title: Chemical cross-linking of the cytosolic and nuclear forms of the Ah receptor in hepatoma cell line 1c1c7.
abstract: Both cytosolic and high salt nuclear extracts were isolated from Hepa 1c1c7 cells incubated with 2-azido-3[125I]iodo-7,8-dibromo-dibenzo-p-dioxin ([125I]N3Br2DpD). The [125I]N3Br2DpD-labeled cytosolic fraction was subjected to chemical cross-linking with dimethyl pimelimidate and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Chemical cross-linking of the cytosolic form of the AhR revealed monomeric (97 kDa), dimeric (185 kDa), trimeric (281 kDa), and tetrameric (327 kDa) complexes. In a time course of exposure to the cross-linking reagent, the largest form given above became the predominant AhR form observed in the cytosolic extracts. The 327 kDa cytosolic species apparently consists of a 97 kDa AhR, an approximately 88 kDa protein, an approximately 96 kDa protein, and an approximately 46 kDa protein. Nuclear extracts from [125I]N3Br2DpD-labeled Hepa 1c1c7 cells were applied to sucrose density gradients. The 6 S nuclear receptor peak fractions were pooled and subjected to chemical cross-linking. Analysis by SDS-PAGE revealed a monomeric (97 kDa) ligand binding protein and a dimeric (182 kDa) complex. This would suggest that the nuclear 6 S AhR consists of a 97 kDa AhR and an approximately 85 kDa protein. These findings would indicate that the AhR exists in cytosol as a tetrameric species, while in the nucleus the AhR exists as a heterodimer.
authors.affiliation: Department of Foods and Nutrition, Purdue University, West Lafayette, Indiana 47907.
authors.forename: G H
authors.initials: GH
authors.lastname: Perdew
mesh_terms: D006528:Carcinoma, Hepatocellular; D002458:Cell Fractionation; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D003432:Cross-Linking Reagents; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008113:Liver Neoplasms; D046911:Macromolecular Substances; D008970:Molecular Weight; D000072317:Polychlorinated Dibenzodioxins / Q000378:metabolism; D011090:Polyenes / Q000494:pharmacology; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D000072317:Polychlorinated Dibenzodioxins; D011090:Polyenes; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug; C100114:hamycin