Purification of a cytosolic enzyme from human liver with phospholipid hydroperoxide glutathione peroxidase activity.
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 micrograms 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 16,100. The turnover number (37 degrees C, pH 7.6) on (beta-(13-hydroperoxy-cis-9, trans-11-octadecadienoyl)-gamma-palmitoyl)-L-alpha-phosphatidylcho line 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Chambers
S J
SJ
Food Molecular Biochemistry Department, Institute of Food Research, (Colney), Norwich, U.K.
Lambert
N
N
Williamson
G
G
D002478:Cells, Cultured; D002852:Chromatography, Ion Exchange; D005979:Glutathione Peroxidase / Q000302:isolation & purification*; D006801:Humans; D015227:Lipid Peroxidation; D008214:Lymphocytes / Q000201:enzymology; D008862:Microsomes, Liver / Q000201:enzymology*; D008970:Molecular Weight; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D011506:Proteins / Q000302:isolation & purification; D051140:Selenoproteins
D011506:Proteins; D051140:Selenoproteins; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D005979:Glutathione Peroxidase