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 Km for both androstenedione and NADPH are similar. Also, the Km for NADPH of the aromatase is approximately the same as the Kmof 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.
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