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

酵母菌Hansenula mrakii IFO 0895具有很强的抗氧化性，能抵抗脂质氢氧化物或超氧阴离子自由基、羟基自由基等活性氧物种引起的氧化应激。这是由于谷胱甘肽过氧化物酶(GPX)，如果细胞暴露在氧化环境中，这种酶是特异诱导的。通过对该酵母菌产生的脂质过氧化氢敏感突变株的生化分析，证明该酶是该酵母适应氧化胁迫所必需的。研究了几种抗氧化酶的活性，并用脂质过氧化氢处理酵母菌时，还诱导了提供NADPH的葡萄糖6-磷酸脱氢酶。对该酵母菌的细胞质膜和线粒体内外膜进行了定位分析，并对该酵母膜进行了纯化，发现该酶对过氧化氢磷脂酰胆碱、胆固醇5α-过氧化氢以及其他有机过氧化氢及其甲酯均有活性。我们试图将GPX与谷胱甘肽(GSH)的合成反应结合起来，但由于我们还没有克隆出H.mrakii GPX的全长基因，所以我们无法构建它。然而，我们通过PCR克隆了GPX基因的一部分片段，它与大鼠GPX-cDNA具有同源性，全长基因的分子克隆正在进行中。然后，我们尝试在固定化细胞体系中使用谷胱甘肽还原酶和葡萄糖6-磷酸脱氢酶偶联的谷胱甘肽循环反应。我们以H.mrakii细胞中依赖谷胱甘肽的反应之一--乙醛酸酶I反应作为模型系统。确定了固定化细胞的固定化、反应和保存的最佳条件。这些条件有望用于使用固定化的H.mrakii细胞系统测定脂质过氧化氢。

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.