INDUCTION AND MODE OF ACTION OF HEPATIC DRUG OXIDASE

该提案包括五个项目，代表着 实验室正在进行的研究。 第一个项目代表了 我们继续努力分离、纯化和表征 肝微粒体细胞色素P-450的其余形式 未处理的大鼠。 到目前为止，我们已经分离出八种形式。 的 使用的方法包括微粒体的去污剂溶解 膜，在疏水柱上分离蛋白质，然后 通过阳离子柱色谱法，羟基磷灰石柱 色谱法和有时阴离子柱色谱法。 纯化的蛋白质的特征在于NH 2-末端 部分氨基酸序列，二维等电聚焦SDS-PAGE 代谢图，以及代谢模式， 内源性底物如类固醇激素。 在项目2中， 维持细胞色素组成形式的稳态 P-450已被探测。 病理生理条件检查，E。 例如，在一个实施例中，糖尿病，以确定对细胞色素P-450升高的影响， 三种形式下降。 胰岛素逆转这些作用。 的作用 丙酮作为介体及其参与古生菌， 将检测糖尿病诱导的P-450的RLM 6的催化作用。细胞 培养条件将用于评估特定的稳态 调节器，e. G.荷尔蒙和化学物质。 蛋白质 将在项目3和项目4中研究相互作用。 项目3 将使用单独的蛋白质改性剂和交联剂 辨别细胞色素P-450与其氧化还原的相互作用模式 伙伴 在项目4中，研究将涉及 P-450系统的作用机制，集中于形成 的电子转移复合物的氧化还原蛋白质，以研究 电子流动模式 第五个项目是一个较短的研究， 评估蛋白质磷酸化作为调节剂的作用， 体内细胞色素P-450。 已知激活蛋白质的条件 磷酸化/去磷酸化将在以下方面进行检查 单个形式的细胞色素P-450的磷酸化， 肝细胞制备物中的催化单加氧酶活性。

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.