Electrophoretic characterization of purified bovine, porcine, murine, rat, and human uterine estrogen receptors.
The calf uterine estrogen receptor (E2R) in the presence of sodium molybdate has been purified, 7,000-fold by a single passage over an estradiol affinity column. A dominant 70,000-dalton band and two minor bands at 50,000 and 30,000 daltons were observed by electrophoretic analysis. These bands had been eluted using estradiol, sodium sulfocyanate, CHAPS, and HEPES (pH 7.4) with insulin as a carrier protein. The identities of the protein bands were initially confirmed by their failure to bind the affinity column when saturated with estradiol. This single step purification procedure was reproducible and rapid, with yields of 10-20%, providing 25% purity. Diffusion blot analysis, with specific 35S- and 125I-labeled monoclonal antibodies to E2R, confirmed that the 70,000-dalton band represented the estrogen receptor. Specificity was demonstrated by inhibition of binding of purified E2R by both estradiol and diethylstilbestrol but not testosterone, progesterone, corticosterone, aldosterone, or hydrocortisone. The relative binding affinity of the purified receptor was: ethynyl estradiol greater than 17 beta estradiol greater than estriol greater than or equal to estrone greater than or equal to 17 alpha-estradiol greater than mestranol. Pig, human, mouse, and rat uterine estrogen receptors were similarly purified with the affinity column. As with the calf uterine preparations, a dominant 70,000-dalton band with minor bands at 50,000 and 30,000 daltons was identified by diffusion blot analysis in all the species examined.
D000818:Animals; D000911:Antibodies, Monoclonal; D002417:Cattle; D002606:Charcoal; D002846:Chromatography, Affinity; D004054:Diethylstilbestrol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004958:Estradiol / Q000494:pharmacology; D004967:Estrogens / Q000378:metabolism; D005260:Female; D005898:Glass; D006801:Humans; D007158:Immunologic Techniques; D051379:Mice; D008970:Molecular Weight; D051381:Rats; D011960:Receptors, Estrogen / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D013552:Swine; D014599:Uterus / Q000032:analysis*
D000911:Antibodies, Monoclonal; D004967:Estrogens; D011960:Receptors, Estrogen; D002606:Charcoal; D004958:Estradiol; D004054:Diethylstilbestrol