Zbaida S, Levine W G
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461.
Biochem Pharmacol. 1990 Dec 1;40(11):2415-23. doi: 10.1016/0006-2952(90)90081-u.
Azo dyes are reduced to primary amines by the microsomal enzymes NADPH-cytochrome P450 reductase and cytochrome P450. Amaranth, a highly polar dye, is reduced almost exclusively by rat liver microsomal cytochrome P450 and the reaction is inhibited almost totally by oxygen or CO. Activity is induced by pretreatment with phenobarbital or 3-methylcholanthrene. In contrast, microsomal reduction of the hepatocarcinogen dimethylaminoazobenzene (DAB), a lipid soluble, weakly polar compound, is insensitive to both oxygen and CO. However, reconstitution of activity with purified NADPH-cytochrome P450 reductase and a partially purified cytochrome P450 preparation indicates that activity is catalyzed almost exclusively by cytochrome P450. Activity is induced by clofibrate but not phenobarbital, beta-naphthoflavone, 3-methylcholanthrene, isosafrol, or pregnenolone-16 alpha-carbonitrile. These observations suggest the existence of at least two classes of azoreductase activity catalyzed by cytochrome P450. To investigate this possibility, the reduction of a number of azo dyes was investigated using microsomal and partially purified systems and the characteristics of the reactions were observed. Microsomal reduction of azo dyes structurally related to DAB required a polar electron-donating substituent on one ring. Activity was insensitive to oxygen and CO if the substrates had no additional substituents on either ring or contained only electron-donating substituents. Introduction of an electron-withdrawing group into the prime ring conferred oxygen and CO sensitivity on the reaction. Substrates in the former group are referred to as insensitive and substrates in the latter group as sensitive. Inhibitors of cytochrome P450 activity depressed reduction of both insensitive and sensitive substrates. In a fully reconstituted system containing lipid, highly purified NADPH-cytochrome P450 reductase and a partially purified cytochrome P450 preparation, rates of reduction of various insensitive substrates varied several-fold, whereas rates of reduction of sensitive substrates varied by three orders of magnitude. Using purified enzymes, each of the insensitive substrates was shown to be reduced by reductase alone, but only at a fraction of the rate seen in the fully reconstituted system, implying that reducing electrons were transferred to the dyes mainly from cytochrome P450. Conversely, there was substantial, in some cases almost exclusive, reduction of sensitive substrates by purified reductase alone and almost no inhibition by CO. Their reduction, however, was inhibited by CO in microsomal systems.(ABSTRACT TRUNCATED AT 250 WORDS)
偶氮染料通过微粒体酶NADPH - 细胞色素P450还原酶和细胞色素P450被还原为伯胺。苋菜红是一种高度极性的染料，几乎完全由大鼠肝脏微粒体细胞色素P450还原，该反应几乎完全被氧气或一氧化碳抑制。用苯巴比妥或3 - 甲基胆蒽预处理可诱导其活性。相比之下，肝癌致癌物二甲基氨基偶氮苯（DAB）是一种脂溶性、弱极性化合物，其微粒体还原对氧气和一氧化碳均不敏感。然而，用纯化的NADPH - 细胞色素P450还原酶和部分纯化的细胞色素P450制剂重建活性表明，该活性几乎完全由细胞色素P450催化。氯贝丁酯可诱导其活性，但苯巴比妥、β - 萘黄酮、3 - 甲基胆蒽、异黄樟素或孕烯醇酮 - 16α - 腈则不能。这些观察结果表明存在至少两类由细胞色素P450催化的偶氮还原酶活性。为了研究这种可能性，使用微粒体和部分纯化系统研究了多种偶氮染料的还原，并观察了反应的特征。与DAB结构相关的偶氮染料的微粒体还原在一个环上需要一个极性供电子取代基。如果底物在两个环上都没有额外的取代基或仅含有供电子取代基，则其活性对氧气和一氧化碳不敏感。在主环中引入吸电子基团会使反应对氧气和一氧化碳敏感。前一组底物称为不敏感底物，后一组底物称为敏感底物。细胞色素P450活性抑制剂会降低不敏感和敏感底物的还原。在一个包含脂质、高度纯化的NADPH - 细胞色素P450还原酶和部分纯化的细胞色素P450制剂的完全重建系统中，各种不敏感底物的还原速率变化几倍，而敏感底物的还原速率变化三个数量级。使用纯化的酶，每种不敏感底物单独被还原酶还原，但速率仅为完全重建系统中的一小部分，这意味着还原电子主要从细胞色素P450转移到染料上。相反，单独使用纯化的还原酶对敏感底物有大量还原，在某些情况下几乎是唯一的还原，且几乎不受一氧化碳抑制。然而，在微粒体系统中它们的还原受到一氧化碳的抑制。（摘要截断于250字）
