Swanson H I, Perdew G H
Department of Foods and Nutrition, Purdue University, West Lafayette, IN 47906.
Toxicol Lett. 1991 Sep;58(1):85-95. doi: 10.1016/0378-4274(91)90194-b.
The Ah receptor was detected in RTG-2 cells (rainbow trout embryonic gonad cells) following the addition of the photoaffinity ligand, [125I]2-azido-3-iodo-7,8-dibromodibenzo-p-dioxin, to cells in culture. Cytosolic and nuclear extracts were prepared and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and revealed one radiolabeled band. Very little non-specific binding was observed under the conditions employed when compared to photoaffinity labeling RTG-2 cytosolic extracts in vitro. The photoaffinity-labeled Ah receptor in RTG-2 cytosol was analyzed by sucrose density centrifugation. The cytosolic form was observed to sediment at approximately 9.8S and the high salt nuclear extract form at approximately 7.5S. The relative molecular weight of the Ah receptor was determined to be 145 kDa under denaturing conditions and is considerably larger than the Ah receptor from mammalian sources. Inhibition of photoaffinity ligand binding to the RTG-2 cytosolic Ah receptor by competing ligands revealed the same rank order of ligand affinity as that previously demonstrated for the mouse Ah receptor.
在培养的细胞中加入光亲和配体[125I]2-叠氮基-3-碘-7,8-二溴二苯并对二恶英后，在虹鳟胚胎性腺细胞（RTG-2细胞）中检测到芳烃受体（Ah受体）。制备细胞溶质和核提取物，并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳进行分析，结果显示出一条放射性标记带。与体外光亲和标记RTG-2细胞溶质提取物相比，在所采用的条件下观察到的非特异性结合极少。通过蔗糖密度离心法分析RTG-2细胞溶质中的光亲和标记Ah受体。观察到细胞溶质形式在约9.8S处沉降，高盐核提取物形式在约7.5S处沉降。在变性条件下，Ah受体的相对分子质量测定为145 kDa，比来自哺乳动物来源的Ah受体大得多。通过竞争配体抑制光亲和配体与RTG-2细胞溶质Ah受体的结合，揭示出与先前在小鼠Ah受体中证明的相同的配体亲和力等级顺序。
