title: Isolation and purification of a hen nuclear oestrogen receptor and its effect on transcription of chick chromatin.
abstract: An oestrogen receptor was isolated, characterized and purified from the nuclear fraction of the hen oviduct. The receptor sediments at 4.6 S on glycerol gradients, has an equilibrium dissociation constant (Kd) of 1.1 X 10(-10)M, an association constant (ka) of 1.4 X 10(-6) M-1.S-1, and a dissociation constant (kd) of 5 x 10(-5) s-1. The receptor chromatographed from DEAE-cellulose as a single peak at 0.15 M-KCl and was not retained by phosphocellulose. Polyacrylamide-gel electrophoresis of the receptor in the presence of sodium dodecyl sulphate demonstrated two subunits with apparent mol.wts. of 74000 and 80000. The overall purification achieved was 90000-fold by using a combination of cell fractionation, (NH4)2SO4 fractionation and affinity chromatography. This represents the first separation, isolation and purification of the highest-affinity binding component (Kd 10(-10)M) of two high-affinity oestrogen-binding proteins present in both chick and hen oviduct cytosol and nuclei. To examine directly the effect of the purified receptor on transcription a reconstituted cell-free system was used, which contained the receptor--oestradiol complex, Escherichia coli RNA polymerase, rifampicin and chromatin prepared from hormone-withdrawn chick tissue. The receptor-hormone complex at a concentration of 0.1 nM stimulated transcription of oviduct chromatin by promoting an increase of 14000 sites for RNA-chain initiation, which is similar to the number of additional sites measured in the oviducts of diethylstilboestrol-stimulated immature chicks [Tsai, Schwartz, Tsai & O'Malley (1975) J. Biol. Chem. 250, 5165-5174]. Oestradiol alone had no effect on transcription. Thus the data demonstrate that the purified nuclear oestradiol-receptor complex can regulate gene transcription in vitro in a manner similar to that observed in target cells in vivo.
mesh_terms: D000818:Animals; D002467:Cell Nucleus / Q000737:chemistry; D002645:Chickens; D002843:Chromatin / Q000378:metabolism; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004958:Estradiol / Q000378:metabolism; D005260:Female; D066298:In Vitro Techniques; D007700:Kinetics; D010057:Oviducts / Q000737:chemistry*; D012313:RNA / Q000096:biosynthesis; D011960:Receptors, Estrogen / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects*
chemical_list: D002843:Chromatin; D011960:Receptors, Estrogen; D004958:Estradiol; D012313:RNA