Kobayashi S, Tobioka N, Samoto T, Kobayashi M, Iwase H, Masaoka A, Kato T
Nihon Naibunpi Gakkai Zasshi. 1984 Feb 20;60(2):110-9. doi: 10.1507/endocrine1927.60.2_110.
The purification of human uterine estrogen receptor (ER) was performed by way of the affinity chromatography technique using immobilized 17 beta-estradiol on Sepharose 4B, which was developed by Sica and Bresciani (Biochemistry, 18: 2369-2378, 1979). The anti-human ER antibody was induced and its immunological reactivity was also studied. About 300 micrograms of ER were obtained with the recovery of 18%. The purification magnitude of ER was 4000 times. The purified ER appeared as a complex with 17 beta-estradiol, and formed aggregated macromolecule which was eluate at void volume in Sephadex G-200 and Sephacryl S-300 gel filtration. In 7% approximately 18% linear gradient polyacrylamide gel electrophoresis after SDS treatment, two equally stained bands were observed at 90,000 and 70,000 MW areas. Although anti ER antiserum formed no detectable immunoreactive perception with any kinds of antigens, the gamma-globulin fraction of the antiserum produced a precipitate with human and cow uterine cytosols or their purified ERs. On the other hand, two other precipitin lines were observed in human uterine cytosol. One of the lines was also prevalently found with human muscle and liver cytosols, and human whole serum. The immunoreactive component in the human serum was detected in the globulin fraction, but not in the albumin fraction. These facts imply the immunological heterogeneity of the antibody induced by the purified human uterine ER using the affinity chromatography technique. The cytosol of human uterus obtained from hormonally treated women contains lesser amounts of ER than that of cow uterus in Dextran coated charcoal assay. The ER could not fully occupy the estradiol immobilized on Sepharose 4B in the purification of human uterine ER. Therefore, the non-specific estrogen binding proteins originated both from serum and cytoplasm such as steroid binding globulin, could bind to the remaining free estradiol-ligands of the affinity columns. Thus the marginal utility of the affinity chromatography using immobilized estradiol in the purification of human uterine ER was suggested.
采用西卡和布雷西亚尼（《生物化学》，18: 2369 - 2378, 1979）开发的亲和色谱技术，以固定在琼脂糖4B上的17β - 雌二醇为介质，对人子宫雌激素受体（ER）进行了纯化。诱导产生了抗人ER抗体，并对其免疫反应性进行了研究。获得了约300微克的ER，回收率为18%。ER的纯化倍数为4000倍。纯化后的ER以与17β - 雌二醇的复合物形式存在，并形成聚集的大分子，在葡聚糖凝胶G - 200和Sephacryl S - 300凝胶过滤中于空体积处洗脱。在SDS处理后的7%至18%线性梯度聚丙烯酰胺凝胶电泳中，在90,000和70,000分子量区域观察到两条等深染色带。尽管抗ER抗血清与任何种类的抗原均未形成可检测到的免疫反应性沉淀，但抗血清的γ - 球蛋白部分与人及牛子宫胞质溶胶或其纯化的ER产生了沉淀。另一方面，在人子宫胞质溶胶中观察到另外两条沉淀线。其中一条线在人肌肉和肝脏胞质溶胶以及人全血清中也普遍存在。人血清中的免疫反应性成分在球蛋白部分被检测到，而在白蛋白部分未被检测到。这些事实表明，使用亲和色谱技术由纯化的人子宫ER诱导产生的抗体具有免疫异质性。在葡聚糖包被活性炭测定中，来自激素处理女性的人子宫胞质溶胶中含有的ER量比牛子宫的少。在人子宫ER的纯化过程中，ER不能完全占据固定在琼脂糖4B上的雌二醇。因此，血清和细胞质中产生的非特异性雌激素结合蛋白，如类固醇结合球蛋白，可与亲和柱中剩余的游离雌二醇配体结合。由此提示，使用固定化雌二醇的亲和色谱在人子宫ER纯化中的应用价值有限。
