Pettersson K S, Vanharanta R M, Söderholm J R
J Steroid Biochem. 1985 Jan;22(1):39-45. doi: 10.1016/0022-4731(85)90139-6.
This study investigated the influence of the degree of concentration of breast tumor cytosols on the apparent estrogen receptor content as measured by the Dextran-charcoal assay. It was found that the dilution of cytosols to 1-2 mg protein/ml frequently but not always causes highly underestimated receptor concentrations. This could not be explained by the protein loss through adsorption to the charcoal. The effect was also studied in the presence of gelatin, sodium molybdate or with limited trypsinization of the incubation mixture. Addition of 1 mg/ml gelatin in the Dextran-charcoal suspension was very useful in most cases in preventing dilution induced losses in receptor sites. Both trypsinization and addition of sodium molybdate produced increases in receptor concentrations that were not as susceptible to inactivation through dilution of the cytosol. These data suggest that the observed high variability in the dilution induced receptor losses can be explained by receptor heterogeneity: some receptor form(s) are either readily absorbed to or "stripped" by the charcoal particles. As a conclusion we recommend that in order to optimize the estrogen receptor assay as regards both binding sites and affinities the cytosol concentrations should be maintained as high as possible and a protein expander be included in the Dextran-charcoal suspension. Though sodium molybdate frequently gives considerable increases in estrogen binding sites it occasionally has an opposite effect. For this reason we hesitate to recommend its use in routine assays of estrogen receptors.
本研究调查了乳腺肿瘤胞质溶胶的浓缩程度对通过葡聚糖-活性炭法测定的表观雌激素受体含量的影响。发现将胞质溶胶稀释至1-2mg蛋白质/ ml时，常常（但并非总是）会导致受体浓度被高度低估。这无法用吸附到活性炭上导致的蛋白质损失来解释。还在明胶、钼酸钠存在的情况下或对孵育混合物进行有限胰蛋白酶消化的情况下研究了这种效应。在葡聚糖-活性炭悬浮液中添加1mg / ml明胶在大多数情况下对于防止稀释引起的受体位点损失非常有用。胰蛋白酶消化和添加钼酸钠都会使受体浓度增加，且这些增加不太容易因胞质溶胶稀释而失活。这些数据表明，观察到的稀释诱导受体损失的高度变异性可以通过受体异质性来解释：某些受体形式要么容易被活性炭颗粒吸附，要么被其“剥离”。作为结论，我们建议，为了在结合位点和亲和力方面优化雌激素受体测定，应尽可能保持胞质溶胶浓度高，并在葡聚糖-活性炭悬浮液中加入蛋白质膨胀剂。尽管钼酸钠常常会使雌激素结合位点大幅增加，但它偶尔也会产生相反的效果。因此，我们在常规雌激素受体测定中犹豫是否推荐使用它。
