Nelson K, Pavlik E J, van Nagell J R, Hanson M B, Donaldson E S, Flanigan R C
Biochemistry. 1984 Jun 5;23(12):2565-72. doi: 10.1021/bi00307a005.
The estrogenicity of coumestrol, a fluorescent phytoestrogen, has been examined in murine uteri. Coumestrol competed with 17 beta-[3H]estradiol for binding to cytoplasmic estrogen receptors, caused cytoplasmic estrogen receptors to associate with chromatin in the nucleus, and induced progesterone receptors. By use of size-exclusion high-performance liquid chromatography (SEHPLC), the interaction of coumestrol with estrogen receptors was examined directly by monitoring the fluorescence associated with macromolecules having properties characteristic of estrogen receptors. These analyses were made possible by the addition of dimethylformamide to the elution buffer, at a concentration (7.5%) which improved recoveries but did not interfere with estrogen receptor binding. It was possible to detect fluorescent coumestrol at approximately 0.5 nM. All determinations were performed with preparations in which estrogen receptor activity was 3-10 nM. Exposure of these preparations to coumestrol (50 nM) resulted in the elution of increased fluorescent activity in the regions where estrogen receptors eluted during SEHPLC. This fluorescent activity was reduced when diethylstilbestrol, 17 beta-estradiol, hexestrol, or tamoxifen was present as a competitor (2 microM) but was unaffected by testosterone or progesterone. Diethylstilbestrol reduced fluorescence below endogenous base lines and thereby displayed a fluorescence quench property which was not observed with other ligands. When hepatic and renal estrogen receptor preparations were used, the injected receptor activity was observed to be the major limiting factor in detecting the interaction of coumestrol with estrogen receptors. These observations are relevant to attempts to visualize estrogen receptors in tumor cells and demonstrate that accepted biochemical criteria for ligand-receptor interaction can be satisfied when fluorescent ligands are examined.
已在小鼠子宫中检测了一种荧光植物雌激素香豆雌酚的雌激素活性。香豆雌酚与17β-[³H]雌二醇竞争结合细胞质雌激素受体，使细胞质雌激素受体与细胞核中的染色质结合，并诱导孕激素受体。通过尺寸排阻高效液相色谱法（SEHPLC），通过监测与具有雌激素受体特性的大分子相关的荧光，直接检测香豆雌酚与雌激素受体的相互作用。通过在洗脱缓冲液中添加二甲基甲酰胺，使其浓度为7.5%，可以实现这些分析，该浓度提高了回收率，但不干扰雌激素受体结合。可以检测到约0.5 nM的荧光香豆雌酚。所有测定均使用雌激素受体活性为3 - 10 nM的制剂进行。将这些制剂暴露于香豆雌酚（50 nM）后，在SEHPLC过程中雌激素受体洗脱的区域出现荧光活性增加的洗脱峰。当己烯雌酚、17β-雌二醇、己烷雌酚或他莫昔芬作为竞争者（2 μM）存在时，这种荧光活性降低，但不受睾酮或孕激素的影响。己烯雌酚使荧光降低至内源性基线以下，从而表现出其他配体未观察到的荧光猝灭特性。当使用肝脏和肾脏雌激素受体制剂时，注射的受体活性被观察到是检测香豆雌酚与雌激素受体相互作用的主要限制因素。这些观察结果与在肿瘤细胞中可视化雌激素受体的尝试相关，并表明当检测荧光配体时，可以满足配体 - 受体相互作用公认的生化标准。
