Polycyclic Aromatic Hydrocarbon Binding Macromolecules. Identification, Characterization and Temperature Activation of a 4.5 S Binding Nucleoprotein

Abstract

A macromolecule binding 3H-methyIcholanthrene (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 10−3 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 stronghly reduced by urea 8 M and by guanidine. HCl 4 M (99 and 70 % respectively). Thin layer chromatography of the ehtyl 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 andC 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 °C. After 2.5 min at 65 °C, binding sites were completely destroyed. The same temperature-induced « activation » was obtained also by prewarming the cytosol at 37 °C in the absence of ligands.