An assay for the detection of specific binding of 3-methylcholanthrene to rat liver cytosolic proteins using DEAE-cellulose.
A method for the detection of the specific binding of 3-methylcholanthrene to rat liver cytosolic proteins is described. The separation of the protein-bound 3-methylcholanthrene from the free 3-methylcholanthrene was achieved using a batch DEAE-cellulose technique. Extraction of the DEAE-cellulose with 0.3 M KCl allowed the selective release and measurement of the amount of protein-bound 3-methylcholanthrene. The assay was optimized for the following parameters: time of incubation with DEAE-cellulose, time required for salt extraction, protein concentration, the concentration of KCl required to elute the specific binding proteins, the amount of DEAE-cellulose required to bind the specific binding proteins, and ligand specificity. The sedimentation properties of those 3-methylcholanthrene-binding proteins which were extracted with salt from DEAE-cellulose were examined on 5 to 20% sucrose gradients; the major binding species sedimented as a broad peak at 4.5 S.
D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism*; D003636:DEAE-Cellulose; D008099:Liver / Q000378:metabolism*; D008297:Male; D008748:Methylcholanthrene / Q000378:metabolism*; D011189:Potassium Chloride / Q000494:pharmacology; D011485:Protein Binding; D051381:Rats; D011919:Rats, Inbred Strains
D008748:Methylcholanthrene; D011189:Potassium Chloride; D003636:DEAE-Cellulose; D003577:Cytochrome P-450 Enzyme System