Assay of total estradiol receptor in tissue homogenate and tissue fractions by exchange with sodium thiocyanate at low temperature.
After injection of radioactive estradiol to ovariectomized rats, the [3H]estradiol--receptor complex transferred to the nuclei can be solubilized by low concentrations of NaSCN. The extraction by NaSCN is significantly more efficient than that obtained by KCl and is, in fact, complete; i.e., no radioactivity can be found in the nuclei after extraction. Since NaSCN also induces the exchange of receptor-bound estradiol with free hormone [Sica, V., Puca, G. A., Molinari, A. M., Buonaguro, F. M., & Bresciani, F. (1980) Biochemistry 19, 83], a simple assay method has been set up which measures receptor in tissue and tissue fractions, including nuclei and whole homogenate, at 0--4 degrees C, irrespective of whether the receptor is or is not interacting with endogenous hormone. The procedure consists of a simple incubation step at 0--4 degrees C overnight (16 h) of the nuclear fraction, cytosol, and a total homogenate in the presence of excess radioactive estradiol and 0.5 M NaSCN. This method is very easy to carry out, accurate, and precise and avoids the loss of binding sites which results from the heating procedures utilized in other methods. The ability to measure the binding in both the soluble and the particulate fractions of rat uterus permits the determination of the rate of the cytoplasmic to nuclear transfer of estrogen after injection of various hormone concentrations. No nuclear transfer was observed after administration of other nonestrogen hormones such as progesterone, testosterone, or hydrocortisone while a nonsteroid antiestrogen, tamoxifen, was able to translocate the receptor. It was found that 2 h after injection of estradiol into ovariectomized rats total receptor content of uterus shows a decrease which is proportional to the amount of hormone injected. After injection of a hyperphysiological dose of 17 beta-estradiol, a certain amount of the receptor--hormone complex remains in the cytosol for at least 4 h. The nuclear turnover of estradiol receptor related to the progesterone receptor induction has been studied. Actinomycin D and cycloheximide prevent nuclear processing.
D000818:Animals; D002369:Castration; D002467:Cell Nucleus / Q000032:analysis / Q000187:drug effects; D003513:Cycloheximide / Q000494:pharmacology; D003593:Cytoplasm / Q000032:analysis; D003609:Dactinomycin / Q000494:pharmacology; D004958:Estradiol / Q000378:metabolism* / Q000494:pharmacology; D004986:Ether; D005260:Female; D011189:Potassium Chloride; D051381:Rats; D011960:Receptors, Estrogen / Q000032:analysis* / Q000187:drug effects; D013696:Temperature; D013861:Thiocyanates; D014599:Uterus / Q000032:analysis
D011960:Receptors, Estrogen; D013861:Thiocyanates; D004986:Ether; D003609:Dactinomycin; D004958:Estradiol; C024553:sodium thiocyanate; D011189:Potassium Chloride; D003513:Cycloheximide