ACETONE-INDUCIBLE CYTOCHROME-P-450 - PURIFICATION, CATALYTIC ACTIVITY, AND INTERACTION WITH CYTOCHROME-B5
ACETONE-INDUCIBLE CYTOCHROME-P-450 - PURIFICATION, CATALYTIC ACTIVITY, AND INTERACTION WITH CYTOCHROME-B5
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DOI：
10.1016/0003-9861(86)90373-5
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发表时间：
1986-12-01
影响因子：
3.9
通讯作者：
YANG, CS
中科院分区：
文献类型：
--
作者：
PATTEN, CJ;NING, SM;YANG, CS
本文报道了从丙酮处理的大鼠肝微粒体中纯化丙酮诱导型细胞色素P-450（P-450 ac）的方法。含有16.0至16.5 nmol P-450/mg蛋白质的P-450 ac制剂在十二烷基硫酸钠存在下凝胶电泳时以单一蛋白质条带移动，估计分子量为52，000。铁P-450 ac在25 ℃下在394 nm处显示出最大吸收。这表明它主要以高自旋形式存在。它也存在于低自旋的形式，特别是在较低的温度下，如在412 nm区域的吸收最大值所示。用NADPH复溶后：细胞色素P-450还原酶和磷脂，P-450 ac有效地催化N-亚硝基二甲胺（NDMA）的脱甲基和脱亚硝化，Vmax值分别为23.8和2.3 nmol min-1 nmol P-450-1。P-450 ac的催化活性受到细胞色素b5的极大影响，细胞色素b5使这些反应的Km值降低了10倍，并使Vmax值增加。细胞色素b5似乎与P-450以1：1的摩尔比相互作用，并且这种相互作用需要完整的细胞色素b5结构。在所研究的底物中，NDMA的脱甲基化受细胞色素b5的影响最大，且速率最高。P-450 ac还催化N-亚硝基甲基乙胺和苯胺的氧化，细胞色素b5的活性略有增强。细胞色素b5没有增强其他药物底物，如苄非他明，氨基比林，乙基吗啡的P-450 ac催化代谢。P-450 ac的性质似乎与先前研究的大鼠P-450 et（乙醇诱导型）、大鼠P-450 j（异烟肼诱导型）和兔P-450 LM 3a（乙醇诱导型）相似。这些P-450物种代表了一类新的P-450同工酶，它们在许多内源性和外源性物质的代谢中很重要。
A procedure was developed for the purification of an acetone-inducible form of cytochrome P-450 (P-450ac) to electrophoretical homogeneity from liver microsomes of acetone-treated rats. The P-450ac preparation containing 16.0 to 16.5 nmol P-450/mg protein moved as a single protein band with an estimated molecular weight of 52,000 upon gel electrophoresis in the presence of sodium dodecyl sulfate. The ferric P-450ac showed an absorption maximum at 394 nm at 25.degree. C, suggesting that it exists mainly in the high-spin form. It also existed in the low-spin form, especially at lower temperatures, as indicated by the absorption maximum in the 412-nm region. Upon reconstitution with NADPH: cytochrome P-450 reductase and phospholipid, P-450ac efficiently catalyzed both the demethylation and denitrosation of N-nitrosodimethylamine (NDMA) showing Vmax values of 23.8 and 2.3 nmol min-1 nmol P-450-1, respectively. The catalytic activity of P-450ac was greatly affected by cytochrome b5 which decreased the Km values of these reactions by a factor of 10 and increased the Vmax values. Cytochrome b5 appeared to interact with P-450 at a molar ratio of 1:1 and an intact cytochrome b5 structure was required for such interaction. Among the substrates studied, the demethylation of NDMA was affected the most by cytochrome b5 and showed the highest rate. P-450ac also catalyzed the oxygenation of N-nitrosomethylethylamine and aniline and the activity was enhanced slightly by cytochrome b5. Cytochrome b5 did not enhance the P-450ac-catalyzed metabolism of other drug substrates such as benzphetamine, aminopyrine, and ethylmorphine. P-450ac appeared to be similar in property to the previously studied rat P-450et, (ethanol-inducible), rat P-450j (isoniazid-inducible), and rabbit P-450LM3a (ethanol-inducible). These P-450 species represent a new class of P-450 isozymes that are important in the metabolism of many endobiotics and xenobiotics.