Receptor interconversion model of hormone action. 1. Purification of a factor involved in conferring estradiol binding properties to the estrogen receptor.
We previously demonstrated that the chick oviduct estrogen receptor exists in three interconvertible forms. Two of these forms bind estradiol with high but distinct affinities. A third form exists as a non-estrogen binding recyclable form, Rnb, which upon treatment with ATP/Mg2+ is quantitatively converted to the lower affinity estradiol binding form. We now describe the isolation from chick oviduct cytosol of a factor involved in this conversion and its 1100-fold purification by ammonium sulfate fractionation, DEAE ion-exchange chromatography, and size-exclusion HPLC. The factor elutes from the size-exclusion column with an apparent molecular weight of 40,000. This highly purified factor potentiates estradiol binding in a dose-dependent manner in the presence of ATP/Mg2+. Its activity is destroyed by heating or by trypsin treatment but is relatively stable to freezing and thawing and is inert to treatment with reducing agents. ATP is an essential nucleotide substrate; GTP and cyclic nucleotides are inactive. Studies of cation dependence demonstrate that Mg2+ is also essential; Ca2+ alone is completely ineffective in catalyzing receptor potentiation and does not synergize with Mg2+. In the presence of excess ATP/Mg2+ and a fixed concentration of Fy, the Km for potentiation of estradiol binding is approximately 0.4 nM.
McNaught
R W
RW
Department of Urology, Baylor College of Medicine, Houston, Texas 77030.
Dayani
N
N
Smith
R G
RG
D000645:Ammonium Sulfate; D000818:Animals; D001685:Biological Factors / Q000302:isolation & purification*; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000032:analysis; D004958:Estradiol / Q000378:metabolism*; D008954:Models, Biological; D010057:Oviducts / Q000032:analysis*; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D011960:Receptors, Estrogen / Q000378:metabolism*
D001685:Biological Factors; D011960:Receptors, Estrogen; D004958:Estradiol; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D000645:Ammonium Sulfate