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CA Bradfield, E Glover and A Poland
McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison 53706.
The Ah receptor is a presumed member of the superfamily of steroid/thyroid hormone receptors, a trace soluble protein present in a wide variety of vertebrate species that mediates the biological effects of halogenated aromatic hydrocarbons. In this paper, we report the purification to homogeneity of this protein (from the liver of C57BL/6J mice) and its N-terminal amino acid sequence. Selective covalent labeling of the Ah receptor in hepatic cytosol with the photoaffinity ligands 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin simplified identification and quantitation of the receptor and permitted purification under denaturing conditions. Photoaffinity-labeled hepatic cytosol was applied to a phosphocellulose column at 80 mM NaCl, and the fraction enriched with the Ah receptor eluted with 225 mM NaCl. The eluate was diluted to 150 mM NaCl and applied to a DEAE-cellulose column, and the enriched fraction eluted with 300 mM. These two ion exchange chromatography steps usually gave approximately 100-fold enrichment and 40-50% recovery of Ah receptor. The dilute protein in the eluate was precipitated with n-propanol/trichloroacetic acid and solubilized in formic acid. The sample was then subjected to three successive rounds of high performance liquid chromatography on C4 reverse phase columns. The final, shallow-gradient chromatography was able to resolve the unlabeled 95-kDa receptor protein from the later eluting 125I-photoaffinity-labeled protein. The pooled high performance liquid chromatography fractions subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels contained only the 95-kDa band upon staining with Coomassie blue R250 or silver. Using the above protocol, the Ah receptor was purified greater than 150,000-fold, to apparent homogeneity, with an overall yield of 3-5%. The N-terminal amino acid sequence of the purified peptide was determined to be ala/asp-ser-Arg- Lys-arg-Lys-Pro-Val-Gln-Lys-Thr-Val-Lys-Pro-Ile-Pro-Ala- Glu-Gly--Ile- Lys-ser-Asn-Pro-ser-Lys- (where the lowercase indicates a residue determined with less confidence).
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