Acceptor sites for the oestrogen receptor in hen oviduct chromatin.
Acceptor sites for the oestrogen receptor in hen oviduct chromatin.
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母鸡输卵管染色质中雌激素受体的受体位点。
DOI：
10.1042/bj2100905
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发表时间：
1983
期刊：
影响因子：
--
通讯作者：
Spelsberg,TC
中科院分区：
文献类型：
--
作者：
Ruh,TS;Spelsberg,TC
体外实验表明，部分纯化的母鸡输卵管雌激素受体能与纯化的母鸡输卵管染色质特异性结合。在含有0.1M-KCl和0.5 mM苯甲烷磺酰氟的Tris缓冲液中，0℃下60min内结合最大。[~H]雌二醇-受体复合体与完整的纯化染色质的结合是饱和的，而受体与母鸡DNA的结合保持线性。在相同的[~3H]雌二醇浓度下，与[~H]雌二醇受体相比，[~3H]雌二醇加200倍的雌二醇与受体的最小受体结合进一步证明了饱和。Scatchard分析表明，在DNA水平以上，雌二醇受体与染色质的结合是高亲和力结合，但结合容量较低。此外，核结合是组织特异性的，因为与母鸡脾染色质的结合可以忽略不计。为了进一步揭示鸡输卵管染色质的特异性受体位点，通过增加盐酸胍浓度(1-7M)从鸡输卵管染色质中去除蛋白质。用3-7M-盐酸胍提取的残余组分的受体活性最高(在DNA水平以上)，而5M-盐酸胍的活性最高。在3M-氯化钠存在下，将输卵管染色质与羟基磷灰石结合，然后用1-7M-盐酸胍依次提取蛋白质组分，以进一步确定雌激素受体的结合部位。每一组分通过反梯度透析法重组为纯净的母鸡DNA。[~3H]雌二醇受体与5M-盐酸胍蛋白提取物的重组组份结合程度最大。重组核酸酸性蛋白(NAP)可与[~3H]-雌二醇受体饱和结合，而与母鸡DNA结合不饱和。雌激素受体与NAP结合的高亲和力、低容量和特异性与完整染色质相似。因此，雌激素受体的染色质受体蛋白已在母鸡输卵管中部分分离和鉴定，并显示出与已报道的孕激素受体受体蛋白相似的性质。
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.