Photoaffinity labeling of the Ah receptor.
A series of halodibenzo-p-dioxins with the photolabile aryl azide functional group were synthesized and screened as potential photoaffinity labels for the Ah receptor, and 2-azido-3-iodo-7,8-dibromodibenzo-p-dioxin was selected for radiosynthesis with 125I (specific activity 2176 Ci/mmol, equilibrium dissociation constant, KD = 0.76 nM). Following incubation of this 125I-labeled photoaffinity ligand with the protamine sulfate-precipitated fraction of C57BL/6J mouse liver cytosol, and irradiation with long wavelength ultraviolet light, the radiolabeled macromolecules were precipitated with acetone and analyzed by denaturing gel electrophoresis and autoradiography. Among the labeled products, two peptides with apparent molecular masses of 95,000 and 70,000 daltons had the following properties: 1) they were selectively labeled at low ligand concentrations; 2) they were labeled in approximately a 1:1 ratio; 3) co-incubation with receptor agonists inhibited the photoaffinity labeling of both peptides to a similar extent, and structure activity relationship for inhibition of labeling by these agonists corresponded to that for their binding affinity to the Ah receptor; 4) upon nondenaturing chromatographic separation of photoaffinity labeled cytosol on high performance liquid chromatography size exclusion and anion exchange columns, the 95- and 70-kDa peptides coelute; 5) the migration of these peptides upon denaturing electrophoresis is the same in the presence or absence of a thiol reducing agent; and 6) proteolysis of the 95- and 70-kDa peptides produces a similar pattern of cleavage peptides. The simplest structure of the Ah receptor in mouse liver cytosol, appears to be a dimer composed of two noncovalently linked subunits of apparent molecular masses of 95 and 70 kDa, which have homologous structure and similar ligand binding sites, but other possibilities are discussed.
D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D010777:Photochemistry; D010782:Photolysis; D000072317:Polychlorinated Dibenzodioxins / Q000378:metabolism; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug / Q000378:metabolism*; D012697:Serine Endopeptidases; D013056:Spectrophotometry, Ultraviolet
D000345:Affinity Labels; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug; D010450:Endopeptidases; D012697:Serine Endopeptidases; D002918:Chymotrypsin; C028344:glutamyl endopeptidase