Correlation of induction of aryl hydrocarbon hydroxylase in cultured rat hepatocytes with saturable high-affinity binding of 3-methylcholanthrene to a 4S cytoplasmic protein.
The binding of 3-methylcholanthrene (3-MC), a potent inducer of aryl hydrocarbon hydroxylase activity, to cytoplasmic proteins of a cloned rat hepatocyte culture, RL-PR-C, was studied by sucrose gradient centrifugation. Time course and dose-binding experiments performed on late-passage aryl hydrocarbon hydroxylase-inducible cultures indicate the presence of a saturable pool of high-affinity (average Kd, 3.6 nm) binding sites in the cytosol of these cells. The number of binding sites varied from 20,000 to 80,000 per late-passage hepatocyte with a total capacity of approximately 2.2 pmol of 3-MC bound per mg of cytosolic protein. The complex sedimented at 4.0 +/- 0.2S regardless of the ionic strength of the homogenization buffer or gradient solutions. It was sensitive to denaturation by sodium dodecyl sulfate and trypsin but not by DNase I, RNase A, or the nonionic detergent Nonidet P-40. The binding of 3-MC to the protein was inhibited by 1,2-benzanthracene, benzo(a)pyrene, 5,6-benzoflavone, and 7,8-benzoflavone but not by a series of steroids, aflatoxin B1, phenobarbital, or Aroclor 1254. Elevating the temperature of cultures cells to 37 degrees after the standard ligand-binding incubation at 4 degrees resulted in a rapid decrease in cytoplasmic saturable binding and a concomitant increase in nuclear- and chromatin-associated ligand. A portion of this nuclear-associated ligand was extractable with 400 mM KCl. Adsorption of the [3H]-3-MC binding complex by nuclei in vitro suggested that the 4S binding protein facilitated the entry of 3-MC into the nucleus. The presence of the 4S binding species correlated with the level of inducibility of aryl hydrocarbon hydroxylase throughout its development in RL-PR-C and therefore may be involved in the process of induction of this enzyme.
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000096:biosynthesis*; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D004790:Enzyme Induction; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008748:Methylcholanthrene / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D013696:Temperature
C027326:3-methylcholanthrene-binding protein, rat; D002352:Carrier Proteins; D008748:Methylcholanthrene; D001189:Aryl Hydrocarbon Hydroxylases