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REGULATION OF HUMAN ESTROGEN SYNTHETASE

人类雌激素合成酶的调节

基本信息

批准号：
3313779
负责人：
FRANCIS L BELLINO
金额：
$ 19.29万
依托单位：
STATE UNIVERSITY OF NEW YORK AT BUFFALO
依托单位国家：
美国
项目类别：
财政年份：
1981
资助国家：
美国
起止时间：
1981-09-29 至 1990-05-31
项目状态：
已结题

项目摘要

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，并在几个病理过程，如老年男性的雌激素依赖性癌症和心脏病。尽管大量的研究描述了雌激素的调节，促性腺激素在人类固醇生成组织中的合成酶（芳香酶）类固醇和cAMP，很少有研究涉及芳香化酶的机制活动监管。该项目的最终目标是确定这种细胞色素P-450酶系统的调节机制，来自雌激素分泌的细胞培养物中的生理因子人体组织为了实现这一目标，需要获得必要的工具，以适当地进行明确的研究，这一重要的系统，即，纯化的芳香酶蛋白组分和单特异性的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经纯化至大于99%，该项目的同质性和单特异性（多克隆和单克隆）如果有针对这种蛋白质的抗体，我们将集中精力在芳香酶P-450组分的纯化至均质方面，已经用手头的兔抗血清部分纯化。两蛋白质组分将被表征，芳香化酶的研究，以确定Km值和如何敏感性碳一氧化碳抑制作用取决于两种蛋白质的相对比例件. 芳香化酶P-450单克隆抗体将使用一个独特的检测方案，利用 3 H从非挥发性化合物转移到挥发性化合物，同种抗原我们还提出了合成混合序列基于部分氨基的合成寡核苷酸探针（15-至25-聚体）蛋白质组分的氨基酸序列。由于道德限制，人类实验，我们打算充分开发人类细胞培养，排卵前和排卵期的足月胎盘、颗粒细胞和卵泡膜细胞卵泡，子宫内膜和脂肪组织基质细胞，最终用于确定芳香酶调节的机制。研究使用抗胎盘芳香化酶的抗体测定交叉反应性针对非胎盘芳香化酶进行。