Domin B A, Philpot R M
Arch Biochem Biophys. 1986 Apr;246(1):128-42. doi: 10.1016/0003-9861(86)90456-x.
The content of cytochrome P-450, isozyme 6, in the rabbit pulmonary microsomal fraction was estimated by immunochemical methods to be 1 to 3% of the total cytochrome P-450. Following treatment of rabbits with 2,3,7,8-tetrachlorodibenzo-p-dioxin, the pulmonary microsomal concentration of isozyme 6 increased 16-fold. Isozyme 6 was also detected by immunochemical methods, but not by electrophoresis and staining for protein, in preparations of isozyme 5 isolated from the pulmonary microsomal fraction of untreated rabbits. The metabolism of benzo[a]pyrene in these preparations was found to be catalyzed by isozyme 6, not by isozyme 5 as previously concluded. Cytochrome P-450, isozyme 4, was not detected in the pulmonary microsomal fraction from untreated or 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rabbits. Although benzo[a]pyrene and 7-ethoxyresorufin are both substrates for isozyme 6, the pulmonary microsomal metabolism of these compounds was not increased to the same extent by treatment of rabbits with 2,3,7,8-tetrachlorodibenzo-p-dioxin (about 13-fold for 7-ethoxyresorufin and less than 2-fold for BP). However, lack of agreement between increases in isozyme 6 content and activity, and between the relative increases of the activities with the two substrates, was overcome by the addition of purified NADPH-cytochrome P-450 reductase to the microsomal incubations. When alpha-naphthoflavone, at the minimum concentration required for greater than 90% inhibition of isozyme 6 catalysis, was present in the incubations, no increases in activity were obtained by the addition of purified reductase. The turnover numbers of isozyme 6 in microsomal preparations incubated with purified reductase were similar to those of the purified isozyme in a reconstituted monooxygenase system. The relevance of our results to determinations of the substrate specificities and the microsomal concentrations and activities of isozymes of cytochrome P-450 is discussed. In addition, these parameters are used to assess the extent to which the catalytic potential of isozyme 6 is expressed in the rabbit pulmonary microsomal fraction.
采用免疫化学方法估算，兔肺微粒体组分中细胞色素P - 450同工酶6的含量占细胞色素P - 450总量的1%至3%。用2,3,7,8 - 四氯二苯并 - p - 二恶英处理兔子后，同工酶6在肺微粒体中的浓度增加了16倍。在从未经处理的兔子肺微粒体组分中分离得到的同工酶5制剂中，通过免疫化学方法检测到了同工酶6，但蛋白质电泳和染色未检测到。发现这些制剂中苯并[a]芘的代谢由同工酶6催化，而非如先前结论所述由同工酶5催化。在未经处理或经2,3,7,8 - 四氯二苯并 - p - 二恶英处理的兔子的肺微粒体组分中未检测到细胞色素P - 450同工酶4。虽然苯并[a]芘和7 - 乙氧基试卤灵都是同工酶6的底物，但用2,3,7,8 - 四氯二苯并 - p - 二恶英处理兔子后，这些化合物在肺微粒体中的代谢增加幅度不同（7 - 乙氧基试卤灵约为13倍，苯并[a]芘小于2倍）。然而，通过向微粒体孵育体系中添加纯化的NADPH - 细胞色素P - 450还原酶，克服了同工酶6含量与活性增加之间以及两种底物活性相对增加之间缺乏一致性的问题。当在孵育体系中存在大于90%抑制同工酶6催化所需的最低浓度的α - 萘黄酮时，添加纯化的还原酶未使活性增加。与纯化的还原酶一起孵育的微粒体制剂中同工酶6的转换数与重组单加氧酶体系中纯化的同工酶的转换数相似。讨论了我们的结果与细胞色素P - 450同工酶底物特异性、微粒体浓度和活性测定的相关性。此外，这些参数用于评估同工酶6的催化潜力在兔肺微粒体组分中表达的程度。
