PROSTAGLANDIN 19- & 20-HYDROXYLATION BY CYTOCHROME P450

当这项研究项目在1984年启动时，实验的重点是 关于前列腺素的纯化和性质的研究-- 羟基酶在怀孕兔的肺中诱导了100多倍的反应， 首先由鲍威尔和他的合作研究员[J.Biol]观察。化学，253， 6711-6716(1978)]。本实验室[Williams，et al，J.Biol，Chem.， 259,14600-14608(1984年)]，与日本Kusunose的情况一致 [Yamamoto等，J.Biochem，(Tokyo)，96,593-603(1984)]提纯 催化这一活性的酶，鉴定为细胞色素P450。 该酶已被归类为细胞色素P450基因的成员 CYP 4A，并设计了CYP 4A4，表明其与 大鼠肝月桂酸W-羟基酶的克隆和Hardwick等的测序。 作者声明：[J.Biol.化学，262,801-810(1987)]。现在至少有12名成员 ，其中几个已经被克隆，测序和 与埃里克·约翰逊博士的实验室合作表达。这个 这些克隆在非洲绿猴肾细胞(COS-1)中的表达 已经允许测定相对底物的特异性，但是 而不是建立催化效率或物理性质。 许多这些CyP4A蛋白已被证明催化w- 花生四烯酸羟基化合成20-羟基二十碳四烯酸 (20-HETE)，一种有效的肾弓状动脉和主动脉的血管收缩因子 戒指。这项提案的具体目标旨在解决这些问题 这个细胞色素P450基因亚家族的各个成员的性质， P450 4A4(肺)和P450 4A5、4A6和4A7(肾)，这赋予了 底物特异性并确定结构-功能关系， 并鉴定这些细胞色素P450催化形成 花生四烯酸代谢产物产生特定的生理效应。 具体目的是：1)表达细胞色素P450 4A4、4A5、4A6和4A7 在大肠杆菌中，为了获得足够量的动力学， 光谱和生物物理表征；2)利用歧视性 抑制性抑制剂，被设计为基于机制的“自杀底物”， 它既可以在体内应用，也可以在体外应用，以探索其功能 3)通过定点突变，确定 细胞色素P450 4A4、4A5、 4A6和4A7，利用在我们实验室获得的信息 嵌合结构和在结构中获得的任何附加信息- 这些和其他细胞色素P450的功能研究；4)在 原位杂交法用于差异定位的研究 细胞色素P450 4A蛋白，并建立细胞培养模型 将模仿荷尔蒙治疗对整个动物的诱导作用， 例如黄体酮和地塞米松，以研究肺的调节 和肾脏CyP4A基因亚家族成员，这两个基因是不同的 以及5)开发转基因动物模型以确定 肺(P450 4A4)和肾(P450 4A5、4A6、4A7)的功能(S) 酶，例如在维持和调节血流动力学方面 动态平衡。这些研究旨在解决这些特性 这个密切相关的基因亚家族的成员决定了他们的 底物的特异性，从而确定它们的功能 意义。特别值得注意的是，各种CYP 4A 蛋白质受激素治疗的差异调节 动物。花生四烯酸对肾脏血流动力学的调节作用 酸性代谢物是当前研究的热点。 关于获得性和遗传决定的病理生理学 高血压。

When this research project was initiated in 1984, the experimental focus was on the purification and characterization of the prostaglandin w- hydroxylase induced more than 100-fold in the lungs of pregnant rabbits, first observed by Powell and his co-investigator [J. Biol. Chem., 253, 6711-6716(1978)]. This laboratory [Williams, et al., J. Biol, Chem., 259, 14600-14608 (1984)], coincident with that of Kusunose in Japan [Yamamoto, et al., J. Biochem, (Tokyo), 96, 593-603 (1984)] purified the enzyme catalyzing this activity and characterized it as cytochrome P450. This enzyme has been classified as a member of the cytochrome p450 gene subfamily, CYP 4A, and designed CYP 4A4, indicating its homology with the rat liver laureate w-hydroxylase cloned ad sequenced by Hardwick, et al. [J. Biol. Chem, 262, 801-810 (1987)]. There are now at least 12 members of this gene subfamily, several of which have been cloned, sequenced and expressed in collaboration with Dr. Eric Johnson's laboratory. The expression of these clones in African green monkey kidney cells (COS-1) has permitted the determination of relative substrate specificities but not the establishment of catalytic efficiencies or physical properties. A number of these CYP 4A proteins have been shown to catalyse the w- hydroxylation of arachidonic acid to form 20-hydroxyeicosatetraenoic acid (20-HETE), a potent vasoconstrictor of renal arcuate arteries and aortic rings. The Specific Aims of this proposal are designed to address those properties of the various members of this cytochrome p450 gene subfamily, p450 4A4 (lung) and P450s 4A5, 4A6, and 4A7 (kidney), which confer the substrate specificities and determine structure-function relationships, and to identify those cytochromes P450 catalyzing the formation of arachidonic acid metabolites producing specific physiological effects. Specific Aims are: 1) to express cytochromes P450 4A4, 4A5, 4A6, and 4A7 in E. coli in order to obtain sufficient quantities for kinetic, spectral, and biophysical characterization; 2) to utilize discriminatory inhibitory inhibitors, designed as mechanism-based, "suicide substrates", which can applied in vivo, as well as in vitro, to probe the functions of these enzymes; 3) to determine, by site-directed mutagenesis, the specificity-conferring determinants/domains of cytochromes P450 4A4, 4A5, 4A6, and 4A7, utilizing the information obtained in our laboratory with chimeric constructs and any additional information obtained in structure- function studies of these and other cytochromes P450; 4) to perform in situ hybridization for differential determination of the localization of the cytochrome P450 4A proteins and to develop cell culture models which will mimic the inductive effects of hormonal treatments of whole animals, e.g., progesterone and dexamethasone, to study the regulation of the lung and kidney CYP 4A gene subfamily members, which are differentially induced; and 5) to develop transgenic animal models to determine the function(s) of the lung (P450 4A4) and kidney (P450s 4A5, 4A6, 4A7) enzymes, for example in maintaining and regulating hemodynamic homeostasis. These studies are designed to address those properties of the members of this closely related gene subfamily which determine their substrate specificities and, thus, to ascertain their functional significance. It is particularly noteworthy that the various CYP 4A proteins are differentially regulated by hormonal treatment of whole animals. The regulation of hemodynamics in the kidney by arachidonic acid metabolites is a subject of current interest and intense research regarding the pathophysiology of acquired and genetically determined hypertension.