Gray W G, Gorski J
Department of Biochemistry, University of Wisconsin-Madison 53706-1569, USA.
Biochemistry. 1996 Sep 10;35(36):11685-92. doi: 10.1021/bi960068k.
Cytosolic proteins from uteri of 19-day-old rats were analyzed by an electrophoresis mobility shift assay (EMSA) using a 31 base pair DNA probe containing an estrogen-responsive element (ERE) from the vitellogenin A2 gene. EMSA identified three distinct cytosolic protein-DNA complexes that are separable by Q-Sepharose anion exchange chromatography into an estrogen receptor (ER)-containing fraction (150 mM NaCl eluate) and a non-ER-containing fraction (250 mM NaCl eluate). We thus refer to the non-ER fraction as the ERE binding protein (ERE-BP). The ERE-BP-containing fraction was repressed to 40-50% of its normal levels following a single injection of estradiol. In addition, ERE-BP levels were repressed to the same extent (greater than 50%) by day 20 of the rat's gestational period. Examination of the expression pattern of ERE-BP shows that this activity is differentially expressed in both estrogen-responsive and nonresponsive tissues, with the highest levels of expression occurring in the pituitary. We next examined the specificity of ERE-BP binding by competition analysis using DNA sequences corresponding to binding sites of several known transcription factors. ERE-BP was found to be specific for both the ER binding site (ERE) and TATA binding protein binding sites. Furthermore, saturation analysis demonstrated that ERE-BP binds to the ERE and TATA binding protein sequences with an apparent Kd of 1.2 and 0.12 nM, respectively. Partial purification of ERE-BP using three chromatography steps (Q-Sepharose, hydroxyapatite, and Sephacryl S300) followed by sodium dodecyl sulfate analysis indicated the presence of three major protein bands (p102, p81, and p48) as judged by Coomassie staining. UV cross-linking of the ERE-BP/DNA complex followed by sodium dodecyl sulfate analysis-polyacrylamide gel electrophoresis analysis indicates that the 48 kDa band seen in the final, partially purified fraction correlates with the ERE-BP activity. Thus, this study has identified a unique uterine cytosolic protein that binds to the ER binding site and may influence ER binding.
使用来自卵黄蛋白原A2基因的包含雌激素反应元件（ERE）的31个碱基对的DNA探针，通过电泳迁移率变动分析（EMSA）对19日龄大鼠子宫的胞质蛋白进行分析。EMSA鉴定出三种不同的胞质蛋白-DNA复合物，它们可通过Q-琼脂糖阴离子交换色谱法分离为含雌激素受体（ER）的部分（150 mM NaCl洗脱液）和不含ER的部分（250 mM NaCl洗脱液）。因此，我们将不含ER的部分称为ERE结合蛋白（ERE-BP）。单次注射雌二醇后，含ERE-BP的部分被抑制至其正常水平的40-50%。此外，在大鼠妊娠期第20天时，ERE-BP水平也被抑制至相同程度（超过50%）。对ERE-BP表达模式的检查表明，这种活性在雌激素反应性和非反应性组织中均有差异表达，在垂体中表达水平最高。接下来，我们通过使用与几种已知转录因子结合位点相对应的DNA序列进行竞争分析，研究了ERE-BP结合的特异性。发现ERE-BP对ER结合位点（ERE）和TATA结合蛋白结合位点均具有特异性。此外，饱和分析表明，ERE-BP与ERE和TATA结合蛋白序列的结合表观解离常数（Kd）分别为1.2和0.12 nM。使用三个色谱步骤（Q-琼脂糖、羟基磷灰石和Sephacryl S300）对ERE-BP进行部分纯化，随后进行十二烷基硫酸钠分析，考马斯亮蓝染色显示存在三条主要蛋白带（p102、p81和p48）。对ERE-BP/DNA复合物进行紫外交联，随后进行十二烷基硫酸钠分析-聚丙烯酰胺凝胶电泳分析表明，在最终的部分纯化级分中看到的48 kDa条带与ERE-BP活性相关。因此，本研究鉴定出一种独特的子宫胞质蛋白，它与ER结合位点结合并可能影响ER结合。
