Structural characterization of the 9-10S estradiol receptor from calf uterus.
An affinity chromatography-based method has been developed for estrogen receptor isolation which requires the inclusion of sodium molybdate in purification buffers for maintaining the large 9-10S form of the receptor. The protein products obtained from affinity chromatography of calf uterine receptor extracts or from extracts presaturated with estradiol have been analyzed by gel electrophoresis under denaturing conditions. Major estrogen sensitive proteins were peptides with Mr approximately 90,000, 65,000 and 50,000. Two additional proteins (60,000 and 53,000) of lower abundance and with demonstrated estrogen sensitivity were also observed. Affinity labeling with [3H]tamoxifen aziridine identified the Mr 65,000 protein as the estrogen receptor and suggested that the Mr 60,000, 53,000 and 50,000 peptide components were derived proteolytically from this parent unit. The 90,000 mol. wt component was readily dissociated from heparin-sepharose immobilized estrogen receptor by elution with low salt buffers without molybdate. Peptide mapping experiments indicated that the 90,000 mol. wt component was not related to the Mr 65,000 and 50,000 estrogen receptors, but confirmed the smaller binding unit to be a proteolytic fragment of the 65,000 mol. wt receptor. The results suggest that the 90K protein associates non-covalently with the Mr 65,000 estrogen binding unit as a nonhormone binding component of the 9-10S receptor.
Ratajczak
T
T
Department of Obstetrics and Gynaecology, University of Western Australia, King Edward Memorial Hospital for Women, Subiaco.
Comber
M
M
Hähnel
R
R
D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D005260:Female; D008970:Molecular Weight; D008982:Molybdenum / Q000494:pharmacology; D010449:Peptide Mapping; D011959:Receptors, Estradiol / Q000032:analysis*; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000378:metabolism; D014599:Uterus / Q000032:analysis*
D011959:Receptors, Estradiol; D013629:Tamoxifen; C044659:molybdate; C031859:tamoxifen aziridine; D008982:Molybdenum