PURIFICATION OF A CYTOSOLIC ENZYME FROM HUMAN LIVER WITH PHOSPHOLIPIDHYDROPEROXIDE GLUTATHIONE-PEROXIDASE ACTIVITY

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a selenop rotein which inhibits peroxidation of microsomes. The human enzyme, wh ich may play an important role in protecting the cell from oxidative d amage, has not been purified or characterized. PHGPx was isolated from human liver using ammonium sulphate fractionation, affinity chromatog raphy on bromosulphophthalein-glutathione-agarose, gel filtration on S ephadex G-50, anion exchange chromatography on Mono Q resin and high r esolution gel filtration on Superdex 75. The protein was purified abou t 112,000-fold, and 12 mu g was obtained from 140 g of human liver wit h a 9% yield. PHGPx was active on hydrogen peroxide, cumene hydroperox ide, linoleic acid hydroperoxide and phosphatidylcholine hydroperoxide . The molecular weight, as estimated from non-denaturing gel filtratio n, was 16,000. The turnover number (37 degrees C, pH 7.6) or (beta-(13 -hydroperoxy-cis-9, noyl)-gamma-palmitoyl)-L-alpha-phosphatidylcholine was 91 mol mol(-1) s(-1). As reported for pig PHGPx, activity of the enzyme from human liver on cumene hydroperoxide and on linoleic acid h ydroperoxide was inhibited by deoxycholate. In the presence of glutath ione, the enzyme was a potent inhibitor of ascorbate/Fe induced lipid peroxidation in microsomes derived from human B lymphoblastic AHH-1 TK +/- CHol cells but not from human liver microsomes. Human cell line m icrosomes contained no detectable PHGPx activity. However, microsomes prepared from human liver contained 0.009 U/mg of endogenous PHGPx act ivity, which is 4-5 times the activity required for maximum inhibition of lipid peroxidation when pure PHGPx was added back to human lymphob lastic cell microsomes. PHGPx from human liver exhibits similar proper ties to previously described enzymes with PHGPx activity isolated from pig and rat tissues, but does not inhibit peroxidation of human liver microsomes owing to a high level of PHGPx activity already present in these microsomes.