Abstract

Microsomal cytochromes P-450 play key roles in steroid biosynthesis in testis and adrenal gland and xenobiotic metabolism in liver. We propose to perform structure-function studies on the 17-Alpha-hydroxylase/C17, 20-lyase enzyme involved in androgen biosynthesis in testis and on the 17-Alpha-hydroxylase and 21-hydroxylase enzymes from adrenal gland. All three enzymes will be studied in pig as a model system. Structural studies will utilize the microsequencing methodology developed in our laboratory to obtain partial amino acid sequences and molecular cloning technology to obtain cDNA sequences. cDNA sequences will be used to align and complete amino acid sequences. cDNA sequences will be used to align and complete amino acid sequences. Specific active site modification by bromoacetoxysteroid derivaties will be used on all three enzymes. The kinetics of inactivation will be performed by Dr. Peter Hall at the Worcester Foundation (Shrewsbury, Mass.), and the sequencing of modified peptides by us. Since very little is known about the structures of microsomal cytochromes P-450, this work should provide a basis for the comparison of a number of these key enzymes.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD014900-03
Application #
3312846
Study Section
Biochemical Endocrinology Study Section (BCE)
Project Start
1984-04-01
Project End
1987-07-31
Budget Start
1986-04-01
Budget End
1987-07-31
Support Year
3
Fiscal Year
1986
Total Cost
Indirect Cost

  Institution

Name
City of Hope/Beckman Research Institute
Department
Type
DUNS #
City
Duarte
State
CA
Country
United States
Zip Code
91010

  Publications

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

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