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.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD016593-08
Application #
3313779
Study Section
Biochemical Endocrinology Study Section (BCE)
Project Start
1981-09-29
Project End
1990-05-31
Budget Start
1988-12-01
Budget End
1990-05-31
Support Year
8
Fiscal Year
1989
Total Cost
Indirect Cost
Name
State University of New York at Buffalo
Department
Type
Schools of Arts and Sciences
DUNS #
038633251
City
Buffalo
State
NY
Country
United States
Zip Code
14260
Sethumadhavan, K; Bellino, F L (1991) Human placental estrogen synthetase (aromatase). Effect of environment on the kinetics of protein-protein and substrate-protein interactions and the production of 19-oxygenated androgen intermediates in the purified reconstituted cytochrome P450 enzyme sy J Steroid Biochem Mol Biol 39:381-94
Sethumadhavan, K; Bellino, F L; Thotakura, N R (1991) Estrogen synthetase (aromatase). The cytochrome P-450 component of the human placental enzyme is a glycoprotein. Mol Cell Endocrinol 78:25-32
Sethumadhavan, K; Bellino, F L (1990) Estrogen synthetase (aromatase) Affinity purification of antibody against the cytochrome P450 component. J Steroid Biochem 36:295-9
Lobo, J O; Bellino, F L (1989) Estrogen synthetase (aromatase) activity in primary culture of human term placental cells: effects of cell preparation, growth medium, and serum on adenosine 3',5'-monophosphate response. J Clin Endocrinol Metab 69:868-74
Lobo, J O; Bellino, F L (1989) Oestrogen synthetase (aromatase) and hormone secretion in primary cultures of human placental trophoblast cells. Effects of cyclic AMP addition at the start of culture in attached and unattached cell populations. Placenta 10:377-85
Bellino, F L; Holben, L (1989) Placental estrogen synthetase (aromatase): evidence for phosphatase-dependent inactivation. Biochem Biophys Res Commun 162:498-504
Huang, J R; Bellino, F L; Osawa, Y et al. (1989) Immunologic identification of the aromatase enzyme system in human endometrium. J Steroid Biochem 33:1043-7
Bellino, F L; Tseng, L; Lobo, J O (1987) Antisera against estrogen synthetase from human placental microsomes. Antibody characterization and cross-reactivity studies in other organs. Mol Cell Endocrinol 52:143-50
Bellino, F L; Lobo, J O (1987) Estrogen synthetase (aromatase) in cultured human term placental cells and neoplastic human trophoblast. Steroids 50:73-87
Bellino, F L; Hussa, R O (1985) Estrogen synthetase stimulation by hemin in human choriocarcinoma cell culture. Biochem Biophys Res Commun 127:232-8

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