Purification and characterization of three male-specific and one female-specific forms of cytochrome P-450 from rat liver microsomes.
Purification and characterization of three male-specific and one female-specific forms of cytochrome P-450 from rat liver microsomes.
复制标题
从大鼠肝微粒体中纯化和表征三种雄性特异性和一种雌性特异性细胞色素 P-450。
DOI：
10.1093/oxfordjournals.jbchem.a121842
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发表时间：
1986
影响因子：
2.7
通讯作者：
T. Omura
中科院分区：
文献类型：
--
作者：
T. Matsumoto;Y. Emi;S. Kawabata;T. Omura
分别从未经处理的雄性和雌性大鼠的肝微粒体中纯化了三种形式的细胞色素 P-450，暂定为 P-450(M-1)、P-450(M-2) 和 P-450(M-3)，以及一种形式的细胞色素 P-450，P-450(F-1)。每种纯化形式的细胞色素在 SDS 聚丙烯酰胺凝胶电泳上显示单个蛋白条带，P-450(M-1) 的最小分子量为 51,000，P-450(M-2) 为 48,000，P-450(M-3) 为 49,000，P-450(F-1) 为 50,000。还原后的 P-450(M-1)、P-450(M-2)、P-450(M-3) 和 P-450(F-1) 的一氧化碳差值光谱分别在 451、451、448 和 449 nm 处显示最大吸收。从绝对吸收光谱来看，细胞色素P-450的四种形式在氧化形式中均属于低自旋型。在 Ouchterlony 双扩散试验中，针对 P-450(M-2) 的抗体不与其他形式发生交叉反应，而免疫扩散试验显示 P-450(M-1) 和 P-450(F-1)、P-450(M-1) 和 P-450(M-3) 以及 P-450(M-3) 和 P-450(F-1) 之间存在免疫交叉反应。四种形式的 NH2 末端氨基酸序列证实它们是不同的分子种类，尽管 P-450(M-1)、P-450(M-3) 和 P-450(F-1) 之间存在显着的同源性。通过定量免疫沉淀法对肝微粒体中的 P-450(M-1) 和 P-450(F-1) 进行定量，证实这两种形式的细胞色素 P-450 分别在雄性和雌性大鼠中被诱导发育。 P-450(M-2) 也在雄性大鼠中被诱导发育。在含有 NADPH 和 NADPH-细胞色素 P-450 还原酶的重建系统中，P-450(M-1) 以高速率氧化苯异丙胺，而其他形式对苯异丙胺的活性较低。四种形式均未表现出对苯并(a)芘的高活性。 P-450(M-1) 在 16 α 和 2 α 位催化睾酮羟基化，而 P-450(M-2) 催化同一底物的 15 α 羟基化。
Three forms of cytochrome P-450, tentatively designated P-450(M-1), P-450(M-2), and P-450(M-3), and one form of cytochrome P-450, P-450(F-1), were purified from the liver microsomes of untreated male and female rats, respectively. Each purified form of the cytochrome showed a single protein band on SDS-polyacrylamide gel electrophoresis, and gave a minimum molecular weight of 51,000 for P-450(M-1), 48,000 for P-450(M-2), 49,000 for P-450(M-3), and 50,000 for P-450(F-1). The carbon monoxide-difference spectra of reduced P-450(M-1), P-450(M-2), P-450(M-3), and P-450(F-1) showed an absorption maximum at 451, 451, 448, and 449 nm, respectively. Judging from the absolute absorption spectra, the four forms of cytochrome P-450 were of low-spin type in the oxidized forms. The antibodies against P-450(M-2) did not crossreact with the other forms in the Ouchterlony double diffusion test, whereas the immunodiffusion test showed immunocrossreactivity between P-450(M-1) and P-450(F-1), P-450(M-1) and P-450(M-3), and P-450(M-3) and P-450(F-1). The NH2-terminal amino acid sequences of the four forms confirmed that they were different molecular species, although significant homology was noticed among P-450(M-1), P-450(M-3), and P-450(F-1). The quantitation of P-450(M-1) and P-450(F-1) in liver microsomes by quantitative immunoprecipitation confirmed that these two forms of cytochrome P-450 were developmentally induced in male and female rats, respectively. P-450(M-2) was also developmentally induced in male rats. In a reconstituted system containing NADPH and NADPH-cytochrome P-450 reductase, P-450(M-1) oxidized benzphetamine at a high rate, whereas the other forms had low activity toward benzphetamine. None of the four forms showed high activity toward benzo(a)pyrene. P-450(M-1) catalyzed the hydroxylation testosterone at the 16 alpha and 2 alpha positions, whereas P-450(M-2) catalyzed the 15 alpha hydroxylation of the same substrate.