Characterization of molybdate-stabilized estrogen receptors by hydrophobic interaction HPLC: resolution of two 8S subunits.
This report describes the separation and characterization of estrogen receptors (ER) according to their degree of hydrophobicity and surface charges. Molybdate-stabilized [3H]ER from rabbit uterine cytosol was sequentially purified by passage through a size-exclusion pre-column, an anion-exchange column, and a hydrophobic interaction column. With fresh cytosol, a major radioactive peak was eluted from the DEAE columns; a major peak and a minor, less hydrophobic, peak were eluted from the hydrophobic column. In contrast, ER from frozen cytosol showed one peak in the DEAE-column and exhibited four radioactive peaks in the hydrophobic column. By sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis, [3H] tamoxifen aziridine(TA)-labelled ER showed radioactive bands at 62 and 48 kd. The subunits which were characterized by these radioactive bands were successfully separated by the hydrophobic column; the more hydrophobic subunit corresponded to the 62 kd band. The HPLC-purified [3H]TA-labelled ER subunits sediment at a 7.4-8.5S region in a low-salt sucrose gradient. These results show that differential negative surface charge and hydrophobic areas exist in the holo-receptor and its subunits, and the hydrophobic interaction HPLC column separates the two major 8S steroid binding subunits of ER.
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000032:analysis; D004563:Electrochemistry; D004721:Endoplasmic Reticulum / Q000032:analysis; D005260:Female; D008970:Molecular Weight; D008982:Molybdenum; D011487:Protein Conformation; D011817:Rabbits; D011960:Receptors, Estrogen / Q000302:isolation & purification*; D014599:Uterus / Q000032:analysis
D011960:Receptors, Estrogen; D008982:Molybdenum; C024687:sodium molybdate(VI)