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A Poland and E Glover
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Two allelic forms of the Ah receptor have been previously identified by covalent labeling of the hepatic cytosol fractions of inbred strains of mice with the photoaffinity ligand 2-azido-3-[125I] iodo-7,8- dibromodibenzo-p-dioxin and resolution of the labeled protein by denaturing gel electrophoresis: 1) a Mr 95,000 protein encoded by the Ahb-1 allele carried by the C57 and C58 family of mice, and 2) a Mr 104,000 protein encoded by the Ahb-2 allele present in other common inbred strains that are responsive to aromatic hydrocarbons (e.g., C3H/He, BALB/cBy, and A). In this report, 125I-photoaffinity labeling is used to characterize two further murine variants and the strains that carry them: 1) the low affinity Ah receptor (Ahd allele) in strains that are nonresponsive to aromatic hydrocarbons and 2) a newly identified, high affinity variant (Ahb-3) found in several strains recently derived from feral mice. The low affinity Ah receptor has been recently characterized by reversible ligand binding by Okey et al. [Mol. Pharmacol. 35:823-830 (1989)], through the inclusion of sodium molybdate in the buffers during tissue preparation and ligand incubation to stabilize the receptor. Examination of the Ah receptor in hepatic cytosol from 18 strains of mice carrying the Ahd allele, by preparation in molybdate and photoaffinity labeling, revealed that all strains express a Mr 104,000 protein. Tissue preparation in 20 mM sodium molybdate and subsequent dilution of the molybdate to approximately 0.5 mM during ligand incubation was found to enhance photoaffinity labeling of the high and low affinity allelic forms of the Ah receptor. A new variant of the receptor (Ahb-3) expressing a Mr 105,000 protein was detected in Mus molossinus, hortulanus, pahari, spretus, and caroli but was absent from the strains of Mus musculus or domesticus that were examined. Allelic variants were also distinguishable by thermolability, i.e., the half-life of specific ligand binding capacity upon incubation at 35 degrees. For the Ahb-1 allele (Mr 95,000) the t 1/2 (thermostability) is 20-30 min, for the Ahb-2 allele (Mr 104,000) the t 1/2 is 3-6 min, and for the Ahb-3 allele (Mr 105,000) the thermolability is intermediate.
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