Sakai H, Hino Y, Minakami S
Biochem J. 1983 Oct 1;215(1):83-89. doi: 10.1042/bj2150083.
Three forms of cytochrome P-450 were purified to homogeneity from liver microsomes of Wistar-strain rats treated with phenobarbital. They had minimum mol.wts. of 52 000, 53 000 and 54 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and are designated as P-450(L), P-450(M) and P-450(H) respectively. They were shown to be immunoidentical by Ouchterlony double-diffusion analysis. Several criteria, such as isoelectric points, substrate specificities and sensitivities to tryptic digestion, however, indicated that these cytochromes are distinct isoenzymes of cytochrome P-450. Whereas P-450(L) was highly active on various substrates, P-450(H) had generally low catalytic activities, except on aminopyrine. The cytochromes purified by immunoaffinity chromatography using anti-P-450(L) showed a marked variation in their distribution depending on the strain and colony of rat. Limited tryptic digestion of P-450(H) gave one tryptic peptide showing the same mobility as P-450(L) by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and their primary structures were very similar. The result suggests a possibility that such limited proteolysis is involved in the post-translational modification of the cytochrome or its destruction.
从用苯巴比妥处理的Wistar品系大鼠的肝微粒体中纯化出三种细胞色素P - 450，使其达到均一性。通过十二烷基硫酸钠/聚丙烯酰胺凝胶电泳测定，它们的最小分子量分别为52000、53000和54000，分别被命名为P - 450(L)、P - 450(M)和P - 450(H)。通过双向免疫扩散分析表明它们具有免疫同一性。然而，一些标准，如等电点、底物特异性和对胰蛋白酶消化的敏感性，表明这些细胞色素是细胞色素P - 450的不同同工酶。P - 450(L)对各种底物具有高活性，而P - 450(H)除对氨基比林外，催化活性普遍较低。使用抗P - 450(L)通过免疫亲和色谱法纯化的细胞色素，其分布因大鼠的品系和群体而有显著差异。对P - 450(H)进行有限的胰蛋白酶消化后，得到一个胰蛋白酶肽段，通过十二烷基硫酸钠/聚丙烯酰胺凝胶电泳显示其迁移率与P - 450(L)相同，并且它们的一级结构非常相似。该结果表明这种有限的蛋白水解可能参与了细胞色素的翻译后修饰或其破坏过程。
