Detection of the Ah receptor in rainbow trout: Use of 2-azido-3-[¹²⁵I]iodo-7,8-dibromodibenzo-P-dioxmin in cell culture

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 dodecyi 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 ∼9.8S and the high salt nuclear extract form at -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 agonist affinity as that previously demonstrated for the mouse Ah receptor.