Rapid vertical tube rotor gradient assay for binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin to the Ah receptor.
The Ah receptor, which regulates induction of aryl hydrocarbon hydroxylase (cytochrome P1-450), can be assayed using [3H] 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as the radioligand. Sucrose density gradient (SDG) centrifugation is the most reliable method for detecting and quantitating Ah receptor; however, previous gradient assays in swinging bucket rotors (SBR) have required overnight centrifugation to achieve adequate separation of specific receptor peaks from nonspecific binding to other cytosolic components. These lengthy runs limit the number of assays which can be performed and could permit excessive dissociation of radioligand from the receptor. Thus, we tested brief (2-h) SDG centrifugation assays in a vertical tube rotor (VTR) as a possible alternative to prolonged SBR runs. Two-hour VTR runs yield gradient profiles very similar to those obtained with 16-h runs in an SBR and the calculated sedimentation coefficient (in low ionic strength buffer) is about 9.6 S for the Ah receptor assayed in either rotor. The concentration of Ah receptor in cytosols from liver, lung, kidney, and testis of C57BL/6J mice is the same by VTR analysis as when measured by SBR analysis. Thus, extensive dissociation of [3H]TCDD from the Ah receptor does not appear to occur in cytosols from a variety of tissues even with run times up to 16 h. Assays performed by rapid VTR centrifugation appear equally valid to those obtained by overnight SBR centrifugation.
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics*; D001665:Binding Sites; D002499:Centrifugation, Density Gradient / Q000379:methods; D003600:Cytosol / Q000378:metabolism; D004147:Dioxins / Q000378:metabolism*; D005809:Genes, Regulator; D008099:Liver / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D000072317:Polychlorinated Dibenzodioxins / Q000378:metabolism*
D004147:Dioxins; D000072317:Polychlorinated Dibenzodioxins; D001189:Aryl Hydrocarbon Hydroxylases