PROSTAGLANDIN 19- & 20-HYDROXYLATION BY CYTOCHROME P-450

这项研究提案中描述的实验方法是 选择阐明细胞色素P-450 omega-1的功能(S) 肺微粒体中的羟基酶(P-450 pg-omega) 由妊娠、黄体酮和其他荷尔蒙调节所致。这 独特的血红素蛋白对二十碳烷类化合物具有底物专一性 在omega-6位置具有含氧官能团，并且 在欧米伽-(或末端)区域选择性地羟化这些化合物 碳。这种酶在兔体内的胎龄依赖性 引发了从全肺灌注到确定代谢物的研究 P-450PG-的分离、纯化和性质研究 分子水平上的omega。正在测试的假设是P-450 PG-omega发挥保护作用和/或参与触发 在申请人的实验室中正在进行的研究显示分娩 前列腺素在全肺代谢中的明确差异 怀孕和未怀孕的动物。这些分子探测器 首席调查员的实验室现在手中将允许 进一步探索器官的功能意义、阐明 特异性、细胞定位、酶机制和代谢 控制这些反应。为此，我们进行了以下研究 设计：1)催化这些反应的细胞色素P-450的定位 免疫组织化学和免疫组织化学检测不同器官和细胞的反应 免疫电子显微镜技术和原位杂交；2) 测定各种二十烷类化合物(前列腺素， 羟基二十碳四烯酸和血栓烷)按区域和细胞类型分类 肝、肺、肾、胎盘；3)底物测定 特异性，通过特别设计的基于机制的抑制剂进行抑制， 以及分离共价结合组织或纯化的酶加合物；4) P-450PG-omega及其同源物的生理功能评价 血流动力学和心血管测量；以及5)全长cDNA 用于筛选基因组克隆、测序cDNA和基因组的手工 克隆，并在哺乳动物细胞中表达。利用嵌合基因和 定点突变将允许序列的确定 导致P-450PG-omega独特特异性的差异 以及它的同源物，如脂肪酸欧米伽羟基酶。

The experimental approaches described in this research proposal have been selected to elucidate the function(s) of the cytochrome P-450 omega- hydroxylase (P-450 PG-omega) in pulmonary microsomes which is highly induced by pregnancy, progesterone and other hormonal manipulations. This unique heme protein exhibits a substrate specificity toward eicosanoids with oxygen-containing functional groups at the omega-6 position and regioselectively hydroxylates these compounds at the omega- (or terminal) carbon. The gestational age dependence of this enzyme in the rabbit has prompted studies ranging from whole lung perfusion to determine metabolite profiles to isolation, purification, and characterization of the P-450 PG- omega at the molecular level. The hypothesis being tested is that P-450 PG-omega plays a protective role and/or participates in the triggering of parturition since ongoing studies in the Applicant's laboratory show definitive differences in whole lung metabolism of prostaglandins between the pregnant and non-pregnant animals. The molecular probes which the Principal Investigators's laboratory now has in hand will permit the further search for functional significance, elucidation of organ specificity, cellular localization, enzymatic mechanism, and metabolic control of these reactions. To this end, the following studies have been designed: 1) Localization of the cytochromes P-450 which catalyze these reactions in various organs and cells by immunohistochemical and immunoelectron microscopic techniques and in situ hybridization; 2) Measurement of the metabolism of the various eicosanoids (prostaglandins, hydroxyeicosatetraenoic acids, and thromboxanes) by regions and cell types of liver, lung, kidney, and placenta; 3) Determinations of substrate specificity, inhibition by specially designed mechanism-based inhibitors, and isolation of covalently bound tissue or purified enzyme adducts; 4) Assessment of physiological function of P-450 PG-omega or its orthologs in hemodynamic and cardiovascular measurements; and 5) full-length cDNAs in hand for screening of genomic clones, sequencing of cDNA and genomic clones, and expression in mammalian cells. The use of chimeric genes and site-directed mutagenesis will permit the determination of the sequence differences which contribute to the unique specificity of P-450 PG-omega and its orthologs, such as the fatty acid omega-hydroxylases.