REGULATION OF HUMAN ESTROGEN SYNTHETASE

人类雌激素合成酶的调节

• 批准号: 3313777
• 负责人: FRANCIS L BELLINO
• 金额: $ 16.91万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-09-29 至 1989-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3313777
• 关键词: androgens; chorioallantoic membrane; cyclic AMP; cytochrome P450; enzyme structure; estrogens; gel electrophoresis; gonadotropins; hormone biosynthesis; human tissue; immunochemistry; ligase; microsomes; placenta; prostaglandins; radiotracer; steroids; tissue /cell culture; tritium

The regulation of estrogen secretion plays a crucial role in many physiological processes, particularly reproduction, fetal development, sexual behavior, and in several pathological processes, such as estrogen-dependent carcinomas and heart disease in aging males. Despite the large number of studies describing the regulation of estrogen synthetase (aromatase) in human steroidogenic tissues by gonadotropins steroids and cAMP, very few studies address the mechanism of aromatase activity regulation. The eventual goal of this project is to determine the mechanisms by which this cytochrome P-450 enzyme system is regulated by physiological factors in cell cultures derived from estrogen-secreting human tissues. To accomplish this objective access is required to the tools necessary to properly carry out definitive studies of this important system, i.e., purified aromatase protein components and monospecific antibodies against these components. Since the NADPH-cytochrome P-450 reductase component of aromatase is already purified to greater than 99% homogeneity on this project, and monospecific (polyclonal and monoclonal) antibodies against this protein are available, our efforts will concentrate on the purification to homogeneity of the aromatase P-450 component, already partially purified with rabbit antiserum against it on hand. Both protein components will be characterized and the reconstitution of aromatase studied to determine Km values and how the sensitivity to carbon monoxide inhibition depends on the relative ratios of the two protein components. Monoclonal antibodies against the aromatase P-450 component will be procurred using a unique detection scheme, that utilized the transfer of 3H from a non-volatile to a volatile compound in the absence of homogeneous antigen. We also propose to synthesize mixed sequence synthetic oligonucleotide probes (15- to 25-mer) based on a partial amino acid sequence of the protein components. Because of ethical limitations on human experimentation, we intend to fully develop human cell cultures from term placenta, granulosa and theca cells from pre- and peri-ovulatory ovarian follicles, and endometrial and adipose tissue stromal cells for eventual use in determining the mechanism of aromatase regulation. Studies using antibodies against placental aromatase to determine cross-reactivity against non-placental aromatase will be conducted.

雌激素分泌的调节在许多疾病中起着至关重要的作用 生理过程，特别是生殖，胎儿发育， 性行为，以及在几个病理过程中，例如 老年男性的雌激素依赖型癌症和心脏病。尽管 描述雌激素调节的大量研究 促性腺激素在人类固醇合成组织中的合成酶(芳香酶) 类固醇和cAMP，很少有研究涉及芳香酶的机制。 活动监管。这个项目的最终目标是确定 细胞色素P-450酶系统的调控机制 雌激素分泌来源的细胞培养中的生理因素 人体组织。要实现这一目标，需要访问 适当地对这一重要问题进行明确研究所需的工具 系统，即纯化的芳香酶蛋白组分和单特异性 针对这些成分的抗体。由于NADPH-细胞色素P-450 芳香酶的还原酶组分已提纯到99%以上 该项目的同质性和单特异性(多克隆和单克隆) 针对这种蛋白的抗体已经可用，我们将集中力量 芳香酶P-450组分的纯化及均一性研究 已经用兔抗血清进行了部分纯化。两者都有 将对蛋白质组分进行表征，并重新构建 研究了芳香酶测定Km值以及如何对碳的敏感性 一氧化碳抑制取决于两种蛋白质的相对比例。 组件。抗芳香酶P-450组分的单抗 将使用唯一的检测方案进行获取，该方案利用 在不存在的情况下，将~3H从非挥发性化合物转移到挥发性化合物 同质抗原。我们还提出了合成混合序列的方法 基于部分氨基的人工合成寡核苷酸探针(15-25-mer) 蛋白质组分的酸序列。因为道德上的限制 人体实验，我们打算从 排卵前期和围产期足月胎盘、颗粒和膜细胞 卵巢滤泡、子宫内膜和脂肪组织基质细胞 最终用于确定芳香酶的调节机制。研究 用抗胎盘芳香酶抗体判断交叉反应性 抗非胎盘性芳香酶将进行。