PARTICIPATION OF CYTOCHROME B5 IN ANESTHETIC METABOLISM

本建议的长期目标是通过以下方式了解该机制： 哪些药物代谢和微粒体生物合成反应调节其中之一 另 这一过程是通过两个微粒体之间的相互作用发生的。 细胞色素P-450和细胞色素b5的电子传递链是 件. 有证据表明，这种相互作用发生在微粒体中， 在使用纯化的酶的重构系统中。 我的实验室 证明细胞色素b5在微粒体和 纯化的重组细胞色素P-450系统，用于代谢 挥发性麻醉剂甲氧氟烷。 我实验室的初步数据 也表明甲氧氟烷抑制胆固醇的合成， 已知需要细胞色素b5的物质， 严重的体内后果。 该项目的近期目标有三个方面。 第一， 细胞色素b5在挥发性麻醉剂，氟烷， 将探索恩氟烷和异氟烷。 的卤酸代谢产物 挥发性麻醉剂将衍生化;卤酸衍生物将 通过气相色谱法与干扰物质分离并定量 通过质谱分析。 第二个目标是阐明化学 基于蛋白质修饰剂对细胞色素b5的灭活， 焦碳酸二乙酯。这些实验将首先确定 其中氨基酸被修饰，然后确定 蛋白质中的氨基酸。 第三个也是最后一个短期目标是 确定我们观察的分子基础， 细胞色素b5抑制甲氧氟烷代谢更容易， 苯巴比妥诱导的肝微粒体比肺微粒体更明显。 细胞色素b5和 将从肝和肺中纯化细胞色素P-450 LM 2，表征 和比较 完成前面概述的实验将 大大有助于我们的知识的因素，支配 细胞色素P-450和细胞色素b5之间的相互作用。

The long term objective of this proposal is to understand the mechanism by which drug metabolism and microsomal biosynthetic reactions modulate one another. The process occurs via an interaction of the two microsomal electron transport chains of which cytochrome P-450 and cytochrome b5 are components. Evidence exists that such interaction occurs in microsomes and in a reconstituted system employing purified enzymes. My laboratory has demonstrated that cytochrome b5 is required both in microsomes and in a purified reconstituted cytochrome P-450 system for the metabolism of the volatile anesthetic methoxyflurane. Preliminary data from my laboratory also indicate that methoxyflurane inhibits the synthesis of cholesterol, a substance which is known to require cytochrome b5 with unknown but possibly substantial in vivo consequences. The immediate goals of this project are three-fold. First, the role of cytochrome b5 in the metabolism of the volatile anesthetics, halothane, enflurane and isoflurane will be explored. The haloacid metabolites of the volatile anesthetics will be derivatized; the haloacid derivatives will be separated from interfering substances by gas chromatography and quantitated by mass spectrometry. The second objective is to elucidate the chemical basis of the inactivation of cytochrome b5 by the protein modifying agent, diethylpyrocarbonate. The experiments will be designed to first determine which amino acid(s) is modified and then determine the exact location of the amino acid in the protein. Our third and final short term aim is to determine the molecular basis for our observation that antibodies to cytochrome b5 inhibit methoxyflurane metabolism much more readily in phenobarbital induced liver than in lung microsomes. Cytochrome b5 and cytochrome P-450 LM2 from liver and lung will be purified, characterized and compared. Completion of the previously outlined experiments will significantly contribute to our knowledge of the factors which govern the interactions between cytochrome P-450 and cytochrome b5.