Korytowski W, Geiger P G, Girotti A W
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, 53226, USA.
Biochemistry. 1996 Jul 2;35(26):8670-9. doi: 10.1021/bi960522k.
Phospholipid hydroperoxide glutathione peroxidase (PHGPX) is a selenoenzyme that can catalyze the direct reduction of various membrane lipid hydroperoxides and by so doing could play a vital role in cytoprotection against peroxidative damage. The activity of purified testicular PHGPX on several photochemically-generated cholesterol hydroperoxide (ChOOH) species was investigated, using high-performance liquid chromatography with electrochemical detection for peroxide analysis and thinlayer chromatography with 14C-radiodetection for diol product analysis. The following ChOOH isomers were monitored: 5 alpha-OOH, 6 alpha-OOH, 6 beta-OOH (singlet oxygen adducts), and unresolved 7 alpha,7 beta-OOH (derived from 5 alpha-OOH rearrangement). Apparent first-order rate constants for GSH/PHGPX-induced peroxide loss (or diol accumulation) in Triton X-100 micelles, unilamellar liposomes, or erythrocyte ghost membranes increased in the following order: 5 alpha-OOH < 6 alpha-OOH approximately equal to 7 alpha,7 beta-OOH < 6beta-OOH. A similar trend was observed when the peroxides were incubated with Triton Iysates of Se-replete L1210 or K562 cells, implicating PHGPX in these reactions. Consistent with this, there was little or no ChOOH reduction if GSH was omitted or if lysates from Se-deprived cells were used. Liposomal 5 alpha-OOH was found to be much more cytotoxic than equimolar liposomal 6 beta-OOH, producing a 50% loss of L1210 clonogenicity at approximately 1/5 the concentration of the latter. Faster uptake of 5 alpha-OOH was ruled out as the basis for greater cytotoxicity, suggesting that relatively inefficient metabolism by the GSH/PHGPX system might be the reason. As supporting evidence, it was found that cells accumulate the diol reduction product of 5 alpha-OOH more slowly than that of 6 beta-OOH during incubation with the respective peroxides. Slow detoxification coupled with rapid formation makes 5 alpha-OOH potentially the most damaging ChOOH to arise in cells exposed to singlet oxygen.
磷脂氢过氧化物谷胱甘肽过氧化物酶（PHGPX）是一种含硒酶，它能够催化各种膜脂氢过氧化物的直接还原反应，因此在细胞抵御过氧化损伤的过程中可能发挥着至关重要的作用。利用高效液相色谱 - 电化学检测法进行过氧化物分析，以及利用含¹⁴C放射性检测的薄层层析法进行二醇产物分析，研究了纯化的睾丸PHGPX对几种光化学产生的胆固醇氢过氧化物（ChOOH）的活性。监测了以下ChOOH异构体：5α - OOH、6α - OOH、6β - OOH（单线态氧加合物），以及未解析的7α,7β - OOH（由5α - OOH重排产生）。在Triton X - 100胶束、单层脂质体或红细胞血影膜中，谷胱甘肽/PHGPX诱导的过氧化物损失（或二醇积累）的表观一级速率常数按以下顺序增加：5α - OOH < 6α - OOH ≈ 7α,7β - OOH < 6β - OOH。当过氧化物与富含硒的L1210或K562细胞的Triton裂解物一起孵育时，观察到了类似的趋势，这表明PHGPX参与了这些反应。与此一致的是，如果省略谷胱甘肽或使用缺硒细胞的裂解物，则几乎没有ChOOH的还原。发现脂质体5α - OOH的细胞毒性比等摩尔的脂质体6β - OOH大得多，在大约后者浓度的1/5时，导致L1210克隆形成能力损失50%。排除了5α - OOH更快摄取作为更大细胞毒性基础的可能性，这表明谷胱甘肽/PHGPX系统相对低效的代谢可能是原因。作为支持证据，发现在与各自的过氧化物孵育期间，细胞积累5α - OOH的二醇还原产物比6β - OOH的慢。缓慢的解毒作用加上快速的形成使得5α - OOH可能是暴露于单线态氧的细胞中产生的最具破坏性的ChOOH。
