期刊
出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jchromb.2005.03.045
关键词
peroxidase activity; thin-layer chromatography; phospholipid hydroperoxide; cholestrol hydroperoxide
资金
- NCI NIH HHS [CA70823, CA72630] Funding Source: Medline
Thiol-dependent peroxidases catalyzing the reductive detoxification of lipid hydroperoxides (LOOHs) are crucial antioxidant components of mammalian cells. There is a growing interest in manipulating expression of such enzymes to better understand their biological roles. A new approach for determining their cellular activity is described, whereby LOOH reduction kinetics are tracked by high performance thin layer chromatography with peroxide-sensitive tetramethyl-p-phenylenediamine detection (HPTLC-TPD). The approach was tested on a tumor cell transfectant clone (7G4) over-expressing selenoperoxidase GPx4. Timed incubation of Triton-solubilized 7G4 cells with GSH and peroxidized phosphatidylcholine (PCOOH), followed by lipid extraction, HPTLC-TPD and densitometry revealed an exponential decay of PCOOH at a rate similar to 80-times greater than that for GPx4-deficient controls (VC). A TPD-detectable cholesterol hydroperoxide (7 alpha-00H) was also reduced much faster by 7G4 than VC extracts. Spraying with H(2)SO(4) after TPD revealed both 7 alpha-00H loss and resolved diol product (7 alpha-OH) accumulation, the kinetics of which were identical. The approach described is relatively convenient, highly specific, and much more sensitive than conventional assays for cellular LOOH reducing enzymes. (c) 2005 Elsevier B.V. All rights reserved.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据