The binding components for 2,3,7,8-tetrachlorodibenzo-p-dioxin and polycyclic aromatic hydrocarbons. Separation from the rat and mouse hepatic cytosol and characterization of a light density component.
Using sucrose density gradient centrifugation in a vertical rotor, we have separated three major binding components contained in hepatic cytosols from C57BL/6 mice and Sprague-Dawley rats. Using this preparative method we have obtained, after a 3-h run of 2.4 ml of crude cytosol from 1,4-bis[2-(3,5-dichlorodipyridyloxy)]benzene-treated C57BL/6 mice (approximately 50 mg of protein: 10,000 fmol of Ah receptor) 50 and 75% yields of isolated Ah receptor and carcinogen-binding protein (4 S binding protein), respectively. Both binding components may be kept at -70 degrees C for several months without loss of activity. A third binding component, which did not sediment in a sucrose density gradient (5-20%), even after a 4-h run at 63,000 rpm, was recovered from the top fractions of gradients. When applied to Sephacryl S-300 columns this component was eluted in the void fraction. Resistant to the direct degradative action of nucleases and proteases, this large complex was sequentially converted to its subcomponents by lipoprotein-lipase, proteinase K, and phospholipases. Only the phospholipases are able to abolish the binding capacity of this light density component (LDC) for [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin: hence, we conclude that phospholipids are the true binders of this radioligand. In vitro, this lipoprotein irreversibly binds many hydrophobic radioligands (2,3,7,8-tetrachlorodibenzo-p-dioxin,3-methylcholanthrene, benzo(a)pyrene, 7,12-dimethylbenz(a)anthracene, and dexamethasone). Using single vertical spin density gradient ultracentrifugation, the major part (80%) of LDC was characterized as a very low-density lipoprotein, and a minor part (20%) as a low-density lipoprotein. This conclusion was supported by the size of LDC particles (about 25-75 nm) observed in electron microscopy.
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D003600:Cytosol / Q000378:metabolism; D004147:Dioxins / Q000378:metabolism*; D066298:In Vitro Techniques; D008079:Lipoproteins, VLDL / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D051379:Mice; D000072317:Polychlorinated Dibenzodioxins / Q000378:metabolism*; D011083:Polycyclic Compounds / Q000378:metabolism*; D051381:Rats
D002352:Carrier Proteins; D004147:Dioxins; D008079:Lipoproteins, VLDL; D000072317:Polychlorinated Dibenzodioxins; D011083:Polycyclic Compounds