Regioselective progesterone hydroxylation catalyzed by eleven rat hepatic cytochrome P-450 isozymes

• Published in: Biochemistry (Easton) 1987-11, Vol.26 (22), p.7073-7083
• Main Authors: Swinney, D. C, Ryan, D. E, Thomas, P. E, Levin, W
• Format: Article
• Language: English
• Subjects: Aging; Analytical, structural and metabolic biochemistry; Animals; Aryl Hydrocarbon Hydroxylases; Biological and medical sciences; Cytochrome P-450 Enzyme System - metabolism; cytochrome P450; Cytochrome P450 Family 2; Enzymes and enzyme inhibitors; Female; Fundamental and applied biological sciences. Psychology; Hydroxylation; Isoenzymes - metabolism; liver; Liver - growth & development; Male; Microsomes, Liver - metabolism; Oxidoreductases; progesterone; Progesterone - metabolism; Rats; Sex Factors; Steroid 16-alpha-Hydroxylase
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00396a032

Quantitative high-pressure liquid chromatographic assays were developed that separate progesterone and 17 authentic monohydroxylated derivatives. The assays were utilized to investigate the hydroxylation of progesterone by 11 purified rat hepatic cytochrome P-450 isozymes and 8 different rat hepatic microsomal preparations. In a reconstituted system, progesterone was most efficiently metabolized by cytochrome P-450h followed by P-450g and P-450b. Seven different monohydroxylated progesterone metabolites were identified. 16 alpha-Hydroxyprogesterone, formed by 8 of the 11 isozymes, was the only detectable metabolite formed by cytochromes P-450b and P-450e. 2 alpha-Hydroxyprogesterone was formed almost exclusively by cytochrome P-450h, and 6 alpha-hydroxyprogesterone and 7 alpha-hydroxyprogesterone were only formed by P-450a. 6 beta-hydroxylation of progesterone was catalyzed by four isozymes with cytochrome P-450g being the most efficient, and 15 alpha-hydroxyprogesterone was formed as a minor metabolite by cytochromes P-450g, P-450h, and P-450i. None of the isozymes catalyzed 17 alpha-hydroxylation of progesterone, and only cytochrome P-450k had detectable 21-hydroxylase activity. 16 alpha-Hydroxylation catalyzed by cytochrome P-450b was inhibited in the presence of dilauroylphosphatidylcholine (1.6-80 microM), while this phospholipid either stimulated (up to 3-fold) or had no effect on the metabolism of progesterone by the other purified isozymes. Results of microsomal metabolism in conjunction with antibody inhibition experiments indicated that cytochromes P-450a and P-450h were the sole 7 alpha- and 2 alpha-hydroxylases, respectively, and that P-450k or an immunochemically related isozyme contributed greater than 80% of the 21-hydroxylase activity observed in microsomes from phenobarbital-induced rats.