Osawa Y, Yoshida N, Fronckowiak M, Kitawaki J
Endocrine Biochemistry Department, Medical Foundation of Buffalo, NY 14203-1196.
Steroids. 1987 Jul-Sep;50(1-3):11-28. doi: 10.1016/0039-128x(83)90058-2.
Microsomal estrogen synthetase (aromatase) cytochrome P-450 was purified from fresh human placental microsomes by monoclonal anti-aromatase P-450 antibody-Sepharose 4B chromatography. The purified P-450 showed a single band of 55 kDa on SDS-polyacrylamide gel electrophoresis and the aromatase specific activity on reconstitution was 70 nmol/min/mg protein. The purified P-450 was stable with a t 1/2 of approximately 2 years on storage at -90 degrees C and showed Km = 43 nM for androstenedione aromatization. However, it was unstable under spectral measurement conditions in the presence of sodium dithionite and carbon monoxide and the carbon monoxide difference spectra showed a maximum at 450 nm and a specific content of 9.1 nmol of P-450/mg protein, giving a turnover number of approximately 7.7 per min for the purified aromatase. The one-step immunochemical purification method gave a 490-fold increase of specific activity with 55% yield of aromatase activity of the original microsomes. Analysis of androgen metabolism by the purified aromatase and an apparent large kinetic isotope effect found at the secondary positions when using [19(-3)H3, 4(-14)C] androgens revealed metabolic switching from the first 19-hydroxylation to 1 beta- and 2 beta- monohydroxylation by aromatase. Substrate specificity for [19(-3)H3]androstenedione and testosterone was indicated by differences in the extent of metabolic switching (18% and 30%) and in the 2 beta/1 beta ratio (60/40 and 10/90, respectively). The mouse monoclonal antibody used for immunoaffinity purification suppresses aromatase activity of human placenta, but was totally ineffective for aromatase in goldfish brain and rat ovary. Rabbit polyclonal antibodies to human placental aromatase P-450 suppressed both human placental and rat ovarian aromatase but were ineffective for goldfish brain aromatase. The study indicates that they are isozymes of aromatase based on different structures of P-450.
微粒体雌激素合成酶（芳香化酶）细胞色素P-450通过单克隆抗芳香化酶P-450抗体-琼脂糖4B层析从新鲜人胎盘微粒体中纯化得到。纯化后的P-450在SDS-聚丙烯酰胺凝胶电泳上显示出一条55 kDa的单一条带，重组时的芳香化酶比活性为70 nmol/分钟/毫克蛋白。纯化后的P-450很稳定，在-90℃储存时半衰期约为2年，雄烯二酮芳香化反应的Km值为43 nM。然而，在连二亚硫酸钠和一氧化碳存在的光谱测量条件下它不稳定，一氧化碳差光谱在450 nm处有最大值，P-450的比含量为9.1 nmol/毫克蛋白，纯化后的芳香化酶每分钟的转换数约为7.7。一步免疫化学纯化方法使比活性提高了490倍，原微粒体芳香化酶活性的产率为55%。用纯化后的芳香化酶分析雄激素代谢，以及在使用[19(-3)H3, 4(-14)C]雄激素时在二级位置发现明显的大动力学同位素效应，揭示了芳香化酶的代谢从最初的19-羟基化转变为1β-和2β-单羟基化。[19(-3)H3]雄烯二酮和睾酮的底物特异性通过代谢转换程度（分别为18%和30%）以及2β/1β比值（分别为60/40和10/90）的差异来表明。用于免疫亲和纯化的小鼠单克隆抗体抑制人胎盘的芳香化酶活性，但对金鱼脑和大鼠卵巢中的芳香化酶完全无效。兔抗人胎盘芳香化酶P-450多克隆抗体抑制人胎盘和大鼠卵巢的芳香化酶，但对金鱼脑芳香化酶无效。该研究表明，基于P-450的不同结构，它们是芳香化酶的同工酶。
