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‐nor‐androstenedione. 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 2β proton and the 1α proton, rather than the 1β proton, as generally assumed. Copyright © 1986, Wiley Blackwell. All rights reserved