Estrogen synthetase. demonstration that the high molecular weight form of nadph-cytochrome c reductase from human placental microsomes is required for androgen aromatization

Abstract

NADPH-cytochrome c reductase from human placental microsomes was solubilized using deoxycholate and purified by 2',5'ADP-Sepharose 4B affinity chromatography in deoxycholate buffer followed by exclusion chromatography on Bio-Gel A-1.5 m in detergent-free buffer. Two principal peaks of reductase activity elute from the Bio-Gel column: the first peak (A form) comes at the void volume and has a principal monomer molecular weight of 73,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE); the second peak (B form) elutes at 80,000 dallons with a principal monomer molecular weight of 68,000 by SDS-PAGE. The two fractions contain flavoproteins and are immunochemically identical by Ouchterlony double diffusion but apparently differ in net charge since they have different electrophoretic mobilities under non-denaturing conditions. The following results show that only the A form of the purified human placental microsomal NADPH-cytochrome c reductase is necessary for the expression of estrogen synthetase (aromatase) activity in placental microsomes, although aromatase activity was not detected in the purified protein. In a preparation of detergent solubilized placental microsomes which lacked both reductase and aromatase activities, but contained cytochrome P-450, aromatase activity could be regained by adding the A form of the reductase to these microsomes. Also, only the A form stimulated placental microsomal aromatase when mixed with a normal microsomal homogenate containing both reductase and aromatase activities. Rabbit anti-sera prepared against either the A form or a mixture of A and B forms inhibit equally both NADPH-cytochrome c reductase and aromatase activities in placental microsomes but do not inhibit NADH-cytochrome c reductase or estrogen 17β-hydroxysteroid dehydrogenase activities. The other aromatase component protein has not yet been positively identified, but its likely identity as cytochrome P-450 is consistent with these results.