to the oxidative environments. We proved that the enzyme is necessary for this yeast to adapt to the oxidative stress through the biochemical analyzes of lipid hydroperoxide-sensitive mutants derived from this yeast strain. The activities of several antioxidant enzymes were investigated, and glucose 6-phosphate dehydrogenase, which supplies NADPH,was also induced when the yeast was treated by lipid hydroperoxide. Localizaition of GPx was analyzed, and it distributed in the cytoplasmic membrane as well as the inner and outer membrane of mitochondria of this yeast.We purified the GPx from the membrane fractions of this yeast and found that the enzyme was active toward phosphatidylcholine hydroperoxide, cholesterol 5alpha-hydroperoxide as well as other organic hydroperoxides and their methyl esters. We tried to combine GPx with the gluathione (GSH) -synthesizing reaction using E.coli enzymes, however, since we have not yet cloned the full length gene of GPx of H.mrakii, we could not construct it. However, we cloned a partial fragment of GPx gene by PCR.It shared the homology with rat GPx-cDNA.Molecular cloning of the full length gene is now in progress. We then tried to use the GSH-recycling reaction coupled by GPx, glutathione reductase and glucose 6-phosphate dehydrogenase, in immobolized cell system. We used glyoxalase I reaction, which is one of the GSH-dependent reaction in H.mrakii cell , as a model system. We could established the optimal conditions for the immobilization, reaction and storage of the immobilized cell. These conditions are expected to be utilized in the determination of lipid hydroperoxide using the immobolized H.mrakii cell system.

酵母Hansenula mrakii IFO 0895对脂质氢过氧化物或活性氧如超氧阴离子自由基和羟基自由基引起的氧化应激具有高度抗性。这是由于谷胱甘肽过氧化物酶（GPx）的特异性诱导，如果细胞暴露于氧化环境。我们证明，该酶是必要的，该酵母适应氧化应激，通过生化分析的脂质过氧化氢敏感的突变体，从该酵母菌株。研究了几种抗氧化酶的活性，并发现脂质过氧化氢处理酵母时，还诱导产生了葡萄糖6-磷酸脱氢酶，该酶可提供NADPH。GPx的定位分析表明，它分布在该酵母的细胞质膜和线粒体内外膜上，并从该酵母的膜组分中纯化了GPx，发现该酶对磷脂酰胆碱过氧化氢、胆固醇5 α-过氧化氢以及其它有机过氧化氢及其甲酯有活性。本研究尝试将联合收割机GPx与大肠杆菌的谷胱甘肽（GSH）合成反应相结合，但由于还没有克隆到麦氏血吸虫GPx的全长基因，因此无法构建该基因。本研究利用PCR技术克隆了麦氏血吸虫GPx基因的部分片段，该片段与大鼠GPx-cDNA具有同源性。然后，我们尝试在固定化细胞系统中使用由GPx、谷胱甘肽还原酶和葡萄糖6-磷酸脱氢酶偶联的GSH再循环反应。我们使用乙二醛酶I反应（H.mrakii细胞中的GSH依赖性反应之一）作为模型系统。确定了固定化细胞的最佳固定化条件、反应条件和保存条件。预期这些条件可用于使用固定化的麦氏血吸虫细胞系统测定脂质过氧化氢。