Shafie S, Brooks S C
J Lab Clin Med. 1979 Nov;94(5):784-98.
The measurement of E2 receptor (E2R) in human breast cancer cytosol is significantly influenced by conditions usually employed in the dextran-coated charcoal assay. The incubation time and temperature have an influence on the rate of binding and stability of the receptor. Since lower temperatures preserve the integrity of the receptor, a 2 hr incubation at 4 degrees was selected as the standard incubation procedure. These conditions allow for the detection of at least 80% of the E2R. With supernatants from high-speed centrifugation of HBT biopsies or the human breast cancer cell line MCF-7, reducing agents increased the apparent E2R binding in the order: DTT greater than G-SH greater than MTG. The maximum enhancement of specific E2R binding by a given thiol agent was dependent on its concentration in the incubation medium. The optimum DTT level (7.5 mM) for MCF-7 cell homogenization and cytosol equilibration with tritiated E2 increased E2R to two times control (no DTT). For the HBT 150,000 g supernatant, 1 mM DTT was required to optimize the E2R quantitation. The duration of the dextran-coated charcoal extraction of the cytosol-[3H]E2 incubation had no effect on the level of E2R up to 21 hr. Minimum levels of nonspecific binding of [3H]E2 could be obtained after 4 hr extraction. Maximum depletion of specific [3H]E2 binding could be obtained by adding between 200- and 1000-fold molar excess of unlabeled E2. Greater amounts of unlabeled steroid displaced the radioactive E2 from the dextran-coated charcoal, thereby artifactually increasing the apparent nonspecific binding. This phenomenon may be overcome by utilizing more dextran-coated charcoal in the extraction. However, there was a 9% loss of specifically bound [3H]E2 per milligram of dextran-coated charcoal (1:10 dextran to charcoal by weight) when the cytosol protein was below 90 microgram per incubation. Supplementation with 200 microgram or more albumin per incubation prevented this loss. The dextran:charcoal ratio also prevented E2R loss in the order: 1:1 greater than 1:10 greater than 1:100. One milligram of dextran-coated charcoal (1:10) has the capacity to adsorb 0.3 to 0.4 microgram of free E2. Other unlabeled competitors are capable of displacing [3H]E2 on the receptor. Although DES was as effective as E2, U11,100A and estrone were inefficient competitors. It appeared that the levels of these two estrogen analogues required to maximally displace [3H]E2 on receptor also eluted labeled E2 from the dextran-coated charcoal. DES, however, was unable to displace significant quantities of the [3H] E2 from dextran-coated charcoal even at a molar excess of 50,000:1.
人乳腺癌胞质溶胶中E2受体（E2R）的测量受到葡聚糖包被活性炭测定中常用条件的显著影响。孵育时间和温度对受体的结合速率和稳定性有影响。由于较低温度可保持受体的完整性，因此选择在4℃孵育2小时作为标准孵育程序。这些条件可检测到至少80%的E2R。使用HBT活检组织或人乳腺癌细胞系MCF-7高速离心后的上清液时，还原剂按以下顺序增加表观E2R结合：二硫苏糖醇（DTT）＞谷胱甘肽（G-SH）＞巯基甘油（MTG）。给定硫醇试剂对特异性E2R结合的最大增强取决于其在孵育介质中的浓度。用于MCF-7细胞匀浆和用氚标记E2平衡胞质溶胶的最佳DTT水平（7.5 mM）可使E2R增加至对照（无DTT）的两倍。对于HBT 150,000 g上清液，需要1 mM DTT来优化E2R定量。胞质溶胶-[3H]E2孵育的葡聚糖包被活性炭提取持续时间长达21小时对E2R水平无影响。提取4小时后可获得最低水平的[3H]E2非特异性结合。加入200至1000倍摩尔过量的未标记E2可实现特异性[3H]E2结合的最大消耗。大量未标记的类固醇将放射性E2从葡聚糖包被活性炭上置换下来，从而人为增加表观非特异性结合。这种现象可通过在提取中使用更多葡聚糖包被活性炭来克服。然而，当每次孵育的胞质溶胶蛋白低于90微克时，每毫克葡聚糖包被活性炭（葡聚糖与活性炭重量比为1:10）会导致特异性结合的[3H]E2损失9%。每次孵育补充200微克或更多白蛋白可防止这种损失。葡聚糖：活性炭比例也按以下顺序防止E2R损失：1:1＞1:10＞1:100。1毫克葡聚糖包被活性炭（1:10）能够吸附0.3至0.4微克游离E2。其他未标记的竞争者能够置换受体上的[3H]E2。虽然己烯雌酚（DES）与E2一样有效，但U-11,100A和雌酮是低效竞争者。似乎最大程度置换受体上[3H]E2所需的这两种雌激素类似物的水平也会使标记的E2从葡聚糖包被活性炭上洗脱下来但DES即使在摩尔过量为50,000:1时也无法从葡聚糖包被活性炭上置换大量的[3H]E2。
