Kitawaki J, Yoshida N, Osawa Y
Endocrine Biochemistry Department, Medical Foundation of Buffalo, New York 14203.
Endocrinology. 1989 Mar;124(3):1417-23. doi: 10.1210/endo-124-3-1417.
Traditionally, aromatase has been quantified as aromatase activity according to its ability to produce estrogen from androgen. We have developed a quantitative assay based on the protein mass of catalytically active aromatase cytochrome P-450. A solid phase sandwich enzyme-linked immunosorbent assay for aromatase cytochrome P-450 has been devised using mouse monoclonal antibody (MAb3-2C2) and rabbit polyclonal antiserum (PAb R-8-2). Two rabbit antisera (PAb R-8-1 and R-8-2) were raised by immunization against human placental aromatase cytochrome P-450 which had been isolated by immunoaffinity chromatography of MAb3-2C2-coupled to Sepharose 4B resin. Both antisera were capable of suppressing human placental aromatase activity with IC50 values of 0.6 and 0.8 microliter/ml incubate, respectively, and showed monospecific to aromatase cytochrome P-450 in the Western blot analyses. Solubilized human placental microsomal samples were incubated in microtiter wells precoated with MAb3-2C2. The unbound proteins were washed out, and the aromatase cytochrome P-450 bound with the MAb3-2C2 in the wells was then reacted with PAb R-8-2, the binding of which was subsequently probed with goat antirabbit immunoglobulin G antibody alkaline phosphatase conjugate. Immunoaffinity-purified aromatase cytochrome P-450 of human placental microsomes was used for the standard, with the current assay detection limit at 1 ng/ml. There was a positive correlation between aromatase activity and the immunoreactive aromatase cytochrome P-450 level in solubilized microsomal samples after preincubation at 22 and 37 C, indicating that the enzyme-linked immunosorbent assay measures the level of aromatase cytochrome P-450 that has catalytic activity. The mean level of aromatase cytochrome P-450 in solubilized human term placental microsomes was 16.4 +/- 10.3 (+/- SD) micrograms/ml, corresponding to 0.38 +/- 0.19% of the original microsomes. The mean specific activity of aromatization of the solubilized samples was 0.650 +/- 0.163 nmol estrogen formed/min.mg protein. These results indicate that aromatase in the solubilized placental microsomal fraction has catalytic ability of 5.3 +/- 1.6 min-1 based on the immunoassayable cytochrome P-450.
传统上，芳香化酶是根据其将雄激素转化为雌激素的能力，作为芳香化酶活性来进行定量的。我们已经开发出一种基于具有催化活性的芳香化酶细胞色素P - 450蛋白量的定量检测方法。利用小鼠单克隆抗体（MAb3 - 2C2）和兔多克隆抗血清（PAb R - 8 - 2），设计了一种用于检测芳香化酶细胞色素P - 450的固相夹心酶联免疫吸附测定法。通过用与偶联到琼脂糖4B树脂上的MAb3 - 2C2进行免疫亲和层析分离得到的人胎盘芳香化酶细胞色素P - 450免疫兔子，制备了两种兔抗血清（PAb R - 8 - 1和R - 8 - 2）。两种抗血清都能够抑制人胎盘芳香化酶活性，其半数抑制浓度（IC50）值分别为每孵育液0.6和0.8微升/毫升，并且在蛋白质免疫印迹分析中对芳香化酶细胞色素P - 450显示出单特异性。将溶解的人胎盘微粒体样品在预先包被有MAb3 - 2C2的微量滴定孔中孵育。洗去未结合的蛋白质，然后使与孔中MAb3 - 2C2结合的芳香化酶细胞色素P - 450与PAb R - 8 - 2反应，随后用山羊抗兔免疫球蛋白G抗体碱性磷酸酶结合物检测其结合情况。用人胎盘微粒体的免疫亲和纯化的芳香化酶细胞色素P - 450作为标准品，当前检测方法的检测限为1纳克/毫升。在22℃和37℃预孵育后，溶解的微粒体样品中的芳香化酶活性与免疫反应性芳香化酶细胞色素P - 450水平之间存在正相关，这表明酶联免疫吸附测定法测量的是具有催化活性的芳香化酶细胞色素P - 450的水平。溶解的足月人胎盘微粒体中芳香化酶细胞色素P - 450的平均水平为16.4±10.3（±标准差）微克/毫升，相当于原始微粒体的0.38±0.19%。溶解样品的平均芳香化比活性为每分钟每毫克蛋白质形成0.650±0.163纳摩尔雌激素。这些结果表明，基于可免疫检测的细胞色素P - 450，溶解的胎盘微粒体组分中的芳香化酶具有5.3±1.6分钟⁻¹的催化能力。
