Nuclear estradiol receptor in the adult rat uterus: a new exchange assay

Abstract

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.

Effect of estradiol on the progesterone receptor and on morphological ultrastructures in the fetal and newborn uterus and ovary of the rat

“…The measurement of specific [3H]R2858 binding in the cytoplasmic and nuclear extracts was performed by the exchange assay in protamine sulphate precipitates (Chamness et al 1975;Zava et al 1976) with the modifications described previously (Sumida & Pasqua¬ lini 1979). Cytosol and nuclear pellets were incubated with 1 x IO"8 mol/1 [3H]R2858, with and without a 100-fold molar excess of unlabelled R2858 at 30°C for 16 h. [3H]5020-specific binding sites in the cytosol and nuclear extracts were measured after incubation with 5 x 10"9 mol/1 of the radioactive steroid in the absence or presence of a 100-fold excess of the unlabelled steroid at 4°C for 16 h. Binding of radioactive steroids to macromolecules was determined after adsorption of the unbound steroid with a 0.1% (wt/vol) dextran-coated 1% (wt/vol) charcoal mixture at 4°C for 10 min.…”

Oxytocin Receptors and Parturition. I. Control of Oxytocin Receptor Concentration in the Rat Myometrium at Term*

“…Estrogen exchange assays on protamine-precipitated receptor were adapted from those of Zava et al (6) for the nuclear fraction (1,000 x g pellet) and Chamness et al (7) for the cytosol (160,000 X g supernatant). Briefly, the nuclear fraction was extracted with 0.6 M KC1.…”

High-affinity Cortisol receptor activity in the liver of the brook trout, Salvelinus fontinalis (Mitchill)

“…A solid phase exchange assay (Zava et al 1976) was employed to determine specific binding of [3~]cortisol to the nuclear extract. Protamine sulfate (250 pL of 1 mg. mL-' TED) was used in each assay tube containing 250 pL nuclear extract.…”