Purification to homogeneity of the heteromeric DNA-binding form of the aryl hydrocarbon receptor from rat liver.
The aryl hydrocarbon receptor (AhR) is a transcriptional enhancer that is activated by the binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related toxic xenobiotics, as well as some naturally occurring compounds. Ligand binding initiates 1) dissociation of the ligand-bound monomeric AhR from the ligand-unoccupied multimeric complex and 2) biochemical and/or conformational changes that enable association of the ligand-bound monomer with other proteins. This heteromeric complex has high affinity for specific elements [dioxin-responsive elements (DREs)] in the regulatory regions of a number of structural genes, the induction and/or repression of which may be a mechanism of toxicity of TCDD. We have developed a relatively simple and rapid procedure that enables purification to homogeneity of a TCDD-bound receptor complex. The final step of purification is based on binding to an oligonucleotide containing the specific DRE sequence that is found in the upstream region of the CYP1A1 structural gene. The purified complex retains in vitro DRE-binding function. Silver staining and Western blot analyses demonstrate that the complex consists of the AhR ligand-binding monomer of approximately 104 kDa, plus two proteins (94 and 96 kDa) that are recognized by antibodies prepared against the AhR nuclear translocator protein. Previous attempts to purify a DRE-binding form of the AhR were unsuccessful because of dissociation of the complex during chromatography; this is the first report of an isolated functional complex. The purified preparation will be valuable in further studies of receptor regulation and function.
Henry
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Department of Environmental Medicine, University of Rochester School of Medicine and Dentistry, New York 14642.
Rucci
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Gasiewicz
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D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D004268:DNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008099:Liver / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D000072317:Polychlorinated Dibenzodioxins / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D018336:Receptors, Aryl Hydrocarbon / Q000302:isolation & purification* / Q000378:metabolism
D004268:DNA-Binding Proteins; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon