INDUCTION AND MODE OF ACTION OF HEPATIC DRUG OXIDASE

该提案包含五个项目，代表继续 实验室中正在进行的研究。第一个项目代表 我们继续努力隔离、提纯和表征 仔猪肝微粒体中细胞色素P-450的残存形态 未经处理的大鼠。到目前为止，我们已经分离了八种形式。这个 使用的方法包括洗涤剂对微粒体增溶 膜，然后在疏水柱上分离蛋白质 阳离子柱层析，羟基磷灰石柱层析 层析法和阴离子柱层析法。 纯化的蛋白质经氨基末端鉴定。 部分氨基酸序列，2D等电聚焦SDS-PAGE 电泳图，以及代谢物模式 内源性底物，如类固醇激素。在项目2中，角色 动态平衡对细胞色素组成形式的调控作用 对P-450进行了探针检测。检查病理生理条件，如。 例如，糖尿病，以确定对细胞色素P-450升高和 三种形式的衰退。胰岛素可以逆转这些效应。的作用 丙酮作为中介体及其在糖异生中的作用 RLM6是一种糖尿病引起的P-450，它的催化作用将被研究。细胞 培养条件将用于评估特定的动态平衡 调节剂，例如荷尔蒙和化学物质。蛋白质-蛋白质 互动将在项目3和4中进行研究。在项目3中 将使用单独的蛋白质修饰剂和交联剂来 细胞色素P-450与其氧化还原作用模式的辨析 合伙人。在项目4中，研究将关注 P-450系统的作用机制，集中在形成上 氧化还原蛋白的电子转移络合物的研究 电子流模式。第五个项目是一个较短的研究 评估蛋白质磷酸化作为一种调节因子的作用 体内细胞色素P-450。已知激活蛋白质的条件 将检查以下方面的磷酸化/去磷酸化 不同形式细胞色素P-450的磷酸化和 肝细胞制备中催化单加氧酶活性的研究。

This proposal contains five projects representing continuation of ongoing studies in the laboratory. The first project represents a continuation of our efforts to isolate, purify, and characterize the remaining forms of cytochrome P-450 of liver microsomes of the untreated rat. We have, to date, isolated eight forms. The methods used involve detergent solubilization of the microsomal membrane, separation of proteins on a jydrophobic column followed by cationic column chromatography, hydroxlapatite column chromatography and, orn occassion, anionic column chromatography. The purified proteins are characterized by NH2-terminal partialamino acid sequence, 2D-isoelectric focusingSDS-PAGE electrophoretograms, as well as by metabolite patterns with endogenous substrates like steriod hormones. In Project 2 the role of homeostasis on manitenance of constitutive forms of cytochrome P-450 is probed. Pathophysiological conditions are examined, e. g., diabetes, to determine effects on cytochrome P-450 rise and three forms decline. Insulin reverses these effects. The role of acetone as a mediator and its involvement in guconeogensis, under catalysis of RLM6, a diabetes induced P-450, will be examined. Cell culture conditions will be used to assess specific homeostatic regulators, e. g. hormones and chemicals. Protein-protein interactions will be studied in Project 3 and 4. In Project 3 individual protein modifiers and crosslinkers will be utilized to discern the mode of interaction of cytchrome P-450 with its redox partners. In Project 4, studies will be concerned with the mechanism of action of the P-450 system, concentrating on formation of electron transfer complexs of the redox proteins to study the electron flow patterns. The 5th project is a shorter study to assess the role of protein phosphorylation as a modulator of cytochrome P-450 in vivo. Conditions known to activate protein phosphorylation/dephosphorylation will be examined with respect to phosphorylation of individual forms of cytochrome P-450 and catalytic monooxygenase activity in hepatocyte cell preparation.