Rapid analysis of estrogen receptor heterogeneity by chromatofocusing with high-performance liquid chromatography.
Chromatofocusing principles have been utilized to develop a high-performance liquid chromatographic technique for the rapid and routine analysis of steroid receptor heterogeneity. Two anion-exchange columns (SynChropak AX-300 and AX-500) were compared for analytical and preparative chromatofocusing of estrogen receptor components. As many as ten different [125I]iodoestradiol-labeled binding proteins were identified in cytosols prepared from mammary gland and uterus. Estrogen receptors were well separated from other cytosolic proteins and recovery of activity routinely exceeded 90%. Parallel analyses of these cytosols to determine receptor size and shape indicated that HPLC chromatofocusing can be used effectively to study receptor isoforms with Stokes radii ranging from 30 A to greater than 70 A. In contrast to isoelectric focusing, this technique is compatible with the inclusion of a commonly used receptor-stabilizing agent, sodium molybdate. Inclusion of molybdate during chromatofocusing of molybdate-stabilized receptor allowed the identification of two acidic receptor species not previously reported.
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange / Q000295:instrumentation; D003600:Cytosol / Q000032:analysis; D005260:Female; D006801:Humans; D007526:Isoelectric Point; D007774:Lactation; D008321:Mammary Glands, Animal / Q000378:metabolism; D011247:Pregnancy; D051381:Rats; D011960:Receptors, Estrogen / Q000302:isolation & purification*; D014599:Uterus / Q000378:metabolism
D011960:Receptors, Estrogen