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

前列腺素 19-

• 批准号: 3279261
• 负责人: BETTIE SUE SILER MASTERS
• 金额: $ 19.37万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 1994-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3279261
• 关键词: arachidonate; catalase; cell free system; cell type; complementary DNA; cytochrome P450; eicosanoid metabolism; enzyme mechanism; genetic transcription; genome; histochemistry /cytochemistry; hydroxylation; immunochemistry; kidney; laboratory rabbit; laboratory rat; liver metabolism; lung; microorganism genetics; microsomes; molecular cloning; postmortem; pregnancy; progesterone; prostaglandin E; prostaglandin F; prostaglandin analogs; thromboxanes; transfection

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.

本研究提案中描述的实验方法已被 选择阐明细胞色素P-450 ω- 羟化酶（P-450 PG-ω）在肺微粒体中的表达， 由怀孕、孕酮和其他激素控制引起的。 这 一种独特的血红素蛋白对类花生酸具有底物特异性 在ω-6位具有含氧官能团， 区域选择性地在ω-（或末端）羟基化这些化合物 carbon. 这种酶在兔子中的胎龄依赖性 促使研究从全肺灌注到确定代谢物 P-450 PG-1的分离、纯化和表征 分子水平上的欧米茄。 正在测试的假设是P-450 PG-ω发挥保护作用和/或参与触发 分娩，因为申请人实验室正在进行的研究表明， 在以下动物之间， 妊娠和非妊娠动物。 分子探针， 首席研究员的实验室现在已经在手，将允许 进一步寻找功能意义，阐明器官 特异性、细胞定位、酶机制和代谢 控制这些反应。 为此，进行了以下研究： 设计：1）催化这些的细胞色素P-450的定位 免疫组织化学法检测各种器官和细胞的反应， 免疫电镜技术和原位杂交; 2） 测量各种类二十烷酸（洋地黄素， 羟基二十碳四烯酸和血栓烷） 肝、肺、肾和胎盘; 3）底物的测定 特异性，通过专门设计的基于机制的抑制剂进行抑制， 以及分离共价结合的组织或纯化的酶加合物; 4） P-450 PG-ω或其直系同源物在小鼠中的生理功能评估 血液动力学和心血管测量;和5）在 用于筛选基因组克隆、cDNA和基因组测序的手 克隆和在哺乳动物细胞中表达。 嵌合基因的使用和 定点诱变将允许确定序列 导致P-450 PG-Ω独特特异性的差异 及其直向同源物，如脂肪酸ω-羟化酶。