Isolation of untransformed bovine estrogen receptor without molybdate stabilization.
A low concentration estrogen-derivatized affinity resin has been used in a rapid, single step purification of the untransformed estrogen receptor from calf uterine cytosols prepared without sodium molybdata. The procedure isolates the Mr 65,000 estrogen receptor in association with the bovine heat shock protein hsp90. Small amounts of proteolyzed receptor ranging in size from Mr 50,000 to 60,000 are also present in the purified extracts. Results from affinity chromatography of receptor cytosols either untreated or presaturated with estradiol suggest that two proteins of Mr 22,000 and 38,000 are co-purified with the untransformed receptor complex and may represent additional nonhormone-binding components of the native receptor form. Some indication of the stability of protein-protein interactions within the oligomeric complex has been derived from differential salt elution studies with heparin-sepharose and affinity gel-immobilized untransformed receptor. On size exclusion high performance liquid chromatography the untransformed complex eluted with a Stokes radius of 75 +/- 2 A (n = 18), but was shown to be sensitive to extended ultracentrifugal analysis dissociating to the receptor homodimer, sedimentation coefficient 5.3 +/- 0.3 s (n = 5). Preliminary data on urea- and heat-induced transformation of the isolated receptor to the DNA-binding state is presented.
Ratajczak
T
T
Department of Obstetrics and Gynaecology, University of Western Australia, King Edward Memorial Hospital for Women, Subiaco.
Hlaing
J
J
Brockway
M J
MJ
Hähnel
R
R
D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000032:analysis; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006360:Heat-Shock Proteins / Q000302:isolation & purification; D008982:Molybdenum; D011960:Receptors, Estrogen / Q000235:genetics / Q000302:isolation & purification*; D014461:Ultracentrifugation; D014599:Uterus / Q000032:analysis*
D006360:Heat-Shock Proteins; D011960:Receptors, Estrogen; C044659:molybdate; D008982:Molybdenum; D004247:DNA