Swinney D C, Watson D M, So O Y
Syntex Discovery Research, Palo Alto, California 94304.
Arch Biochem Biophys. 1993 Aug 15;305(1):61-7. doi: 10.1006/abbi.1993.1393.
Aromatase activity in microsomes from human placenta (HPM) and rat ovary (ROM) was compared by measuring the accumulation of 19-oxygenated intermediates, the effect of tritium substitution upon rates, and the distribution of tritium in products. A considerable accumulation of intermediates (19-hydroxyandrogen and 19-al-androgen) and a lag in product formation (estrogen and water) was observed with ROM but not HPM. Addition of purified NADPH-cytochrome P450 reductase to ROM decreased the accumulation of 19-hydroxyandrostenedione and increased the rate of estrone formation. This difference could not be attributed to the microsomal reductase concentration since its concentration was similar in both tissues. Estrogen formation by aromatase from these tissues was not associated with a significant kinetic isotope effect when androstenedione was labeled with tritium at C-1 and C-2. Isotopically sensitive switching (branching) from the 19-al-androstenedione enzyme complex to form free 19-al-androstenedione rather than estrogen was not observed. These data suggest that for aromatase in both tissues, an enzymatic step exists between the 19-al-androstenedione intermediate and hydrogen abstraction or enolization that has a large commitment to catalysis. The distribution of tritium into the products, water and estrogen, was dependent upon substrate, enzyme source, and position of the label. Incubation of 1 beta, 2 beta-[3H]androstenedione and testosterone with ROM and 1 beta,2 beta-[3H]testosterone with HPM resulted in approximately 50% of the label being retained in the estrogen and 50% being lost in water. The majority of the label was lost in water upon incubation of 1 beta-labeled substrates with microsomes from both sources. Unexpectedly, no label was lost to water upon incubation of the specifically 1 alpha,2 alpha-labeled substrates with either enzyme source. Only incubation of 1 beta,2 beta-[3H]androstenedione with HPM resulted in loss of tritium from the 2-position. These data were interpreted to indicate that, for androstenedione metabolism by ROM and testosterone metabolism by both ROM and HPM, enolization occurs nonspecifically in an isotopically sensitive manner following deformylation, but for HPM metabolism of androstenedione enolization occurs in an enzyme-assisted manner. The studies show that aromatase located in ROM differs from that in HPM by its accumulation of intermediates and inability to selectively remove the 2 beta-tritium from androstenedione.
通过测量19-氧化中间体的积累、氚取代对速率的影响以及氚在产物中的分布，比较了人胎盘微粒体（HPM）和大鼠卵巢微粒体（ROM）中的芳香化酶活性。在ROM中观察到中间体（19-羟基雄激素和19-醛雄激素）的大量积累以及产物形成（雌激素和水）的延迟，而在HPM中未观察到。向ROM中添加纯化的NADPH-细胞色素P450还原酶可减少19-羟基雄烯二酮的积累并增加雌酮形成的速率。这种差异不能归因于微粒体还原酶的浓度，因为其在两种组织中的浓度相似。当雄烯二酮在C-1和C-2处用氚标记时，这些组织中的芳香化酶形成雌激素与显著的动力学同位素效应无关。未观察到从19-醛-雄烯二酮酶复合物向游离19-醛-雄烯二酮而非雌激素的同位素敏感转换（分支）。这些数据表明，对于两种组织中的芳香化酶，在19-醛-雄烯二酮中间体与氢提取或烯醇化之间存在一个对催化有很大贡献的酶促步骤。氚在产物水和雌激素中的分布取决于底物、酶来源和标记位置。用ROM孵育1β,2β-[3H]雄烯二酮和睾酮以及用HPM孵育1β,2β-[3H]睾酮导致约50%的标记保留在雌激素中，50%在水中损失。用两种来源的微粒体孵育1β-标记的底物时，大部分标记在水中损失。出乎意料的是，用特异性1α,2α-标记的底物与任何一种酶来源孵育时，没有标记损失到水中。仅用HPM孵育1β,2β-[3H]雄烯二酮会导致2位的氚损失。这些数据被解释为表明，对于ROM对雄烯二酮的代谢以及ROM和HPM对睾酮的代谢，在脱甲酰化后烯醇化以同位素敏感的方式非特异性发生，但对于HPM对雄烯二酮的代谢，烯醇化以酶辅助的方式发生。研究表明，位于ROM中的芳香化酶与位于HPM中的芳香化酶的不同之处在于其对中间体的积累以及无法选择性地从雄烯二酮中去除2β-氚。
