Polyacrylamide concentration gradient gel electrophoresis of the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) receptor in rat liver cytosol.
An assay based on polyacrylamide concentration gradient gel electrophoresis for the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) receptor in rat liver cytosol was developed. [3H]TCDD-labeled cytosol was treated with dextran-coated charcoal and electrophoresed for 3000 Vh in polyacrylamide concentration gradient gels (T = 2.5-20%). A Tris-borate running buffer (pH 8.35) without salt and detergents was used. When the polyacrylamide concentration gradient was concave a linear calibration curve of log molecular weight vs. migration distance was obtained for the standard proteins used. Under these conditions the Mr value of the TCDD receptor from rat liver cytosol was estimated to be 316,000 +/- 27,000 before, and 246,000 +/- 12,000 after limited proteolysis. The Bmax for the specific binding of [3H]TCDD was between 35 and 43 fmol/mg cytosolic protein and the Kd of the binding ranged from 0.8 to 1.1 nM, as judged by saturation and Scatchard analysis.
Gillner
M
M
Department of Medical Nutrition, Karolinska Institute, Huddinge University Hospital, Sweden.
D000818:Animals; D003600:Cytosol / Q000032:analysis*; D004147:Dioxins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D008099:Liver / Q000032:analysis*; D000072317:Polychlorinated Dibenzodioxins / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug / Q000032:analysis*
D004147:Dioxins; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug