Ruh M F, Singh R K, Mak P, Callard G V
Endocrinology. 1986 Feb;118(2):811-8. doi: 10.1210/endo-118-2-811.
The testicular estrogen receptor of the shark Squalus acanthias is restricted to nuclear subfractions when tissue is homogenized in low salt buffers and adheres tightly to nuclei and DNA-cellulose even when exposed to high salt conditions. Therefore, we examined the binding characteristics of this receptor to chromatin subfractions from homologous and heterologous tissues. Squalus chromatin linked to cellulose and partially deproteinized by 0-8 M guanidine hydrochloride (GuHCl) gave extraction patterns similar to those obtained with mammalian and avian chromatin. Chromatin as prepared in our laboratory contained no bound estrogen receptor. The binding pattern of the [3H]estradiol-labeled nuclear estrogen receptor to chromatin fractions from Squalus testicular zones I/II (containing spermatogonia, spermatocytes, and high receptor levels) revealed maximal binding activity (acceptor sites) on chromatin previously extracted with 2-4 M GuHCl (350% increase over unextracted chromatin), with a 40% decrease from maximal binding at 5-8 M GuHCl. By contrast, binding to chromatin from zone III (containing spermatozoa and little or no detectable receptor) showed no major peak and 4 times less binding at 3 M GuHCl-extracted chromatin. We have previously shown that zones I and II contain the majority of testicular receptors and, presumably, are the primary sites of estrogen action, whereas receptor activity in zone III is minimal (less than 5%), indicating a secondary or nontarget tissue. Squalus testicular [3H]estradiol-receptor complexes bound minimally to rabbit uterine chromatin. Likewise, [3H]estradiol-receptor complexes from rabbit uterus, Squalus oviduct, or mouse testis bound minimally to Squalus testicular chromatin. Thus, maximal binding occurred only with Squalus zones I/II chromatin and Squalus testicular receptor. The binding of [3H]estradiol-receptor complexes to testicular chromatin (zones I/II) was of high affinity (Kd = 1.9 X 10(-10) M) and low capacity and was optimal in the presence of 150 mM KCl, but was unaffected by the addition of urea in an amount similar to that of Squalus body fluids (300 mM). [3H]Estradiol binding to chromatin required undenatured receptor and was competitively inhibited by radioinert estradiol-receptor complexes, confirming saturability of acceptor sites. The affinity of the Squalus estrogen receptor for homologous chromatin was in the same range as that reported for other systems, despite the unusual nuclear extractability characteristics of Squalus receptor. This study provides new evidence for tissue and species specificity of receptor binding to chromatin acceptor sites.
当在低盐缓冲液中对组织进行匀浆时，棘鲨（Squalus acanthias）的睾丸雌激素受体局限于核亚组分，并且即使暴露于高盐条件下，它也会紧密附着于细胞核和DNA纤维素上。因此，我们研究了该受体与同源和异源组织的染色质亚组分的结合特性。与纤维素相连并通过0 - 8M盐酸胍（GuHCl）部分脱蛋白的棘鲨染色质给出的提取模式与用哺乳动物和鸟类染色质获得的模式相似。我们实验室制备的染色质不含有结合的雌激素受体。[3H]雌二醇标记的核雌激素受体与棘鲨睾丸I/II区（含有精原细胞、精母细胞且受体水平高）的染色质组分的结合模式显示，在用2 - 4M GuHCl预先提取的染色质上具有最大结合活性（受体位点）（比未提取的染色质增加350%），在5 - 8M GuHCl时结合活性从最大值下降40%。相比之下，与III区（含有精子且几乎没有或没有可检测到的受体）的染色质结合在3M GuHCl提取的染色质上没有主要峰值且结合减少4倍。我们之前已经表明，I区和II区含有大部分睾丸受体，大概是雌激素作用的主要部位，而III区的受体活性最小（小于5%），表明是次要或非靶组织。棘鲨睾丸[3H]雌二醇 - 受体复合物与兔子宫染色质的结合最少。同样，来自兔子宫、棘鲨输卵管或小鼠睾丸的[3H]雌二醇 - 受体复合物与棘鲨睾丸染色质的结合也最少。因此，最大结合仅发生在棘鲨I/II区染色质和棘鲨睾丸受体之间。[3H]雌二醇 - 受体复合物与睾丸染色质（I/II区）的结合具有高亲和力（Kd = 1.9×10(-10)M）和低容量，在150mM KCl存在下最佳，但不受添加与棘鲨体液量相似（300mM）的尿素的影响。[3H]雌二醇与染色质的结合需要未变性的受体，并受到放射性惰性雌二醇 - 受体复合物的竞争性抑制，证实了受体位点的饱和性。尽管棘鲨受体具有不寻常的核可提取特性，但棘鲨雌激素受体对同源染色质的亲和力与其他系统报道的范围相同。这项研究为受体与染色质受体位点结合的组织和物种特异性提供了新证据。
