A novel 4 S[H-3]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 compe titive binding with H-3-labelled 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) , 3-methylcholanthrene (3-MC) and benzo[a]pyrene (B[a]P) have shown that be ta-NF is a high-affinity ligand for AhR and also for polycyclic aromatic hy drocarbon (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 [H-3]beta-NF to liver cytosolic proteins of female Sprague-Dawle y rats. Treatment of rats with beta-NF, 3-MC, TCDD or alpha-naphthoflavone (alpha-NF) increased the specific [H-3]beta-NF binding to liver cytosol up to 125-fold that of vehicle (corn oil)-treated rats (< 100 fmol/mg of prote in). Sucrose gradients revealed a large 4 S and a small 8 S peak of radioac tivity from [H-3]beta-NF binding to cytosols of beta-NF-, 3-MC-, TCDD- or a lpha-NF-treated rats. Whereas co-incubation with the unlabelled beta-NF eli minated both peaks, co-incubation with 2,3,7,8-tetrachlorodibenzofuran (TCD F) eliminated only the 8 S peak. The sucrose density gradient from [H-3]TCD D binding to cytosol of beta-NF- or TCDD-treated rats yielded a small 4 S a nd a larger 8 S peak; only the latter was abolished by coincubation with TC DF. Thus, the patterns of sedimentation, distribution and elimination of ra dioactivity 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 [H-3]beta-NF binding. The data indicate that potent AhR agonists, TCDD, 3-MC and beta-NF, and to a l esser extent alpha-NF, a weak AhR agonist, induce a 4 S [H-3]beta-NF-bindin g protein in liver cytosol of female rats. alpha-NF, beta-NF and 3-MC were effective competitors (80-85% inhibition) of the [H-3]beta-NF-specific bind ing to the beta-NF-, 3-MC- or TCDD-induced 4 S protein, whereas several PAH s including B[a]P and benzo[e]pyrene were only weak competitors. The increa sed [H-3]beta-NF binding was not associated with glycine N-methyltransferas e activity. Hence, the 4 S [H-3]beta-NF-binding protein described herein di ffers from the constitutive 4 S PAM-binding protein of rat liver cytosols i n 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 a pproximate to 42 kDa for [H-3]beta-NF-bound protein and suggested that it w as 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 [H-3]beta-NF binding activity was not eluted with glutathione S-transferase Ya, aldehyde-3-dehydrogenase or DT-diaphoras e [NAD(P)H: quinone oxidoreductase] activities, which are AhR-controlled an d beta-NF-inducible. Further studies are needed to determine the identity a nd function of this novel protein which may be involved either directly or indirectly las a carrier protein) in xenobiotic metabolism in vivo.