Characterization of the molecular and structural properties of the transformed and nuclear aryl hydrocarbon (Ah) receptor complexes by proteolytic digestion.
Ligand-dependent differences in the molecular properties of the transformed cytosolic and nuclear aryl hydrocarbon receptor (AhR) were investigated using the proteolytic clipping band shift assay. AhR complexes were incubated with [32P]dioxin responsive element (DRE) (26-mer) or bromodeoxyuridine (BrdU)-DRE and the resulting protein-DNA or crosslinked protein-DNA complexes were treated with trypsin or V8 protease and analyzed by electrophoresis. The results showed that for several different AhR ligands including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran, 1,2,7,8-tetrachlorodibenzofuran and alpha-naphthoflavone, the pattern of degraded protein-DNA products were similar using transformed cytosolic or nuclear AhR complexes. In contrast, the proteolytic clipping band shift assay showed that there were significant differences in the pattern of degraded protein-DNA products using nuclear AhR complexes derived from mouse Hepa 1c1c7 cells treated with TCDD or 6-methyl-1,3,8-trichlorodibenzofuran (MCDF). The differences detected in this in vitro assay parallel the in vivo and in vitro activities of these compounds in which TCDD is a potent AhR agonist whereas MCDF is a partial AhR agonist and antagonist.
Santostefano
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Texas A&M University, College Station 77843-4466, USA.
Safe
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D000818:Animals; D001345:Autoradiography; D001483:Base Sequence; D001572:Benzofurans / Q000494:pharmacology; D002478:Cells, Cultured; D004147:Dioxins / Q000494:pharmacology; D008024:Ligands; D008114:Liver Neoplasms, Experimental; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D015394:Molecular Structure; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology*; D011487:Protein Conformation; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D018336:Receptors, Aryl Hydrocarbon / Q000737:chemistry*; D014357:Trypsin / Q000378:metabolism
D001572:Benzofurans; D004147:Dioxins; D008024:Ligands; D000072317:Polychlorinated Dibenzodioxins; D011506:Proteins; D018336:Receptors, Aryl Hydrocarbon; C054923:6-methyl-1,3,8-trichlorodibenzofuran; D014357:Trypsin