PURIFICATION OF A CYTOSOLIC ENZYME FROM HUMAN LIVER WITH PHOSPHOLIPID HYDROPEROXIDE GLUTATHIONE-PEROXIDASE ACTIVITY
PURIFICATION OF A CYTOSOLIC ENZYME FROM HUMAN LIVER WITH PHOSPHOLIPID HYDROPEROXIDE GLUTATHIONE-PEROXIDASE ACTIVITY
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DOI：
10.1016/0020-711x(94)90097-3
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发表时间：
1994-10-01
期刊：
影响因子：
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通讯作者：
WILLIAMSON, G
中科院分区：
文献类型：
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作者：
CHAMBERS, SJ;LAMBERT, N;WILLIAMSON, G
磷脂过氧化氢谷胱甘肽过氧化物酶(PHGPx)是一种硒蛋白，能抑制微粒体的过氧化作用。人类的酶可能在保护细胞免受氧化损伤方面发挥重要作用，但尚未得到纯化或表征。经硫酸铵分级、亲和层析、Sephadex G-50凝胶过滤、Mono Q树脂阴离子交换层析和Superdex 75高分辨凝胶过滤，从人肝中分离得到PHGPx。纯化倍数约为11.2万倍，以9%的得率从140克人肝组织中获得12微克蛋白质。PHGPx对过氧化氢、过氧化氢异丙苯、过氧化氢亚油酸和过氧化氢磷脂酰胆碱均有活性。根据非变性凝胶过滤的估计，其分子量为16,000。周转数(37℃，pH 7.6)或β-(13-羟基过氧基顺式-9，trans-11-octadecadienoyl)-gamma-palmitoyl)-L-alpha-phosphatidylcholine)为91molmo1-1，S(-1)。根据猪PHGPx的报道，脱氧胆酸能抑制人肝脏酶对过氧化氢异丙苯和对亚油酸过氧化氢的活性。在谷胱甘肽存在下，该酶能有效地抑制抗坏血酸/铁诱导的人B淋巴母细胞AHH-1 TK+/-Chol细胞微粒体的脂质过氧化，但不能抑制人肝微粒体的脂质过氧化。人类细胞系微粒体中没有检测到PHGPx活性。然而，从人肝制备的微体含有0.009 U/mg的内源性PHGPx活性，是将纯PHGPx添加到人淋巴母细胞微体中最大限度地抑制脂质过氧化所需活性的4-5倍。人肝PHGPx表现出与先前从猪和大鼠组织中分离的具有PHGPx活性的酶相似的性质，但由于人肝微体中已经存在高水平的PHGPx活性，因此不能抑制这些微体的过氧化。
Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a selenoprotein which inhibits peroxidation of microsomes. The human enzyme, which may play an important role in protecting the cell from oxidative damage, has not been purified or characterized. PHGPx was isolated from human liver using ammonium sulphate fractionation, affinity chromatography on bromosulphophthalein-glutathione-agarose, gel filtration on Sephadex G-50, anion exchange chromatography on Mono Q resin and high resolution gel filtration on Superdex 75. The protein was purified about 112,000-fold, and 12 mu g was obtained from 140 g of human liver with a 9% yield. PHGPx was active on hydrogen peroxide, cumene hydroperoxide, linoleic acid hydroperoxide and phosphatidylcholine hydroperoxide. The molecular weight, as estimated from non-denaturing gel filtration, was 16,000. The turnover number (37 degrees C, pH 7.6) or (beta-(13-hydroperoxy-cis-9, trans-11-octadecadienoyl)-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 hydroperoxide was inhibited by deoxycholate. In the presence of glutathione, 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 microsomes contained no detectable PHGPx activity. However, microsomes prepared from human liver contained 0.009 U/mg of endogenous PHGPx activity, which is 4-5 times the activity required for maximum inhibition of lipid peroxidation when pure PHGPx was added back to human lymphoblastic cell microsomes. PHGPx from human liver exhibits similar properties 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.