DNA binding of the transformed guinea pig hepatic Ah receptor complex: identification and partial characterization of two high-affinity DNA-binding forms.
We have examined and characterized the binding of transformed guinea pig hepatic Ah receptor to its specific DNA recognition site, the dioxin-responsive element (DRE), using gel retardation analysis. Saturation binding analysis of transformed TCDD:AhR complexes were indicative of a single high-affinity binding site (Kd = 2.5 +/- 0.8 nM); however, DNA-binding analysis revealed the presence of two distinct TCDD-inducible protein-DRE complexes. Sucrose gradient centrifugation and subsequent gel retardation analysis of the fractions demonstrated a similarity in the distribution of [3H]TCDD-specific binding and TCDD-inducible protein-DNA complex formation, supporting the presence of the AhR in both complexes. In addition, the formation of both DNA-binding complexes exhibited the same nucleotide specificity previously determined for the AhR complex. Since labeling studies using a radio-iodinated photoaffinity dioxin agonist demonstrated that guinea pig cytosol contains a single ligand binding subunit of 105 kDa, the difference in migration of the complexes is due to other proteins associated with each complex. Overall, our results demonstrate the presence of two distinct high affinity DNA-binding forms of transformed guinea pig AhR complex which exhibit similar DNA-binding affinity and nucleotide specificity.
Bank
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PA
Department of Biochemistry, Michigan State University, East Lansing 48823.
Yao
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Swanson
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HI
Tullis
K
K
Denison
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MS
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D003600:Cytosol / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006168:Guinea Pigs; D008099:Liver / Q000737:chemistry*; D008297:Male; D008969:Molecular Sequence Data; D000072317:Polychlorinated Dibenzodioxins / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D018336:Receptors, Aryl Hydrocarbon / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; D004247:DNA