Miranda C L, Wang J L, Henderson M C, Buhler D R
Department of Agricultural Chemistry, Environmental Health Sciences Center, Corvallis, Oregon 97331.
Arch Biochem Biophys. 1989 Jan;268(1):227-38. doi: 10.1016/0003-9861(89)90584-5.
Purification of cytochrome P450 from liver microsomes of untreated juvenile male rainbow trout yielded five fractions designated LMC1 to LMC5. All fractions, except LMC4 and LMC5, appeared homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed minimum molecular weights of 50,000 (LMC1), 54,000 (LMC2), 56,000 (LMC3), 58,000 (LMC4), and 59,000 (LMC5). Specific contents ranged from 2.8 (LMC3) to 14.9 (LMC5) nmol heme/mg protein. The catalytic activity of LMC1, LMC2, and LMC5 toward various substrates was examined. LMC2 exhibited the highest estradiol 2-hydroxylase activity and progesterone 16 alpha-hydroxylase activity. LMC2 also was most active in the metabolic activation of aflatoxin B1 (AFB1). In contrast, LMC5 was most active in catalyzing the 6 beta- and 16 beta-hydroxylation of testosterone and the 6 beta-hydroxylation of progesterone. LMC1 showed the highest lauric acid hydroxylase activity. The three isozymes tested had low activity (for LMC2 and LMC5) or no activity (for LMC1) toward benzphetamine or benzo[a]pyrene. Polyclonal antibodies to all five isozymes were raised in rabbits and the antibodies were used to examine the contribution of the P450s to microsomal enzyme activities. The results of microsomal enzyme inhibition studies with polyclonal antibodies showed that anti-LMC2 IgG significantly inhibited the oxidative metabolism of testosterone, lauric acid, AFB1, and benzphetamine. Anti-LMC5 IgG inhibited the oxidation of progesterone, estradiol, benzo[a]pyrene, and benzphetamine. Anti-LMC1 IgG slightly inhibited the microsomal hydroxylation of lauric acid. Anti-LMC3 and anti-LMC4 IgG did not inhibit any of the measured microsomal enzyme activities. These findings suggest that individual constitutive isozymes of trout cytochrome P450 have well-defined contributions to the microsomal metabolism of steroids, fatty acids, and xenobiotics.
从未经处理的幼年雄性虹鳟鱼肝微粒体中纯化细胞色素P450，得到了五个组分，分别命名为LMC1至LMC5。除LMC4和LMC5外，所有组分在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳上均呈现均一性，其最小分子量分别为50,000（LMC1）、54,000（LMC2）、56,000（LMC3）、58,000（LMC4）和59,000（LMC5）。比含量范围为2.8（LMC3）至14.9（LMC5）nmol血红素/毫克蛋白质。检测了LMC1、LMC2和LMC5对各种底物的催化活性。LMC2表现出最高的雌二醇2 - 羟化酶活性和孕酮16α - 羟化酶活性。LMC2在黄曲霉毒素B1（AFB1）的代谢活化中也最为活跃。相比之下，LMC5在催化睾酮的6β - 和16β - 羟化以及孕酮的6β - 羟化方面最为活跃。LMC1表现出最高的月桂酸羟化酶活性。所测试的三种同工酶对苄非他明或苯并[a]芘的活性较低（对于LMC2和LMC5）或无活性（对于LMC1）。用兔制备了针对所有五种同工酶的多克隆抗体，并使用这些抗体来研究细胞色素P450对微粒体酶活性的贡献。用多克隆抗体进行微粒体酶抑制研究的结果表明，抗LMC2 IgG显著抑制睾酮、月桂酸、AFB1和苄非他明的氧化代谢。抗LMC5 IgG抑制孕酮、雌二醇、苯并[a]芘和苄非他明 的氧化。抗LMC1 IgG略微抑制月桂酸的微粒体羟化。抗LMC3和抗LMC4 IgG未抑制任何所测的微粒体酶活性。这些发现表明，虹鳟鱼细胞色素P450的各个组成型同工酶对类固醇、脂肪酸和外源性物质的微粒体代谢有明确的贡献。
