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Correlation of Induction of Aryl Hydrocarbon Hydroxylase in Cultured Rat Hepatocytes with Saturable High-Affinity Binding of 3-Methylcholanthrene to a 4S Cytoplasmic Protein¹

Departments of Medical Microbiology [N. H. H., W. I. S.] and Biochemistry [B. T., E. B.], University of Vermont College of Medicine, Burlington, Vermont 05405

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 K_d, 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 cultured cells to 37° after the standard ligand-binding incubation at 4° 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.

¹ This work was supported by Grants CA 12056 and CA 20711 from the National Cancer Institute.

² This work is part of a Ph.D. thesis submitted to the Graduate Faculty of the University of Vermont. Present address: Department of Biochemistry, University of Virginia, School of Medicine, Jordan Medical Education Building, Charlottesville, Va. 22908.

³ To whom requests for reprints should be addressed, at Given Building, Department of Medical Microbiology, University of Vermont College of Medicine, Burlington, Vt. 05405.

Received 6/27/80. Accepted 2/ 3/81.

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