Effects of salt extraction on the quantitation of nuclear estrogen receptors: interference by secondary estrogen binding sites.

Abstract

The effects of salt-extraction on type I and type II estrogen binding sites were examined in uterine nuclei. Injection (10 ug) of estradiol or estriol in adult ovariectomized rate induced maximum numbers (80-100%, integral of 1 pmole/uterus) of 0.4 M KCL resistant type I estrogen complexes at 1 hour. Only estradiol, which sustained these levels for long periods of time (4-24 hours) stimulated true uterine growth. Likewise, a single injection of estradiol, but not estriol, also elevated nuclear type II sites throughout the entire uterine growth period (1 - 48 hours). However extraction of these nuclei from estradiol injected rats with 0.4 M KCL increased the numbers of type II sites from integral of 1 pmole/uterus (non-extracted nuclei) to integral of 8 pmoles/uterus (salt resistant plus salt-extractable fractions). Sixty percent of these sites were resistant to salt-extraction. Continuous exposure to either estradiol or estriol by beeswax implants stimulated nuclear type II sites which were highly resistant (80%) to KCL-extraction, and additional sites were not exposed by high salt. Thus chronic treatment with both estrogens "locked in" nuclear type II sites such that they were resistant to KCL-extraction. This resistance of type II sites to salt-extraction correlated with the ability of estradiol and estriol implants to stimulate true uterine growth. The procedures presented here for nuclear preparation and assay have reduced non-specific binding considerably in the uterine system, and may eliminate the need to perform exchange assays on salt-extracted nuclei in other systems.