Purification of a cytosolic enzyme from human liver with phospholipid hydroperoxide glutathione peroxidase activity |
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| Institution: | 1. University of Sfax Tunisia, Faculty of Sciences, Department of Life Sciences, Laboratory of Biodiversity and Aquatic Ecosystems, Ecology and Planktonology, Unit UR 11 ES 72/Street of Soukra Km 3,5, B.P. 1171, CP 3000, Tunisia;2. University of Sfax Tunisia, Faculty of Sciences, Department of life sciences, Laboratory of Animal Ecophysiology, B.P. 95, 3000, Tunisia;3. Laboratory of Plant Biotechnology, Faculty of Sciences of Sfax, Route Sokra, BP 1171, 3000, Sfax, University of Sfax, Tunisia;4. Animal Physiology Laboratory, UR/11 ES70, Sfax Faculty of Sciences, University of Sfax, BP 1171, Sfax 3000, Tunisia;1. Division of Hematologic Malignancies and Cellular Therapy, Duke University Medical Center, Durham, NC;2. Department of Hematology and Medical Oncology, Mount Sinai Hospital, New York, NY;3. Department of Medical Oncology/Hematology, Rocky Mountain Cancer Centers US Oncology Research Denver, CO;4. Department of Hematology/Oncology, Cedars-Sinai Medical Center, Los Angeles, CA;5. Department of Medical Oncology/Hematology, Maryland Oncology Hematology, US Oncology Research Columbia, MD;6. Department of Hematology, Internal Medicine, and Medical Oncology, Providence Cancer Institute, Southfield, MI;7. Department of Internal Medicine, Medical Oncology, and Hematology, Steeplechase Cancer Center, Somerville, NJ;8. Department of Epidemiology and Biostatistics, University of South Carolina, Columbia, SC;9. Department of Social Sciences and Health Policy, Wake Forest School of Medicine, NC;10. Department of Hematology/Oncology, Mayo Clinic, Jacksonville, FL;11. Myeloma Research Advocate/Advisor, Grand Island, NE;12. Bristol Myers Squibb, Princeton, NJ,;13. Department of Hematology/Oncology, Indiana University, Indianapolis, IN |
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| Abstract: | Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a selenoprotein which inhibits peroxidation ofmicrosomes. 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 μg, 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°C, pH 7.6) on (β-(13-hydroperoxy-cis-9, trans-11-octadecadienoyl)-γ-palmitoyl)-l-α-phosphatidylcholine was 91 mol mo?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. |
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