Perdew G H, Hollenback C E
Department of Foods and Nutrition, Purdue University, West Lafayette, IN 47907, USA.
J Biochem Toxicol. 1995 Apr;10(2):95-102. doi: 10.1002/jbt.2570100206.
The Ah receptor (AhR) was visualized using monoclonal antibody Rpt 1 on protein blots of HeLa cell cytosol; two bands were detected at 104 and 106 kDa. The photoaffinity ligand, 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin, was added to HeLa cells in culture, and after 1 hour the cells were UV irradiated. Cytosolic and high salt nuclear preparations were isolated and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer of the protein to membrane. The AhR was visualized on the membrane, revealing two bands. Alignment of an autoradiogram with the membrane revealed that only the 106 kDa (upper) band was photoaffinity labeled. The nuclear fraction contained only the photoaffinity-labeled 106 kDa form of the AhR. The 104 kDa AhR does not appear to be a proteolytic product of the 106 kDa form. Cyanogen bromide fragmentation revealed that both forms contain the same size N-terminal fragment. Sucrose density gradient analysis of HeLa cell cytosol indicated that both forms cosedimented at 9 S. Both the 106 and 104 kDa AhR bands were detected in four different human cell lines. Together, these results would indicate that the AhR in human cell lines exists in two distinct forms.
利用单克隆抗体Rpt 1在HeLa细胞胞质溶胶的蛋白质印迹上对芳烃受体（AhR）进行可视化；在104 kDa和106 kDa处检测到两条条带。将光亲和配体2-叠氮基-3-[125I]碘-7,8-二溴二苯并对二恶英添加到培养的HeLa细胞中，1小时后对细胞进行紫外线照射。分离胞质溶胶和高盐核提取物，进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE），随后将蛋白质转移到膜上。在膜上对AhR进行可视化，显示出两条条带。将放射自显影片与膜对齐显示，只有106 kDa（上部）条带被光亲和标记。核级分仅含有光亲和标记的106 kDa形式的AhR。104 kDa的AhR似乎不是106 kDa形式的蛋白水解产物。溴化氰片段化显示两种形式都含有相同大小的N端片段。对HeLa细胞胞质溶胶进行蔗糖密度梯度分析表明，两种形式都在9 S处共同沉降。在四种不同的人类细胞系中都检测到了106 kDa和104 kDa的AhR条带。总之，这些结果表明人类细胞系中的AhR以两种不同的形式存在。
