Polycyclic aromatic hydrocarbon binding macromolecules. Identification, characterization and temperature activation of a 4.5 S binding nucleoprotein.
A macromolecule binding 3H-methylcholanthrene (3H-MCA) and 3H-benzo(a)pyrene (3H-BaP) and sedimenting in the 4-5 S region of sucrose gradient (4.5 S) was identified in rat liver cytosol. The binding was displaced by 100-fold molar excess unlabeled ligands whereas 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) was ineffective. The dissociation constant for both polycyclic aromatic hydrocarbons (PAHs) was of the order of 10(-8) M or lower. Both 3H-MCA and 3H-BaP bound to 4.5 S in a non covalent manner, since 92% of the bound radioactivity was extractable with ethyl ether. Furthermore the binding was strongly reduced by urea 8 M and by guanidine. HCl 4 M (99 and 70% respectively). Thin layer chromatography of the ethyl ether-solubilized radioactivity showed a peak comigrating with PAHs used as standards. When chromatographed on Sephadex G-200, 4.5 S was eluted as a sharp peak with an apparent molecular weight of 50-60,000 daltons. Enzyme treatment of liver cytosol showed that the 4.5 S binding sites were destroyed by micrococcal nuclease (92% of inhibition). Papain and phosphodiesterase I and II reduced the binding to 50%, whereas DNase I, DNase II, RNase, phospholipase A2 and C and trypsin were ineffective. These data suggest that the PAHs binding macromolecule of rat liver cytosol is a protein associated with a polynucleotide. The binding of both PAHs was enhanced by increasing the incubation temperature, the maximum being reached after 20-30 min at 37 degrees C. After 2.5 min at 65 degrees C, binding sites were completely destroyed. The same temperature-induced "activation" was obtained also by prewarming the cytosol at 37 degrees C in the absence of ligands.
D000818:Animals; D001564:Benzo(a)pyrene / Q000378:metabolism; D002352:Carrier Proteins / Q000032:analysis*; D002850:Chromatography, Gel; D003600:Cytosol / Q000032:analysis; D005260:Female; D008748:Methylcholanthrene / Q000378:metabolism; D009698:Nucleoproteins / Q000032:analysis / Q000378:metabolism*; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology; D011083:Polycyclic Compounds / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013696:Temperature; D014316:Tritium
D002352:Carrier Proteins; D009698:Nucleoproteins; D000072317:Polychlorinated Dibenzodioxins; D011083:Polycyclic Compounds; D014316:Tritium; D001564:Benzo(a)pyrene; D008748:Methylcholanthrene