Ca2+-dependent proteolysis of the Ah receptor

Arch Biochem Biophys. 1988 Feb 15;261(1):103-11. doi: 10.1016/0003-9861(88)90109-9.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Affinity Labels / metabolism
  • Animals
  • Calcium / pharmacology*
  • Cell Line
  • Cricetinae
  • Cytosol / metabolism
  • Dioxins / metabolism
  • Hydrolysis
  • Liver / metabolism*
  • Mesocricetus
  • Mice
  • Mice, Inbred C57BL
  • Peptide Hydrolases / metabolism
  • Protease Inhibitors / pharmacology
  • Rats
  • Rats, Inbred Strains
  • Receptors, Aryl Hydrocarbon
  • Receptors, Drug / metabolism*


  • Affinity Labels
  • Dioxins
  • Protease Inhibitors
  • Receptors, Aryl Hydrocarbon
  • Receptors, Drug
  • 2-azido-3-iodo-7,8-dibromodibenzo-1,4-dioxin
  • Peptide Hydrolases
  • Calcium