Henry E C, Rucci G, Gasiewicz T A
Department of Environmental Medicine, University of Rochester School of Medicine and Dentistry, New York 14642.
Mol Pharmacol. 1994 Dec;46(6):1022-7.
The aryl hydrocarbon receptor (AhR) is a transcriptional enhancer that is activated by the binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related toxic xenobiotics, as well as some naturally occurring compounds. Ligand binding initiates 1) dissociation of the ligand-bound monomeric AhR from the ligand-unoccupied multimeric complex and 2) biochemical and/or conformational changes that enable association of the ligand-bound monomer with other proteins. This heteromeric complex has high affinity for specific elements [dioxin-responsive elements (DREs)] in the regulatory regions of a number of structural genes, the induction and/or repression of which may be a mechanism of toxicity of TCDD. We have developed a relatively simple and rapid procedure that enables purification to homogeneity of a TCDD-bound receptor complex. The final step of purification is based on binding to an oligonucleotide containing the specific DRE sequence that is found in the upstream region of the CYP1A1 structural gene. The purified complex retains in vitro DRE-binding function. Silver staining and Western blot analyses demonstrate that the complex consists of the AhR ligand-binding monomer of approximately 104 kDa, plus two proteins (94 and 96 kDa) that are recognized by antibodies prepared against the AhR nuclear translocator protein. Previous attempts to purify a DRE-binding form of the AhR were unsuccessful because of dissociation of the complex during chromatography; this is the first report of an isolated functional complex. The purified preparation will be valuable in further studies of receptor regulation and function.
芳烃受体（AhR）是一种转录增强子，可被2,3,7,8-四氯二苯并对二恶英（TCDD）及相关有毒外源性物质以及一些天然存在的化合物激活。配体结合引发：1）配体结合的单体AhR从配体未占据的多聚体复合物中解离；2）生化和/或构象变化，使配体结合的单体与其他蛋白质缔合。这种异源复合物对许多结构基因调控区域中的特定元件[二恶英反应元件（DREs）]具有高亲和力，其诱导和/或抑制可能是TCDD毒性的一种机制。我们开发了一种相对简单快速的方法，能够将与TCDD结合的受体复合物纯化至同质。纯化的最后一步基于与包含在CYP1A1结构基因上游区域发现的特定DRE序列的寡核苷酸结合。纯化的复合物保留体外DRE结合功能。银染和蛋白质印迹分析表明，该复合物由约104 kDa的AhR配体结合单体以及两种可被针对AhR核转运蛋白制备的抗体识别的蛋白质（94和96 kDa）组成。以前纯化AhR的DRE结合形式的尝试均未成功，因为复合物在色谱过程中解离；这是分离出的功能性复合物的首次报道。纯化的制剂在受体调节和功能的进一步研究中将具有重要价值。
