Immunologic inhibition of estrogen binding and action in preputial-gland cells and their subcellular fractions

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To assess the specific binding and actions of estradiol-17β (E 2β) in preputial gland of ovariectomized rats, isolated cells, consisting of parenchymal- and ductal-cell types were prepared by a collagenase procedure. Such cells exhibited substantial oxidation to 14CO 2 of [ 14C]-cholesterol. The latter function was stimulated 15-fold by treatment for 3 h in vitro with 1 × 10 −9M E 2β but not E 2α. Marked inhibition of such estrogen action was elicited by prior exposure of cells to antibodies directed against preputial-gland lysosomal lipoproteins with high-affinity binding activity for E 2β (anti-PHDLP). Retention of E 2β by preputial-gland cells was saturable, with an association constant of 6.4 × 10 8 M −1. At saturation, E 2β receptors corresponded to ~ 10,900 sites per cell. Treatment of 2 × 10 7 cells with anti-PHDLP, but not preimmune, IgG at 20 μg/ml of protein reduced the specific binding of E 2β by effecting a 4- to 5-fold decrease in the magnitude of the binding constant. The subcellular distribution and immunochemical reactivity of E 2β-binding components in their native state was investigated by disruption of preputial-gland cells in the absence of hormone. Cells were homogenized under carefully monitored conditions in buffered isotonic sucrose with Cacl 2; and proteinase inhibitor and fractionated using isotonic media. Fractions were characterized by determinations of selected enzyme activities and DNA. Activities of plasma membrane marker-enzymes as well as specific binding-sites for E 2β were present principally in paniculate fractions. Plasma membranes of low density ( ρ = 1.13–1.16) were purified from crude nuclear fractions. Activities of appropriate marker-enzymes were enriched in plasma membranes to about 10 times that of the homogenate, while binding-sites for E 2β were concentrated to about 11 times the homogenate. Specific binding of E 2β in membranes was saturable, with an association constant of 2.6 × 10 9 M −1. At saturation, E 2β receptors in plasma membrane corresponded to 0.6 pmol/mg of protein. Specific binding of [ 3H]-E 2β in plasma membrane and other major subcellular fractions was reduced by a 200-fold molar excess of unlabeled E 2β or DES, but not by E 2α, testosterone, or progesterone. Binding was also blocked by prior exposure of membranes to trypsin or to 5 min heating to 65°C, but was diminished by no more than 9.8% by extraction of membranes with either hypotonic or high-salt buffers. Significant inhibition of E 2β binding in plasma membrane, microsomerich, mitochondria-lysosome and cytosol fractions was elicited by treatment of each fraction with anti-PHDLP, but not preimmune, IgG. These findings provide evidence of an immunochemical similarity of specific E 2β binding-sites which occur in multiple cellular loci before hormonal stimulation.