Hahn E F, Fishman J
J Biol Chem. 1984 Feb 10;259(3):1689-94.
The terminal hydroxylation in placental estrogen biosynthesis from androgens is at the 2 beta position. The 2 beta-hydroxy-19-oxoandrogen derivative collapses nonenzymatically to estrogen and is therefore the proximate precursor of the female hormone. To establish the role of this pathway in biological aromatization, an immunological approach was employed in which an antibody was obtained which recognizes 2 beta-hydroxy-19-oxygenated androgens but not intermediates oxygenated at C-19 only. Binding of the 2 beta-hydroxy-19-oxo intermediate by the antibody stabilizes it so that its nonenzymatic transformation to estrogen is delayed and results in slower estrogen formation. When placental microsomes were incubated with [1,2-3H]androstenedione in the presence of the antibody antiserum, a 50% decrease in [3H]estradiol formation and 3H2O release was observed when compared with identical incubations containing normal rabbit serum alone. This inhibition is blocked when the antibody is inactivated by presaturation with 2 beta, 19-dihydroxyandrostenedione. Precipitation of immunoglobulins from the incubations followed by heating liberated the 2 beta-hydroxy-19-oxo intermediate (30%) from the antibody, and resulted in its nonenzymatic collapse to estrogen with concomitant release of 3H2O. Control normal rabbit serum or blocked antibody incubations did not show a similar increase in [3H]estradiol or 3H2O yields in the precipitate. Heat treatment (90 degrees C) of the antibody but not normal rabbit serum incubations resulted in a similar increase in [3H]estradiol and 3H2O yields. These results are consistent with the hypothesis that the final and rate-determining hydroxylation in aromatization of androgens is at the 2 beta position and that this pathway is the dominant, if not the sole, route of estrogen biosynthesis by placental aromatase. The antibody probe also permits the characterization of aromatization mechanisms in tissues other than the placenta.
胎盘由雄激素合成雌激素的终末羟基化作用发生在2β位。2β-羟基-19-氧代雄激素衍生物可非酶促地分解为雌激素，因此是雌性激素的直接前体。为确定该途径在生物芳香化过程中的作用，采用了一种免疫学方法，获得了一种抗体，该抗体可识别2β-羟基-19-氧化雄激素，但不能识别仅在C-19位氧化的中间体。抗体与2β-羟基-19-氧代中间体的结合使其稳定，从而延迟了其非酶促转化为雌激素的过程，导致雌激素形成减慢。当胎盘微粒体与[1,2-³H]雄烯二酮在抗体抗血清存在下孵育时，与仅含正常兔血清的相同孵育相比，[³H]雌二醇形成和³H₂O释放减少了50%。当抗体用2β,19-二羟基雄烯二酮预饱和使其失活时，这种抑制作用被阻断。孵育后沉淀免疫球蛋白，然后加热，可使2β-羟基-19-氧代中间体（30%）从抗体中释放出来，并导致其非酶促分解为雌激素，同时释放³H₂O。对照正常兔血清或封闭抗体的孵育在沉淀中未显示出类似的[³H]雌二醇或³H₂O产量增加。对抗体进行热处理（90℃）而非正常兔血清孵育导致[³H]雌二醇和³H₂O产量出现类似增加。这些结果与以下假设一致：雄激素芳香化的最终和限速羟基化作用发生在2β位，并且该途径是胎盘芳香化酶合成雌激素的主要途径（如果不是唯一途径的话）。该抗体探针还可用于表征胎盘以外组织中的芳香化机制。
