Physicochemical characterization of the nuclear form of Ah receptor from mouse hepatoma cells exposed in culture to 2,3,7,8-tetrachlorodibenzo- p-dioxin

Abstract

Molecular properties of nuclear aromatic hydrocarbon ( Ah) receptor from Hepa-1c1c9 (Hepa-1) cells were assessed by velocity sedimentation on sucrose gradients and by gel permeation chromatography on Sephacryl S-300. Nuclear Ah receptor was obtained by exposing intact cells to [ 3H]-2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) for 1 h at 37 °C in culture followed by extraction of receptor from nuclei with buffers containing 0.5 m KCl. The nuclear Ah receptor was compared to the cytosolic Ah receptor from the same cells. Under conditions of low ionic strength, the Ah receptor from Hepa-1 cytosol sedimented as a single 9.4 ± 0.63 S binding peak that had a Stokes radius of 7.1 ± 0.12 nm and an apparent relative molecular mass of 271,000 ± 16,000. After prolonged (24 h) exposure to high ionic strength (0.5 m KCl), cytosol labeled with [ 3H]TCDD exhibited two specific binding peaks. The large form of cytosolic Ah receptor seen under high ionic strength conditions sedimented at 9.4 ± 0.46 S, had a Stokes radius of 6.9 ± 0.19 nm, and an apparent M r 267,000 ± 15,000. The smaller ligand-binding subunit generated by exposing cytosol to 0.5 m KCl sedimented at 4.9 ± 0.62 S, had a Stokes radius of 5.0 ± 0.14 nm, and an apparent M r 104,000 ± 12,000. Nuclear Ah receptor, analyzed under high ionic strength conditions, sedimented at 6.2 ± 0.20 S, had a Stokes radius of 6.8 ± 0.19 nm, and an apparent M r 176,000 ± 7000. Nuclear Ah receptor from rat H4IIE hepatoma cells was analyzed and found to have physicochemical characteristics identical to those of nuclear Ah receptor from the mouse Hepa-1 cells. The molecular mass of Hepa-1 nuclear Ah receptor was found to be statistically different from both the M r ≈ 267,000 cytosolic Ah receptor and the M r ≈ 104,000 subunit which were present in cytosol under high ionic strength conditions. Hepa-1 nuclear Ah receptor could not be converted to a smaller ligand-binding subunit by treatment with alkaline phosphatase, ribonuclease, or sulfhydryl-modifying reagents or prolonged exposure to 1.0 m KCl. Cytosolic Ah receptor from Hepa-1 cells was “transformed” by heating at 25 °C in vitro into a form with high affinity for DNA-cellulose. The transformed cytosolic Ah receptor, when analyzed under conditions of high ionic strength, sedimented at ≈ 6 S, had a Stokes radius of ≈ 6.7 nm, and an apparent M r ≈ 167,000. Thus, upon transformation to a nuclear binding form in cells in culture or upon transformation of cytosolic receptor to a form that has high affinity for DNA-cellulose in vitro, there appears to be a significant change in the relative molecular mass of the Ah receptor; the transformed receptor does not correspond in size to the M r ≈ 104,000 ligand-binding subunit which exists in cytosol under conditions of high ionic strength.