Giulivi C, Cadenas E
Department of Molecular Pharmacology and Toxicology, School of Pharmacy, University of Southern California, Los Angeles 90033, USA.
Chem Biol Interact. 1998 Jun 5;113(3):191-204. doi: 10.1016/s0009-2797(98)00028-3.
The plasma membrane of HT29 human colon carcinoma cells was characterized by EPR spectroscopy as the site for redox activation of 3,6-difluoro-2,5-bis(aziridinyl)-1,4-benzoquinone (F-DZQ). Supplementation of HT29 cells with F-DZQ yielded an EPR signal ascribed to the semiquinone species; the hyperfine splitting constants of the 11-line spectrum were 1.4 and 1.35 G for aN and aF, respectively. The intensity of the EPR signal was inhibited competitively by potassium ferricyanide, a compound which has no access to the intracellular milieu and used to evaluate transmembrane NADH-ferricyanide reductase activity. The extracellular localization of the signal was confirmed by using chromium trioxalate, a membrane-impermeant spin-broadening agent, which abolished in a concentration-dependent manner the semiquinone signal originating from the metabolism of F-DZQ by HT29 cells. The intensity of the semiquinone signal was decreased by agents which block sulfhydryl groups upon alkylation, fluorodinitrobenzene and p-chloromercuribenzoate, presumably acting on plasma membrane dehydrogenases. Other flavin dehydrogenase inhibitors, such as allopurinol, deprenyl or clorgyline, and D-arginine or NG-methyl-L-arginine did not affect the EPR signal. Conversely, the intensity of the semiquinone signal was increased upon supplementation of HT29 cells with glucose and insulin, which may enhance the intracellular levels of electron donors for the transplasma membrane dehydrogenase activity. The extracellular semiquinone signal was abolished by superoxide dismutase by a mechanism implying displacement of the equilibrium of the autoxidation reaction. Formation of oxygen-centered radicals during this redox activity was evaluated by EPR in conjunction with the spin trap 4-POBN. A composite signal consisting of the spin adducts of methyl, hydroxyl and superoxide radicals was observed (the former arising from hydroxyl radical attack on the quinone solvent, dimethylsulfoxide). The formation of these spin adducts was abolished by superoxide dismutase and their detection became impossible in the presence of the line broadening agent chromiun trioxalate, thus indicating their extracellular formation and localization, respectively. The occurrence of a redox site at the plasma membrane of HT29 cells for the activation of this halogenated aziridinylbenzoquinone is discussed in terms of its significance for intracellular processes and a build-up of oxyradicals in the extracellular milieu.
通过电子顺磁共振波谱（EPR）表征，HT29人结肠癌细胞的质膜是3,6 - 二氟 - 2,5 - 双（氮丙啶基）-1,4 - 苯醌（F - DZQ）氧化还原激活的位点。用F - DZQ处理HT29细胞可产生归因于半醌物种的EPR信号；11线谱的超精细分裂常数，aN为1.4 G，aF为1.35 G。铁氰化钾可竞争性抑制EPR信号强度，铁氰化钾是一种无法进入细胞内环境的化合物，用于评估跨膜NADH - 铁氰化物还原酶活性。通过使用三草酸铬（一种膜不透性的自旋展宽剂）证实了该信号的细胞外定位，三草酸铬以浓度依赖性方式消除了HT29细胞中F - DZQ代谢产生的半醌信号。半醌信号强度会被烷基化时阻断巯基的试剂（氟二硝基苯和对氯汞苯甲酸）降低，推测其作用于质膜脱氢酶。其他黄素脱氢酶抑制剂，如别嘌呤醇、司来吉兰或氯吉兰，以及D - 精氨酸或NG - 甲基 - L - 精氨酸均不影响EPR信号。相反，用葡萄糖和胰岛素处理HT29细胞后，半醌信号强度增加，这可能会提高跨质膜脱氢酶活性所需电子供体的细胞内水平。超氧化物歧化酶通过一种涉及自氧化反应平衡位移 的机制消除了细胞外半醌信号。在该氧化还原活性过程中，通过EPR结合自旋捕获剂4 - POBN评估了以氧为中心的自由基的形成。观察到由甲基、羟基和超氧自由基的自旋加合物组成的复合信号（前者源于羟基自由基对醌溶剂二甲基亚砜的攻击）。超氧化物歧化酶消除了这些自旋加合物的形成，并且在存在线展宽剂三草酸铬的情况下无法检测到它们，这分别表明了它们在细胞外的形成和定位。本文从其对细胞内过程的意义以及细胞外环境中氧自由基的积累方面，讨论了HT29细胞质膜上存在用于激活这种卤代氮丙啶基苯醌的氧化还原位点的情况。
