Cytochromes P-450 are key enzymes in the biosynthesis of steroids in the adrenal, testis, ovary, and placenta. We propose a comprehensive study of the structure-function relationships in these enzymes. One focus of our studies will be aromatase, the enzyme responsible for the conversion of androstenedione to estrone and testosterone to estradiol. We have purified and raised antibodies to human placental aromatase and have cloned a cDNA for this enzyme. The human cDNA probe has been used to pick up a clone for the rat enzyme. The rat probe will be used to study aromatase mRNA distribution in rat tissues for adult and neonatal rats. The adult rats will be treated with aromatase inhibitors to study their possible affect on mRNA levels. In neonates we will study mRNA levels in the brains of males and females before and after birth with and without inhibitors. These studies should provide new insight to the role of aromatase in neonatal imprinting on steroid liver metabolism. The metabolism of the aromatase inhibitor 4- hydroxyandrostenedione will also be studied by our collaborator, Wayne Levin at the Roche Institute of Molecular Biology. Bromoacetoxy derivatives of androstenedione will be used to probe the active site of purified aromatase. We will raise monoclonal antibodies to human placental aromatase which will be used to measure aromatase levels in rat tissues, and in a separate study, aromatase levels in breast cancer patients. We will purify and characterize bovine and rate 3-beta-hydroxysteroid dehydrogenase /steroid 5-ene-4-ene isomerase. This enzyme plays a key role in the conversion of pregnenolone to progesterone. Very little is known about the structure or dual enzymatic role of this key steroidogenic enzyme. We will raise antibodies to this enzyme to study its distribution in rat adrenal, testis, and ovaries, and perform active site studies on the NAD+-binding site with FSBA.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD014900-06
Application #
3312848
Study Section
Biochemical Endocrinology Study Section (BCE)
Project Start
1984-04-01
Project End
1992-01-31
Budget Start
1990-08-01
Budget End
1992-01-31
Support Year
6
Fiscal Year
1990
Total Cost
Indirect Cost
Name
City of Hope/Beckman Research Institute
Department
Type
DUNS #
City
Duarte
State
CA
Country
United States
Zip Code
91010
Perkins, Lizabeth A; Holderbaum, Laura; Tao, Rong et al. (2015) The Transgenic RNAi Project at Harvard Medical School: Resources and Validation. Genetics 201:843-52
Rutherfurd, K J; Chen, S A; Shively, J E (1991) Isolation and amino acid sequence analysis of bovine adrenal 3 beta-hydroxysteroid dehydrogenase/steroid isomerase. Biochemistry 30:8108-16
Mellon, S H; Shively, J E; Miller, W L (1991) Human proopiomelanocortin-(79-96), a proposed androgen stimulatory hormone, does not affect steroidogenesis in cultured human fetal adrenal cells. J Clin Endocrinol Metab 72:19-22
Rutherfurd, K J; Chen, S A; Shively, J E (1991) Affinity labeling of bovine adrenal 3 beta-hydroxysteroid dehydrogenase/steroid isomerase by 5'-[p-(fluorosulfonyl)benzoyl]adenosine. Biochemistry 30:8116-23
Pompon, D; Liu, R Y; Besman, M J et al. (1989) Expression of human placental aromatase in Saccharomyces cerevisiae. Mol Endocrinol 3:1477-87
Besman, M J; Yanagibashi, K; Lee, T D et al. (1989) Identification of des-(Gly-Ile)-endozepine as an effector of corticotropin-dependent adrenal steroidogenesis: stimulation of cholesterol delivery is mediated by the peripheral benzodiazepine receptor. Proc Natl Acad Sci U S A 86:4897-901
Chen, S A; Besman, M J; Shively, J E et al. (1989) Human aromatase. Drug Metab Rev 20:511-7
Hickey, G J; Chen, S A; Besman, M J et al. (1988) Hormonal regulation, tissue distribution, and content of aromatase cytochrome P450 messenger ribonucleic acid and enzyme in rat ovarian follicles and corpora lutea: relationship to estradiol biosynthesis. Endocrinology 122:1426-36
Chen, S A; Besman, M J; Sparkes, R S et al. (1988) Human aromatase: cDNA cloning, Southern blot analysis, and assignment of the gene to chromosome 15. DNA 7:27-38
Xu, Q Y; Shively, J E (1988) Microsequence analysis of peptides and proteins. VIII. Improved electroblotting of proteins onto membranes and derivatized glass-fiber sheets. Anal Biochem 170:19-30

Showing the most recent 10 out of 19 publications