A novel 4 S [3H]beta-naphthoflavone-binding protein in liver cytosol of female Sprague-Dawley rats treated with aryl hydrocarbon receptor agonists.
beta-Naphthoflavone (beta-NF) is a widely used inducer of phase-I and phase-II enzymes controlled by aryl hydrocarbon receptor (AhR). Studies of competitive binding with (3)H-labelled 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD), 3-methylcholanthrene (3-MC) and benzo[a]pyrene (B[a]P) have shown that beta-NF is a high-affinity ligand for AhR and also for polycyclic aromatic hydrocarbon (PAH)-binding protein, both soluble proteins of rat liver in 8 S and 4 S fractions, respectively, of sucrose gradients. This study examined binding of [(3)H]beta-NF to liver cytosolic proteins of female Sprague-Dawley rats. Treatment of rats with beta-NF, 3-MC, TCDD or alpha-naphthoflavone (alpha-NF) increased the specific [(3)H]beta-NF binding to liver cytosol up to 125-fold that of vehicle (corn oil)-treated rats (<100 fmol/mg of protein). Sucrose gradients revealed a large 4 S and a small 8 S peak of radioactivity from [(3)H]beta-NF binding to cytosols of beta-NF-, 3-MC-, TCDD- or alpha-NF-treated rats. Whereas co-incubation with the unlabelled beta-NF eliminated both peaks, co-incubation with 2,3, 7,8-tetrachlorodibenzofuran (TCDF) eliminated only the 8 S peak. The sucrose density gradient from [(3)H]TCDD binding to cytosol of beta-NF- or TCDD-treated rats yielded a small 4 S and a larger 8 S peak; only the latter was abolished by co-incubation with TCDF. Thus, the patterns of sedimentation, distribution and elimination of radioactivity from the 8 S fraction of the liver cytosols from beta-NF-, 3-MC-, TCDD- or alpha-NF-treated rats were characteristic for the AhR, whereas those from the 4 S fraction appeared specific for [(3)H]beta-NF binding. The data indicate that potent AhR agonists, TCDD, 3-MC and beta-NF, and to a lesser extent alpha-NF, a weak AhR agonist, induce a 4 S [(3)H]beta-NF-binding protein in liver cytosol of female rats. alpha-NF, beta-NF and 3-MC were effective competitors (80-85% inhibition) of the [(3)H]beta-NF-specific binding to the beta-NF-, 3 MC- or TCDD-induced 4 S protein, whereas several PAHs including B[a]P and benzo[e]pyrene were only weak competitors. The increased [(3)H]beta-NF binding was not associated with glycine N-methyltransferase activity. Hence, the 4 S [(3)H]beta-NF-binding protein described herein differs from the constitutive 4 S PAH-binding protein of rat liver cytosols in the inducibility by beta-NF and 3-MC, ligand-binding characteristics, and lack of glycine N-methyltransferase activity. Gel filtration on Sephacryl of liver cytosols from beta-NF-treated rats indicated a molecular mass of approximately 42 kDa for [(3)H]beta-NF-bound protein and suggested that it was derived from a large mass component that before the radioligand binding was eluted with the void volume of the gel and sedimented in a 7 S fraction of the sucrose gradient. The [(3)H]beta-NF binding activity was not eluted with glutathione S-transferase Ya, aldehyde-3-dehydrogenase or DT-diaphorase [NAD(P)H: quinone oxidoreductase] activities, which are AhR-controlled and beta-NF-inducible. Further studies are needed to determine the identity and function of this novel protein which may be involved either directly or indirectly (as a carrier protein) in xenobiotic metabolism in vivo.
Brauze
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Veterans Affairs Medical Center, One Veterans Drive (151), Minneapolis, MN 55417, USA.
Malejka-Giganti
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D000818:Animals; D001564:Benzo(a)pyrene / Q000494:pharmacology; D001571:Benzoflavones / Q000494:pharmacology; D001572:Benzofurans / Q000494:pharmacology; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism; D003600:Cytosol / Q000737:chemistry* / Q000187:drug effects; D005260:Female; D050938:Glycine N-Methyltransferase; D007700:Kinetics; D008099:Liver / Q000737:chemistry* / Q000166:cytology / Q000187:drug effects; D008748:Methylcholanthrene / Q000494:pharmacology; D008780:Methyltransferases / Q000378:metabolism; D008970:Molecular Weight; D000072317:Polychlorinated Dibenzodioxins / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D018336:Receptors, Aryl Hydrocarbon / Q000819:agonists* / Q000378:metabolism; D019324:beta-Naphthoflavone / Q000378:metabolism* / Q000494:pharmacology
D001571:Benzoflavones; D001572:Benzofurans; D002352:Carrier Proteins; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; D001564:Benzo(a)pyrene; D008748:Methylcholanthrene; C011512:alpha-naphthoflavone; D019324:beta-Naphthoflavone; D019363:Cytochrome P-450 CYP1A1; D008780:Methyltransferases; D050938:Glycine N-Methyltransferase; C014211:2,3,7,8-tetrachlorodibenzofuran