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A protamine exchange assay has been developed to measure uterine nuclear estrogen receptor in mature rats exposed to estradiol (E). After ovariectomized-adrenalectomized mature rats are injected with E, estrogen receptor (RnE) is extracted from uterine nuclei with 0.6 M potassium chloride, diluted, and quantitatively precipitated with protamine sulfate. The precipitate is subjected to a ligand exchange with radiolabeled estradiol (E), with or without unlabeled diethylstilbesterol, to determine nonspecific binding. At 37 degrees C complete exchange of E for E in RnE is observed at 2.5 h; virtually no receptor degradation occurs up to at least 5 h. Exchange does not occur at 4 degrees C. Using the protamine assay, the depletion of cytoplasmic estrogen receptor (Rc) and the accumulation of RnE were studied at various doses of E at specific time points. Increasing doses of E result in a decrease of Rc with an equal increase of RnE. At the highest dose of E (10 mug) Rc is completely depleted within 10 min, by 6 h it is 25% replenished, and by 24 h returns to slightly above control levels. Within 10 min after the injection, RnE increases to 80-90% of the original cytoplasmic level of receptor (approximately 2-3 pmol/mg of DNA or approximately 1.5 pmol/100 mg of uterus). At 6 h RnE is 75% depleted and it is completely absent at 24 h. The protamine assay permits precise quantitative studies of nuclear estrogen receptor and avoids the problems of receptor degradation and excessive nonspecific binding often found in exchange reactions at elevated temperatures.
Zava et al. (Tue,) studied this question.