Macromolecular binding of estradiol by nuclei in a cell-free system

Abstract

Rat uterine cytosol estradiol-binding protein was labeled with radioactive estradiol and then incubated with highly purified nuclei from an estrogen-binding murine tumor. The nuclei were then extracted with 2M NaCl-5 M urea, and macromolecular bound estradiol was separated by gel filtration. Nuclear preparations incubated with estradiol-labeled uterine cytosol showed five to ten times more macromolecular binding than corresponding nuclear preparations incubated with buffer, albumin, heart cytosol or uterine cytosol deficient in biologically active binding protein. The process by which nuclei bind estradiol is temperature dependent. Radioactive estradiol attached to protein can be extracted from the nuclei with solutions of 2 M NaCl-5 M urea. Binding of estradiol to isolated nuclei could be distinguished from the binding of the hormone to isolated microsomes or mitochondria in that only the nuclear binding was markedly reduced by prewarming the particulate fraction. Furthermore, nuclear binding was decreased by incubation with several metabolic inhibitors. Most of the nuclear binding appears to occur with the chromatin fraction from which labeled protein-bound estradiol can be extracted. When the cytosol estradiol-binding protein was partially by gel filtration there was less nuclear binding of estradiol than resulted from using cytosol. The deficiency of the gel filtration preparation was restored by the addition of unfractionated cytosol. Nuclear binding of estradiol seems to depend on some cofactor present in the cytosol. Nuclei from an estrogen-dependent tumor and the uterus had comparable amounts of nuclear binding that was associated with the 5S fraction as determined by ultracentrifugation on sucrose gradients. Nuclei from liver bound 1 3 as much estradiol, which was associated with a 3S macromolecular fraction. The nuclear-binding macromolecule in the liver is different from that in the uterus and the dependent tumor. Prior incubation of nuclei with non-radioactive estradiol, diethylstilbestrol or estriol in uterine cytosol reduced the uptake of the nuclei when subsequently incubated in [ 3H]estradiol containing cytosol. Incubation in cytosol containing MER-25, an anti-estrogen, resulted in no subsequent reduction of [ 3H]estradiol uptake.