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Glucocorticoid binding in the hen oviduct.

ABSTRACT: [(3)H]Triamcinolone acetonide (15nm) was incubated with cytosol (150000g fraction) prepared from oviducts of egg-laying hens. The extent of steroid binding, as determined by charcoal assays, was greatest between 2-4h at 4 degrees C. A similar time curve was obtained when cytosol preparations were first fractionated with (NH(4))(2)SO(4) before labelling. The addition of 10mm-Na(2)MoO(4) or 10mm-ATP during the incubation of hen oviduct cytosol with [(3)H]triamcinolone acetonide lowered the extent of steroid binding. The presence of glycerol (20%), however, increased the extent of [(3)H]triamcinolone acetonide binding in cytosol fractions from chick (330%) and hen (160%) oviducts. The [(3)H]triamcinolone acetonide-receptor complex was stable for over 4h at 4 degrees C, but dissociated rapidly at 37 degrees C, exhibiting a half-life of about 10min. The presence of 10mm-Na(2)MoO(4) and 10mm-ATP or both had a small protective effect on the dissociation of [(3)H]triamcinolone acetonide-receptor complex. The receptor from hen oviduct showed significant affinity for unlabelled triamcinolone acetonide, cortisol, compound R5020 and dihydrotestosterone and, to a lesser extent, for oestradiol, oestrone and progesterone. Diethylstilboestrol treatment of immature chicks appeared to induce a more specific binder, which showed affinity for unlabelled triamcinolone acetonide, cortisol and compound R5020 only. Scatchard analysis of [(3)H]triamcinolone acetonide binding in hen oviduct cytosol revealed a K(d) value of 6.4nm. The steroid-receptor complex sedimented as a 7-8S and a 4S entity on low-salt (0.01m-KCl)- and high-salt (0.3m-KCl)-containing sucrose gradients respectively. The cytosol [(3)H]triamcinolone acetonide-receptor complex showed no affinity for ATP-Sepharose or DNA-cellulose, but acquired this ability on heat activation (23 degrees C, 40min). The data indicate the avian oviduct possesses a high-affinity binding molecule that fulfils the criteria of a glucocorticoid receptor.

SUBMITTER: Moudgil VK

PROVIDER: S-EPMC1163214 | biostudies-other | 1981 Jul

REPOSITORIES: biostudies-other

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Project description:Partially purified hen oviduct oestrogen receptors, charged with [3H]oestradiol, were shown to specifically bind in vitro to purified hen oviduct chromatin. Maximal binding occurred within 60min at 0 degrees C in a Tris buffer containing 0.1 M-KCl and 0.5 mM-phenylmethanesulphonyl fluoride. The binding of the [3H]oestradiol-receptor complexes to intact purified chromatin was saturable, whereas the receptor binding to hen DNA remained linear. Saturation was further demonstrated by the minimal acceptor binding of receptor charged with [3H]oestradiol plus 200-fold oestradiol compared with [3H]oestradiol receptors at equal [3H]oestradiol concentrations. Scatchard analysis of [3H]oestradiol-receptor binding to chromatin above DNA levels gave indications of high-affinity binding with a low capacity. Further, the nuclear binding was tissue-specific since the binding to hen spleen chromatin was negligible. To further uncover the specific acceptor sites, proteins were removed from hen oviduct chromatin by increasing concentrations of guanidine hydrochloride (1-7M). Those residual fractions extracted with 3-7 M-guanidine hydrochloride had the highest acceptor activity (above DNA levels) with the peak activity uncovered by 5 M-guanidine hydrochloride. To further characterize the oestrogen-receptor acceptor sites, oviduct chromatin was bound to hydroxyapatite in the presence of 3 M-NaCl and then protein fractions were extracted sequentially with 1-7 M-guanidine hydrochloride. Each fraction was then reconstituted to pure hen DNA by reverse gradient dialysis. [3H]Oestradiol receptors were found to bind to the greatest degree to the fraction reconstituted from the 5 M-guanidine hydrochloride protein extract. Reconstituted nucleoacidic proteins (NAP) from combined 4-7 M-guanidine hydrochloride protein extracts showed saturable binding by [3H]-oestradiol receptors, whereas binding to hen DNA did not saturate. The high affinity, low capacity, and specificity of binding of oestrogen receptors to NAP was similar to that found in intact chromatin. Thus, chromatin acceptor proteins for the oestrogen receptor have been partially isolated and characterized in the hen oviduct and display properties similar to that reported for the acceptor proteins of the progesterone receptor.

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Glucocorticoid binding in the hen oviduct.

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