INDUCTION AND MODE OF ACTION OF HEPATIC DRUG OXIDASE

这项建议代表了我们继续努力分离，纯化， 并对肝脏内源性细胞色素P-450同工酶进行了表征 未处理动物的微粒体。 这些程序将使用顺序 疏水、阳离子和阴离子柱层析步骤。 两 细胞色素P-450的形式已经得到了表征，还有三种 已经达到了一个纯度阶段，分离程序将加快 用于表征的足够量。 将试图 辨别这些同工酶在维持体内平衡中的作用。 动物;内源性来源的化合物，例如，睾丸激素，孕酮， 雌二醇、胆固醇和脂肪酸将作为底物进行检查。 的 I型结合位点，即底物结合的区域，将被研究以 决定它的尺寸。 将使用尺寸不断增大的基板，直到 观察到一氧化碳结合的限制。 压力研究， 还将执行体积变化测量。 的氨基酸 将使用氨基酸试剂测定I型位点的组成 并测量它们对底物结合和保护免受 试剂通过底物。 P-450系统的作用机制将 分步骤研究，评估铁的自旋状态的影响， 细胞色素和还原酶结合对还原动力学的影响。 氧 还将测量反应的灵敏度。 这些光谱研究 将得到热力学模型和数学模型的发展的支持。 建模 细胞色素b5在P-450单加氧中的作用也将被讨论。 研究并试图解释NADH的机制 协同作用 此外，细胞色素b5对细胞周期的影响 将检查不同底物的NADPH依赖性代谢， 确定代谢物模式是否改变。 将尝试 了解观察到的差异的原因。

This proposal represetns a continuation of our efforts to isolate, purify, and characterize isozymes of cytochrome P-450 which are endogenous to liver microsomes of the untreated animal. Such procedures wil utilize sequential steps of hydrophobic, cationic and anionic column chromatography. Two forms of cytochrome P-450 have already been characterized and three more have reached a stage of purity whereby separation procedures will gear up for sufficient amounts for characterization. Attempts will be made to discern the role of these isozymes in maintaining the homeostasis of the animal; compounds of endogenous origin, e.g., testosterone, progesterone, estradiol, cholesterol and fatty acids will be examined as substrates. The type I binding site, the region where substrates bind, will be studied to determine its dimension. Substrates of increasing size will be used until restriction of carbon monoxide binding is observed. Pressure studies for volume change mesurements will also be performed. The amino acid composition of the type I site will be determined using amino acid reagents and measuring their affect on substrate binding and protection from reagents by substrates. The mechanism of action of the P-450 system will be studied in steps, assessing the influence of spin state of the ferric cytochrome and reductase binding on the kinetics of reduction. The oxygen sensitivity of the reaction will also be measured. These spectral studies will be backed by development of thermodynamic models and mathematical modeling. The roles of cytochrome b5 in P-450 monooxygenation will also be studied and attempts will be made to explain the mechanism of NADH synergism. In addition, differences in influence of cytochrome b5 on the NADPH-dependent metabolism of different substrates will be examined to determine whether metabolite patterns are altered. Attemps will be made to learn the reason for observed differences.