title: Purification and reconstitution properties of human placental aromatase. A cytochrome P-450-type monooxygenase.
abstract: The hemoprotein component of human placental aromatase (estrogen synthetase) has been purified to a high degree of homogeneity by a combination of affinity and adsorption chromatography on aminohexyl-Sepharose, concanavalin-A-Sepharose, and hydroxyapatite. The monomeric form of the enzyme has an Mr of 55000 +/- 1000 as estimated by sodium dodecyl sulfate gel electrophoresis. Its absolute spectrum shows a high-spin Soret band at 394 nm while its reduced, CO-difference spectrum has a maximum at 447 +/- 1 nm. Full reconstitution of aromatase activity was obtained when it was recombined with a homogeneous preparation of the higher-Mr form of either human placental, or bovine hepatic NADPH-cytochrome P-450 reductase. Critical factors for purification of the very unstable, membrane-bound hemoprotein with good retention of activity were, besides the chromatographic sequence, the use of the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps) during the solubilization, and the stabilizing effect of the aromatase substrate, 4-androstene-3,17-dione, throughout the procedure. In the presence of NADPH, the reconstituted enzyme system smoothly aromatizes 19-oxoandrostenedione, 19-hydroxyandrostenedione and androstenedione in this order of reactivity. The same reconstituted system also aromatized testosterone, but it was inactive towards 19-norandrostenedione. Known cytochrome P-450 inhibitors decreased its activity. We conclude: (a) the terminal oxidase of human placental aromatase is indeed a cytochrome P-450-type monooxygenase; (b) the multistep aromatization reaction of C19 androstenes is catalyzed by a single enzyme; (c) aromatization of 19-norsteroids reported by other authors must be due to a different aromatase. Experimental data obtained with the reconstituted enzyme are fully compatible with the concept of a reaction mechanism for the aromatization sequence involving an all-trans, antiparallel elimination of the 19-methyl group, the 2 beta proton and the 1 alpha proton, rather than the 1 beta proton, as generally assumed.
mesh_terms: D000328:Adult; D000818:Animals; D001141:Aromatase / Q000302:isolation & purification*; D047072:Aromatase Inhibitors; D002417:Cattle; D002846:Chromatography, Affinity; D003577:Cytochrome P-450 Enzyme System; D004591:Electrophoresis, Polyacrylamide Gel; D004970:Estrone / Q000096:biosynthesis; D005260:Female; D006801:Humans; D008099:Liver / Q000201:enzymology; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification; D010105:Oxygenases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D011863:Radioimmunoassay; D013053:Spectrophotometry
chemical_list: D047072:Aromatase Inhibitors; D004970:Estrone; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases; D001141:Aromatase; D009251:NADPH-Ferrihemoprotein Reductase