Partial purification and characterization of oestrogen receptors in subfractions of hepatocyte plasma membranes

Abstract

To assess the subcellular distribution of estrogen-binding components in their native state, plasma membrane and other cell fractions were prepared from hepatocytes in the absence of [3H]-17.beta.-estradiol. Cells from livers of ovariectomized rats were disrupted, with submaximal homogenization in buffered isotonic sucrose with CaCl2 and proteinase inhibitor, and fractionated by using isotonic media. Fractions were characterized by determinations of enzyme activities, biochemical constituents and ligand binding. Specific binding of 2 nM-[3H]-17.beta.-estradiol to intact cells and their fractions was determined after equilibration for 1.5 h at 4 C. More than 92% of the radioactivity from representative preparations was verified as authentic estradiol by TLC. Activities of plasma-membrane marker enzymes as well as binding sites for estrogen and for wheat germ agglutinin were present principally in particulate fractions, rather than in 105,000 g-supernatant fractions. However, by using alternative homogenization procedures (i.e., hypotonic media), known to fragment and strip structural components, estradiol-binding sites and activities of plasma-membrane marker enzymes were distributed predominantly into cytosol. By using the more conservative procedures, plasma membranes of low (P = 1.13-1.16) and high (P = 1.16-1.18) density were purified from crude nuclear fractions. A 2nd low-density subfraction of plasma membrane was prepared from microsome-rich fractions. Activities of plasma-membrane marker enzymes were enriched to .apprx. 28 and 4 times that of the homogenate in plasma membranes of low and high density, respectively. Binding sites for wheat germ agglutinin and estradiol were concentrated in low-density plasma membranes to 46-63 times that of the homogenate. Specific binding of estrogen in low-density plasma membranes purified from crude nuclei was saturable, with an apparent association constant of 3.5 nM. At saturation, such estradiol receptors corresponded to 526 f[fento]mol/mg of membrane protein. A Hill plot showed a moderate degree of positive co-operativity in the interaction of hormone with plasma membranes. Specific binding of [3H]-17.beta.-estradiol was reduced by a 200-fold molar excess of unlabeled 17.beta.-estradiol, estriol or diethylstilbestrol, but not by 17.alpha.-estradiol, cortisol, testosterone or progesterone. Binding was also blocked by prior exposure of membranes to trypsin or to 60.degree. C, but remained essentially undiminished by extraction of membranes with either hypotonic or high-salt buffers. Extraction with 0.1% (vol/vol) Triton X-100 partially solubilized the estrogen-binding components(s) of plasma membranes. Particle-free extracts were resolved on 5-20% (wt/vol) sucrose density gradients with either 0.01 M- or 0.4 M-KCl, and the fractions were analyzed by adsorption to hydroxylapatite. In low-salt gradients macromolecule-bound estrogen sedimented at predominantly 7.4 S and binding was 1560 times that of the homogenate. Under high-salt conditions estradiol-binding activity occurred at both 3.6 S and 4.9 S. Nuclear transfer of estrogen-binding sites was investigated by incubating intact hepatocytes with 0.1 nM-estradiol for 10 min at 22.degree. C. A profound increase in estrogen binding in nuclei was coupled with a marked loss of binding in membranes, as compared with controls at 4.degree. C. Collectively, components of hepatocyte plasma-membrane may be integral to the recognition of and response to estrogen.