Isolation of unliganded steroid receptor proteins by high-performance size-exclusion chromatography. An investigation of steroid-dependent structural alterations.
The physicochemical properties of unliganded steroid receptor proteins remain largely unknown primarily due to receptor lability in the absence of specific ligand, especially during prolonged biochemical analyses. We have utilized high-performance size-exclusion chromatography (HPSEC) as a rapid means of investigating the structural properties of cytosolic estrogen receptor proteins in both the presence and absence of [3H]estradiol-17 beta. Cytosols prepared from immature calf uteri were analyzed by HPSEC on an Altex TSK-3000 SW column (600 mm X 7.5 mm I.D.) either before or after incubation with 10 nM [3H]estradiol-17 beta. Postcolumn detection of previously unliganded receptor was accomplished by incubation of fractions (0.38 ml) with 10 nM [3H]estradiol-17 beta for 2-18 h at 0 degrees C. Receptor-bound steroid was separated from free steroid by incubation with small pellets of hydroxylapatite. Nonspecific binding of [3H]estradiol-17 beta in parallel fractions was estimated using an unlabelled competitor (diethylstilbestrol) specific for the estrogen receptor. In low ionic strengths the receptor exists as a single, relatively stable, large form (retention time 34 min). The elution properties of this receptor configuration do not depend on bound steroid ligand. Analysis of receptor at elevated ionic strengths in the presence and absence of steroid ligand suggests that the salt-induced dissociation of receptor components to smaller forms (retention time 47 min) may be partially steroid-dependent. Characterization of receptor in 6 M urea demonstrates the presence of intermediate-sized receptor components (retention time 36-38 min). Analyses of receptor in 6 M urea-0.4 M potassium chloride suggests an inhibition of the more extensive salt-induced dissociation event seen in 0.4 M potassium chloride alone. Furthermore, the intermediate-sized receptor forms seen under these conditions (retention time 41-42 min) are generated in a steroid-dependent manner. The preparation of different molecular forms of biologically active, unliganded estrogen receptor by HPSEC should help further our investigations into the molecular mechanism(s) by which steroid hormones exert their receptor-mediated effects on target cells.
D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification; D002417:Cattle; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002850:Chromatography, Gel; D003600:Cytosol / Q000032:analysis; D004958:Estradiol / Q000302:isolation & purification; D005260:Female; D066298:In Vitro Techniques; D011960:Receptors, Estrogen / Q000302:isolation & purification; D011987:Receptors, Steroid / Q000302:isolation & purification*; D013256:Steroids / Q000032:analysis; D014599:Uterus / Q000032:analysis
D002352:Carrier Proteins; D011960:Receptors, Estrogen; D011987:Receptors, Steroid; D013256:Steroids; C010140:estrophilin; D004958:Estradiol