Purohit A, Oakey R E
Department of Chemical Pathology, School of Medicine, University of Leeds, U.K.
J Steroid Biochem. 1989 Sep;33(3):439-48. doi: 10.1016/0022-4731(89)90335-x.
Much greater quantities of 16 alpha-hydroxyoestrogens (e.g. oestriol) than of 16-deoxyoestrogens (e.g. oestradiol-17 beta) are formed in human pregnancy than might be expected from the relative availability to the placenta of the 16 alpha-hydroxy- and 16-deoxy-C19 precursors. To investigate this further, 16 alpha-hydroxyandrostenedione (16 alpha-OH-A4) and androstenedione (A4) were tested in vitro as substrates and mutual inhibitors of human placental aromatase. It was found that the Km for aromatisation of A4 (mean = 0.26 mumol/l) was very similar to Ki (0.30, 0.35 mumol/l) for the inhibition by A4 of the aromatisation of 16 alpha-OH-A4. Similarly, Km for aromatisation of 16 alpha-OH-A4 (mean = 1.21 mumol/l) had the same value as the Ki (1.0, 1.2 mumol/l) for the inhibition by 16 alpha-OH-A4 of the aromatisation of A4. From graphical analysis of Lineweaver-Burk plots, both inhibitions were characterised as noncompetitive. Hence, it was concluded that the two 16-deoxy- and 16-hydroxy-C19 substrates bind at separate, but interactive, sites and that each substrate on binding inhibits the aromatisation of the other. Additional evidence for the separate but interactive substrate binding sites for the 16-deoxy- and 16-hydroxy-C19 steroids was obtained by use of the suicide inhibitor 4-hydroxyandrostenedione (4-OH-A4), which is recognised as binding to the aromatisation site for A4. Aromatisation of 16 alpha-OH-A4 was found to be inhibited by pre-incubation of the microsomes with 4-OH-A4 (0.1 mumol/l). The presence of A4 (4.6 mumol/l), but not of 16 alpha-OH-A4 (4.0 mumol/l) during the pre-incubation successfully protected the subsequent aromatisation of 16 alpha-OH-A4 from this inhibition. In addition, the Km values, reported here, suggest also that the 16-deoxyandrogens are preferred to the 16 alpha-hydroxyandrogens as oestrogen precursors. In consequence, factors other than substrate affinity and plasma concentrations must be presumed to be involved in the overwhelming production of 16 alpha-hydroxyoestrogens in human pregnancy.
在人类孕期中，16α-羟基雌激素（如雌三醇）的生成量比16-脱氧雌激素（如17β-雌二醇）多得多，这一数量比根据胎盘对16α-羟基和16-脱氧C19前体的相对可利用性所预期的要多。为了进一步研究这一现象，对16α-羟基雄烯二酮（16α-OH-A4）和雄烯二酮（A4）作为人胎盘芳香化酶的底物和相互抑制剂进行了体外测试。结果发现，A4芳香化的Km值（平均值 = 0.26 μmol/L）与A4抑制16α-OH-A4芳香化的Ki值（0.30、0.35 μmol/L）非常相似。同样，16α-OH-A4芳香化的Km值（平均值 = 1.21 μmol/L）与16α-OH-A4抑制A4芳香化的Ki值（1.0、1.2 μmol/L）相同。通过对Lineweaver-Burk图的图形分析，两种抑制作用均被表征为非竞争性抑制。因此，可以得出结论，两种16-脱氧和16-羟基C19底物在不同但相互作用的位点结合，并且每种底物结合时都会抑制另一种底物的芳香化。通过使用自杀性抑制剂4-羟基雄烯二酮（4-OH-A4）获得了16-脱氧和16-羟基C19类固醇存在不同但相互作用的底物结合位点的额外证据，4-OH-A4被认为与A4的芳香化位点结合。发现用4-OH-A4（0.1 μmol/L）预孵育微粒体可抑制16α-OH-A4的芳香化。预孵育期间存在A4（4.6 μmol/L）而非16α-OH-A4（4.0 μmol/L）成功地保护了随后16α-OH-A4的芳香化免受这种抑制。此外，此处报道的Km值还表明，作为雌激素前体，16-脱氧雄激素比16α-羟基雄激素更受青睐。因此，必须假定除底物亲和力和血浆浓度之外的其他因素参与了人类孕期中16α-羟基雌激素的大量产生。
