Estrogen Receptor Characterization Following Selective Sedimentation Separation of Estrogen-Binding Components In Immature Rat Uterine Cytosol

Abstract

Characterization of a specific estrogen receptor (ER) in fetal and early postnatal rat uterine cytosol is complicated by the presence of other high-affinity estrogen-binding components, such as alpha-fetoprotein (AFP). In an attempt to circumvent their influence, we have employed the selective sedimentation of unlabeled cytosol through sucrose gradients, followed by the analysis of [³H]estradiol binding to a pool of fractions comprising the ER region, as well as to individual gradient fractions. As the amount of AFP present in 21-day-old rats is sufficiently low to permit ER characterization by conventional methodology, we have validated the selective sedimentation method by comparing its results with those obtained conventionally. Though conventional gradient analysis revealed only one estrogen-binding component, saturation and binding inhibition analyses indicated the presence of multiple components, identified as AFP and the ER. These conclusions were supported by results from labeling individual gradient fractions obtained following selective sedimentation of unlabeled cytosol. Further, when unlabeled 7–9 S gradient fractions were pooled and assayed by saturation and binding inhibition analyses, only one binding component, with ER characteristics, was revealed. These results validate selective sedimentation as an effective method for separating multi-component estrogen-binding systems and suggest its applicability to similar systems.