Ca2+-dependent proteolysis of the Ah receptor.
We previously reported (J. Biol. Chem. (1986) 261, 6352-6465) that the photoaffinity ligand for the Ah receptor, [125I]-2-azido-3-iodo-7,8-dibromodibenzo-p-dioxin, upon incubation with the liver cytosol fraction from C57BL/6 mice, labeled in a 1:1 ratio two peptides that had apparent molecular masses of 95 and 70 kDa and similar proteolytic fragmentation patterns. In the cytosolic fraction of Hepa 1 cells, a cloned murine hepatoma cell line, the product of photoaffinity labeling is almost exclusively a 95-kDa peptide which is rapidly hydrolyzed by a Ca2+-dependent proteinase to a 70-kDa peptide as well as other fragments. Thus, the ligand binding unit of the Ah receptor in C57BL/6 mouse liver and Hepa 1 cell is a 95-kDa peptide, and the 70-kDa fragment is a proteolytic artifact. The Ca2+-dependent proteinase which hydrolyzes the 95-kDa peptide has the properties of calpain II: (i) an absolute requirement for Ca2+, with maximal activity at 0.5 to 1.0 mM Ca2+; (ii) a pH optimum of 7.5 to 8.0; (iii) inhibition by EDTA, iodoacetamide, leupeptin and L-trans-epoxysuccinylleucylamido(4-guanidino)butane, but not by soybean trypsin inhibitor, aprotinin, or phenylmethanesufonyl fluoride. Upon chromatographic separation of the liver cytosol of C57BL/6 mice on DEAE-Sephacel, Ca2+-dependent proteinase activity (using casein or the labeled 95-kDa peptide as substrates) elutes with 0.25 M NaCl, and a specific proteinase inhibitor elutes with 0.15 M NaCl. Ca2+-dependent proteinase activity that hydrolyzes the 95-kDa peptide is found in the liver cytosols of several mammalian species.
Poland
A
A
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Glover
E
E
D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002460:Cell Line; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D004147:Dioxins / Q000378:metabolism; D006868:Hydrolysis; D008099:Liver / Q000378:metabolism*; D008647:Mesocricetus; D051379:Mice; D008810:Mice, Inbred C57BL; D010447:Peptide Hydrolases / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug / Q000378:metabolism*
D000345:Affinity Labels; D004147:Dioxins; D011480:Protease Inhibitors; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug; C054916:2-azido-3-iodo-7,8-dibromodibenzo-1,4-dioxin; D010447:Peptide Hydrolases; D002118:Calcium