Solubilization of estrogen synthetase from human term placental microsomes using detergents.

Abstract The microsomal fraction of term placenta was incubated with a range of concentrations of several bile acids and non-ionic detergents (deoxycholate, cholate, taurocholate, Triton X-100, Tween 80, Lubrol PX, Lubrol WX and digitonin) and then centrifuged at 120,000 g for 1 h. Aromatase activity was progressively depleted from the pellet with increasing detergent concentration for each detergent except Tween 80 (up to 5% concentration). Since a cytochrome P-450 monoxygenase system is assumed to be involved in androgen aromatization, two detergents, deoxycholate and Triton X-100, were also examined for their ability to remove NADPH cytochrome c reductase activity and cytochrome P-450 from the microsomes. Although the cytochrome was removed from microsomes at detergent concentrations similar to those for aromatase, approximately 2- to 3-fold higher detergent concentrations were required to deplete the microsomes of reductase activity. Each detergent used inhibited aromatase activity in the microsomal homogenate in a concentration-dependent manner that roughly paralleled the concentration-dependent depletion of aromatase activity from the pellet. Aromatase activity was measured in the supernatant (120,000 g -h) from the cholate- and deoxycholate-treated microsomes by diluting the supernatant to lower detergent concentrations. Significant amounts of aromatase activity could not be measured in the 120,000 g -h supernatant from non-ionic detergent-treated microsomes even after dilution to lower detergent concentrations. Dilution of the cholate- and deoxycholate-soluble prepaations to lower detergent concentrations resulted in an aromatase-active precipitate with a 3-fold higher specific activity than the original microsomes. Deoxycholate- and cholate-solubilized aromatase activity is included in a Sepharose 6B column, eluting at an approximate molecular weight of 100,000 to 120,000 daltons.