title: Effects of SH-modifying reagents on the rat hepatic Ah receptor: inhibition of ligand binding and transformation, and disruption of the ligand-receptor complex.
abstract: The importance of sulfhydryl (SH) groups in maintenance of physicochemical properties of the rat hepatic Ah receptor was demonstrated using a variety of sulfhydryl (SH)-modifying reagents. Inhibition of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) specific binding was approximately equivalent by 5,5'-dithiobis(2-nitrobenzoate), mersalyl, N-ethylmaleimide, and p-chloromercuriphenylsulfonate, whose inhibition curves were steep in the concentration range close to that of nonprotein SH groups in cytosol (ED50 values 50-200 microM or 13-48 nmol/mg cytosolic protein). Inhibition by p-hydroxymercuribenzoate (PHMB), although exhibiting a lower ED50, was more gradual over this range; iodoacetamide was an order of magnitude less potent. The ability of dithiothreitol to reverse binding inhibition induced by 150 microM (approximately 60 nmol/mg protein) mersalyl diminished with time; it decreased more rapidly in the simultaneous presence of TCDD and mersalyl than when mersalyl was present alone, consistent with increased accessibility of key SH group(s) due to conformational changes attending TCDD-receptor complex formation. Brief exposure of unoccupied receptor to mersalyl prior to TCDD binding caused slower sedimentation of the complex in 0-KCl sucrose gradients and alterations in its elution profiles on DEAE- and DNA-Sepharose suggestive of some impairment of the transformation process. When reagents were added to the transformed TCDD-receptor complex, loss of binding was observed only at concentrations which were an order of magnitude higher than those inhibiting TCDD binding. Loss of binding by each reagent was biphasic, and except for that caused by mersalyl, was not complete even after 6-8 h. Dithiothreitol was able to reverse the effects of mersalyl or PHMB only partially and only if added during the early phase (10-30 min) of binding loss. Mersalyl was much more potent in disrupting the untransformed than the transformed TCDD receptor complex. Physical alteration of the mersalyl-treated TCDD-receptor complex was evident from gel filtration, sucrose gradients, and DNA- and DEAE-Sepharose chromatography. Our results are in striking contrast to the effects of these reagents on steroid receptors, whose bound steroid hormone ligand is rapidly and reversibly displaced by lower concentrations of reagent.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Biophysics, University of Rochester Medical Center, NY 14642.
authors.forename: E C
authors.initials: EC
authors.lastname: Henry
authors.forename: J E
authors.initials: JE
authors.lastname: Kester
authors.forename: T A
authors.initials: TA
authors.lastname: Gasiewicz
mesh_terms: D000818:Animals; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D008634:Mersalyl / Q000037:antagonists & inhibitors; D010084:Oxidation-Reduction; D000072317:Polychlorinated Dibenzodioxins / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D013499:Surface Properties
chemical_list: D004268:DNA-Binding Proteins; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug; D013439:Sulfhydryl Reagents; D008634:Mersalyl; D004247:DNA; D004229:Dithiothreitol