Isoforms of estrogen receptors by high-performance ion-exchange chromatography.
SynChropak AX-300, AX-500 and AX-1000 columns were used to separate ionic forms of estrogen receptors by high-performance ion-exchange chromatography. Cytosols from hormone-responsive tissues were incubated for 4-10 h with 3-4 nM [16 alpha-125I]iodoestradiol-17 beta, cleared of unbound ligand and applied to an anion-exchange column. Components were eluted at pH 7.4 using a gradient of phosphate buffer at 4-6 degrees C. Non-specific binding components were identified by chromatographing, the identical cytosol, incubated with [125I]iodoestradiol and a 500-fold excess of diethylstilbestrol, which blocks specific binding sites. [125I]Iodoestradiol was applied to the column in the absence of cytosol and eluted normally to determine the behavior of free ligand. Each column exhibited a different elution pattern for the estrogen receptor. The various isoforms of estrogen receptor were eluted differently from each column usually in the 15-120 mM and 180-250 mM region of the gradient. Often one non-specific binding component was not retained whereas other non-specific species were retained and eluted from the column in a salt-dependent manner; their position in the gradient varied from column to column. Similarly, free [125I]iodoestradiol was eluted at different positions in the gradients, dependent upon which column was employed. In general, the high flow-rates, reproducibility, good recovery and the apparent differential selectivity of each of the columns appear valuable in the investigation of the nature and subunit composition of the estrogen receptor molecule.
D000818:Animals; D000837:Anion Exchange Resins; D001943:Breast Neoplasms / Q000032:analysis; D002848:Chromatography, DEAE-Cellulose; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000032:analysis; D004717:Endometrium / Q000032:analysis; D005260:Female; D006801:Humans; D008321:Mammary Glands, Animal / Q000032:analysis; D051381:Rats; D011960:Receptors, Estrogen / Q000032:analysis*; D014599:Uterus / Q000032:analysis
D000837:Anion Exchange Resins; D011960:Receptors, Estrogen