Purification of the molybdate-stabilized 9-10 S estradiol receptor from calf uterus.
The molybdate-stabilized calf uterine estradiol receptor has been purified to near-homogeneity by a three-step procedure. Initial purification by heparin-Sepharose chromatography provides a concentrated receptor extract in 40% yield with a 5-10-fold increase in purity. The inclusion of molybdate in phosphate-buffered cytosol enhances 9-10 S receptor stability in high salt and allows elution of the oligomeric receptor complex from heparin-Sepharose with 0.4 M KCl. A second affinity step utilizing estrone carboxymethyloxime coupled to diaminoethyl bis(2-hydroxypropoxy)butane-Sepharose Cl-4B increases purification by a further 1600-fold. High performance liquid chromatography gives homogeneous receptor which migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a polypeptide of Mr approximately 89,000. The purified molybdate-stabilized receptor sediments at 9.3 +/- 0.2 S (n = 4) in glycerol gradients and has a Stokes radius of 74 +/- 3 A (n = 2) giving a calculated Mr approximately 290,000. These properties and the steroid-binding specificity of the purified receptor bear a close similarity to those found for the 9-10 S receptor in crude cytosol.
D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000378:metabolism; D004958:Estradiol / Q000378:metabolism; D005260:Female; D066298:In Vitro Techniques; D008970:Molecular Weight; D008982:Molybdenum; D011487:Protein Conformation; D011959:Receptors, Estradiol / Q000302:isolation & purification* / Q000378:metabolism; D011960:Receptors, Estrogen / Q000302:isolation & purification*; D014599:Uterus / Q000378:metabolism*
D011959:Receptors, Estradiol; D011960:Receptors, Estrogen; D004958:Estradiol; D008982:Molybdenum