Bank P A, Yao E F, Swanson H I, Tullis K, Denison M S
Department of Biochemistry, Michigan State University, East Lansing 48823.
Arch Biochem Biophys. 1995 Mar 10;317(2):439-48. doi: 10.1006/abbi.1995.1186.
We have examined and characterized the binding of transformed guinea pig hepatic Ah receptor to its specific DNA recognition site, the dioxin-responsive element (DRE), using gel retardation analysis. Saturation binding analysis of transformed TCDD:AhR complexes were indicative of a single high-affinity binding site (Kd = 2.5 +/- 0.8 nM); however, DNA-binding analysis revealed the presence of two distinct TCDD-inducible protein-DRE complexes. Sucrose gradient centrifugation and subsequent gel retardation analysis of the fractions demonstrated a similarity in the distribution of [3H]TCDD-specific binding and TCDD-inducible protein-DNA complex formation, supporting the presence of the AhR in both complexes. In addition, the formation of both DNA-binding complexes exhibited the same nucleotide specificity previously determined for the AhR complex. Since labeling studies using a radio-iodinated photoaffinity dioxin agonist demonstrated that guinea pig cytosol contains a single ligand binding subunit of 105 kDa, the difference in migration of the complexes is due to other proteins associated with each complex. Overall, our results demonstrate the presence of two distinct high affinity DNA-binding forms of transformed guinea pig AhR complex which exhibit similar DNA-binding affinity and nucleotide specificity.
我们已运用凝胶阻滞分析技术，对转化的豚鼠肝脏芳烃受体（Ah受体）与其特定DNA识别位点——二噁英反应元件（DRE）的结合情况进行了检测和表征。对转化的2,3,7,8-四氯二苯并-对-二噁英（TCDD）:AhR复合物的饱和结合分析表明存在单一的高亲和力结合位点（解离常数Kd = 2.5 ± 0.8 nM）；然而，DNA结合分析揭示了存在两种不同的TCDD诱导型蛋白质-DRE复合物。蔗糖梯度离心及随后对各组分的凝胶阻滞分析表明，[³H]TCDD特异性结合的分布与TCDD诱导型蛋白质-DNA复合物形成的分布相似，这支持了两种复合物中均存在Ah受体。此外，两种DNA结合复合物的形成均表现出先前确定的AhR复合物相同的核苷酸特异性。由于使用放射性碘化光亲和二噁英激动剂的标记研究表明豚鼠细胞溶质含有一个105 kDa的单一配体结合亚基，复合物迁移的差异是由于与每个复合物相关的其他蛋白质所致。总体而言，我们的结果表明存在两种不同的转化豚鼠AhR复合物的高亲和力DNA结合形式，它们表现出相似的DNA结合亲和力和核苷酸特异性。
