INDUCTION AND MODE OF ACTION OF HEPATIC DRUG OXIDASE

该提案包含五个项目，代表正在进行的 实验室里的研究。第一个项目代表了 我们努力分离、提纯和鉴定剩下的形式 未治疗大鼠肝微粒体细胞色素P-450的表达。我们必须，要 日期，隔离了八个表格。使用的方法涉及洗涤剂 微粒体膜的增溶，蛋白质的分离 疏水柱和阳离子柱层析， 羟基磷灰石柱层析和阴离子色谱柱 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，以及内源性代谢物模式 像类固醇激素这样的底物。在项目2中，动态平衡在 探讨了细胞色素P-450的组成形式。 检查病理生理条件，例如糖尿病，以确定 对细胞色素P-450的影响上升，三种形态下降。胰岛素逆转 这些影响。丙酮作为介体的作用及其参与 在RLM6，一种糖尿病诱导的P-450的催化下，糖原的生成将是 检查过了。细胞培养条件将用于评估特定的 动态平衡调节剂，如激素和化学物质。蛋白质-蛋白质 互动将在项目3和项目4中研究。在项目3中个人 蛋白质修饰剂和交联剂将被用来识别 细胞色素P-450与其氧化还原伙伴的相互作用。在项目4中， 研究将关注P-450系统的作用机制， 专注于氧化还原电子转移络合物的形成 研究蛋白质的电子流模式。第五个项目是较短的 评估蛋白质磷酸化作为调节因子的作用的研究 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的磷酸化及其催化作用 肝细胞制剂中的单加氧酶活性。

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 hydrophobic 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 focusing SDS-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.