Partial resolution of the placental microsomal aromatase complex

Incubation of placental microsomes with digitonin under defined conditions results in a solubilization of 15-20% of the total aromatase activity. The aromatase activity remains in the non-turbid supernatant after prolonged centrifugation at 148,000 g, exhaustive dialysis, and column chromatography on Bio-Gel P-10. The particulate and solubilized enzymes are indistinguishable in that the apparent K_m for both androstenedione and NADPH are similar. Also, the K_m for NADPH of the aromatase is approximately the same as the K_mof NADPH-cytochrome c reductase in both the microsomal and solubilized enzyme. Cytochrome P-450 recovered in the digitonin supernatant retained its native spectral characteristics as well as its substrate specificity. The solubilized aromatase can be purified about five-fold (20% recovery). Under these conditions, cytochrome P-450 can be purified about three to five-fold (15% recovery) and NADPH-cytochrome c reductase about 30-fold (100% recovery). Cytochrome P-450 and the reductase could be partially resolved into two fractions by batchwise adsorption on DEAE-cellulose. The unadsorbed fraction contained cytochrome P-450 while that obtained by extraction with 1.0M KCl contained the reductase and a trace of cytochrome P-450. Both of these fractions contained minimal aromatase activity. When these fractions were recombined, however, the rate of aromatization was increased by about two-fold. This observation is consistent with a requirement for both cytochrome P-450 and NADPH-cytochrome c reductase for aromatization of androstenedione.