Mendelson C R, Wright E E, Evans C T, Porter J C, Simpson E R
Arch Biochem Biophys. 1985 Dec;243(2):480-91. doi: 10.1016/0003-9861(85)90525-9.
Aromatase cytochrome P-450 (P-450AROM) was partially purified from human placental microsomes by hydrophobic affinity chromatography using Phenyl-Sepharose and ion-exchange chromatography on DEAE-cellulose. The resulting preparation had a specific activity of 2 nmol/mg protein with respect to cytochrome P-450 content and displayed a type I difference spectrum upon addition of the substrate androstenedione. When the cytochrome P-450-enriched fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie blue, there was an enrichment of two proteins having apparent molecular weights of 50,000 and 55,000. The bands containing these proteins were removed from unstained polyacrylamide gels and injected separately or together into three rabbits. An aliquot of the serum or an immunoglobulin (IgG) fraction prepared from the serum of the rabbit injected with the 55-kDa band or with both the 50- and 55-kDa bands inhibited aromatase activity of human placental microsomes by 80%; this IgG had no effect on 17 alpha-hydroxylase or 21-hydroxylase activities of human fetal adrenal microsomes. In contrast, the serum of the rabbit injected with the 50-kDa band had little capacity to inhibit placental aromatase activity. By immunoblot analysis, it was found that the IgG from the serum of the rabbit immunized with the 55-kDa protein bound specifically to a protein of 55 kDa in human placental microsomes. Monoclonal antibodies were prepared from a hybridoma cell line derived from the spleen cells of mice immunized against the 55-kDa protein. The monoclonal IgG was covalently linked to a Sepharose 4B column and was used for immunoaffinity chromatography of cytochrome P-450AROM. The finding that cytochrome P-450 and the 55-kDa protein were selectively retained by the affinity column and eluted with NaCl (2 M) and glycine (0.2 M, pH 3.0) and that this fraction contained aromatase activity upon reconstitution with purified NADPH-cytochrome P-450 reductase and phospholipid, is indicative that the 55-kDa protein is indeed cytochrome P-450AROM. These findings are also indicative that both the monoclonal and polyclonal IgGs are specific for human cytochrome P-450AROM.
通过使用苯基琼脂糖进行疏水亲和层析以及在DEAE - 纤维素上进行离子交换层析，从人胎盘微粒体中部分纯化了芳香化酶细胞色素P - 450（P - 450AROM）。所得制剂相对于细胞色素P - 450含量的比活性为2 nmol/mg蛋白质，并且在加入底物雄烯二酮后呈现I型差异光谱。当富含细胞色素P - 450的组分进行十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳并用考马斯亮蓝染色时，有两种表观分子量分别为50,000和55,000的蛋白质得到了富集。从未染色的聚丙烯酰胺凝胶中切下含有这些蛋白质的条带，分别或一起注射到三只兔子体内。用55 kDa条带或50 kDa和55 kDa条带一起注射的兔子血清或从该血清制备的免疫球蛋白（IgG）组分的等分试样可抑制人胎盘微粒体的芳香化酶活性达80%；这种IgG对人胎儿肾上腺微粒体的17α - 羟化酶或21 - 羟化酶活性没有影响。相比之下，用50 kDa条带注射的兔子血清几乎没有抑制胎盘芳香化酶活性的能力。通过免疫印迹分析发现，用55 kDa蛋白质免疫的兔子血清中的IgG与人胎盘微粒体中55 kDa的蛋白质特异性结合。从免疫55 kDa蛋白质的小鼠脾细胞衍生的杂交瘤细胞系制备了单克隆抗体。单克隆IgG与琼脂糖4B柱共价连接，并用于细胞色素P - 450AROM的免疫亲和层析。细胞色素P - 450和55 kDa蛋白质被亲和柱选择性保留，并用NaCl（2 M）和甘氨酸（0.2 M，pH 3.0）洗脱，并且该组分在用纯化的NADPH - 细胞色素P - 450还原酶和磷脂重构后具有芳香化酶活性，这表明55 kDa蛋白质确实是细胞色素P - 450AROM。这些发现还表明单克隆和多克隆IgG对人细胞色素P - 450AROM均具有特异性。
