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Nuclear binding of progesterone in hen oviduct. Role of acidic chromatin proteins in high-affinity binding.

ABSTRACT: The multiple classes of binding sites for the progesterone-receptor complex in hen oviduct muclei were found to be of chromatin origin. The highest-affinity, and presumably most physiologically important class, is localized in oviduct chromatin and contains approx. 6000-10000 sites per nucleus. None of these sites is detected in spleen chromatin. Two new techniques were used for assaying rapidly the binding of steroid-receptor complexes to soluble deoxyribonucleoproteins in vito. The extent of high-affinity binding by the nucleo-acidic protein fraction from spleen chromatin is as great as that by the nucleo-acidic protein from oviduct chromatin. Consequently the tissue-specific nuclear binding of the progesterone receptor is found not to be a consequence of the absence of the nuclear binding sites (acceptors) from chromatin of non-target tissue (spleen), but rather a result of complete masking of these sites. In the target-tissue (oviduct) chromatin, approx. 70% of the high-affinity acceptor sites are also masked. Acidic proteins, and not histones, appear to be responsible for the masking of these acceptor sites. In addition, acidic proteins represent (or at least are an essential part of) these high-affinity sites in the oviduct nucleus. Pure DNA displays a few high-and many low-affinity binding sites. In support of previous work with immature chicks, the acidic protein fraction of the nucleo-acidic results thus support the hypotheis that protein complexed with DNA, and not DNA alone, represent the high-affinity binding sites for the steroid-receptor complexes in nuclear chromatin. The lower-affinity classes of binding sites may represent DNA and/or other nuclear components.

SUBMITTER: Webster RA

PROVIDER: S-EPMC1163762 | biostudies-other | 1976 May

REPOSITORIES: biostudies-other

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Project description:[(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.

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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Nuclear binding of progesterone in hen oviduct. Role of acidic chromatin proteins in high-affinity binding.

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