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

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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.