An exchange assay for estrogen receptors in cell nuclei of the adult rat brain

Abstract

A nuclear exchange assay was developed for brain estrogen receptors. The assay employs an extraction procedure which solubilizes essentially all of the estradiol from brain cell nuclei: purified cell nuclei are evenly dispersed in a hypotonic buffer prior to the addition of an equal volume of 0.8M KC1. Experiments with an in vivo injection of 3H-estradiol established that this procedure extracts even normally salt-resistant binding. For exchange, aliquots of the extract are incubated with 3H-estradiol or 3h-estradiol plus 100-fold excess unlabeled estradiol. Bound 3H-estradiol is separated from free 3H-estradiol on Sephdex LH-20 columns. Loss of estradiol binding activity can occur with brain nuclear extracts under conditions required for exchange. This loss of binding is inhibited by the addition of bacitracin to the incubation buffer. The exchange is complete within 5 hrs at 25 degrees C and specific binding activity is stable for at least 16 hrs. The assay was validated by comparing levels of macromolecular-bound radioactivity after an in vivo injection of 3H-estradiol and levels determined by exchange after an injection of unlabeled estradiol. Scatchard analysis confirmed the high affinity nature of the binding measured by exchange.