Isolation and purification of a hen nuclear oestrogen receptor and its effect on transcription of chick chromatin.
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the receptor--oestradiol complex, Escherichia coli RNA polymerase, rifampicin and chromatin prepared from hormone-withdrawn chick tissue.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.
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*
D002843:Chromatin; D011960:Receptors, Estrogen; D004958:Estradiol; D012313:RNA