Nuclear Ah receptor from mouse hepatoma cells: effect of partial proteolysis on relative molecular mass and DNA-binding properties.
The nuclear Ah receptor from mouse hepatoma (Hepa-1c1c9) cells is a 176-kDa multimeric protein which is stable under conditions of up to 1 M KCl. Under denaturing conditions, the Hepa-1 nuclear receptor can be dissociated into a ligand-binding subunit of Mr approximately 91,000. The identity of subunits that compose the nuclear Ah receptor is currently unknown. We used partial proteolysis under nondenaturing conditions as an approach to study the domain organization of the nuclear form of Ah receptor from Hepa-1c1c9 cells treated with [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in culture. Low concentrations of trypsin (0.5 microgram/mg nuclear protein) generated heterogeneous fragments with the main fragment having a Stokes radius (Rs) approximately 6 nm. More discrete ligand-binding fragments of Mr approximately 84,000 (Rs approximately 4 nm/approximately 5 S) and Mr approximately 16,000 (Rs approximately 2 nm/approximately 2 S) could be generated using higher concentrations of trypsin (5 micrograms/mg nuclear protein). The relative concentration of the 84 and 16-kDa fragment was dependent on duration of protease treatment; formation of the 16-kDa fragment was accompanied by some loss in [3H]TCDD binding. Treatment of nuclear Ah receptor with alpha-chymotrypsin (1 microgram/mg nuclear protein) generated a single, apparently homogeneous ligand-binding fragment of Mr approximately 101,000 (Rs approximately 5 nm/approximately 5 S). When analyzed by DNA-cellulose chromatography, the chymotryptic fragment eluted at a significantly higher KCl concentration (462 mM) compared to native untreated nuclear Ah receptor (385 mM). Despite this increased affinity for DNA-cellulose columns, the ligand-binding fragment generated by chymotrypsin treatment was unable to interact with a dioxin responsive element in a gel retardation assay. DNA-cellulose binding ability, therefore, does not appear to be a reliable indicator of specific DNA interactions for these protease-modified fragments.
Prokipcak
R D
RD
Department of Pediatrics, Hospital for Sick Children, Toronto, Ontario, Canada.
Denison
M S
MS
Okey
A B
AB
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D002482:Cellulose / Q000031:analogs & derivatives; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002918:Chymotrypsin / Q000378:metabolism / Q000494:pharmacology*; D004247:DNA; D004268:DNA-Binding Proteins / Q000378:metabolism*; D008114:Liver Neoplasms, Experimental / Q000378:metabolism*; D046911:Macromolecular Substances; D051379:Mice; D008970:Molecular Weight; D000072317:Polychlorinated Dibenzodioxins / Q000378:metabolism; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug / Q000302:isolation & purification / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism / Q000494:pharmacology*
D004268:DNA-Binding Proteins; C033190:DNA-cellulose; D046911:Macromolecular Substances; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug; D002482:Cellulose; D004247:DNA; D002918:Chymotrypsin; D014357:Trypsin