Chemical reagents, such as pyridoxal 5’-phosphate, 1,2-cyclohexanedione, ethoxyformic anhydride, and rose bengal, which have been shown to serve as probes for specific amino acid residues in polypeptides may modify the actives sites of the hepatic glucocorticoid receptor. Reactivity of pyridoxal 5’- phosphate demonstrated that lysine residues participate at the DNA-binding site and possibly at another site on the receptor. At elevated temperatures, this chemical probe can affect the dissociation of ligand. At a concentration of 100 mM, 1,2-cyclohexanedione, a probe for arginyl residues, inhibited ligand binding to receptor by 82%. The ligand-binding site could be protected by a saturating concentration of steroid. Under this condition, 1,2-cyclohexanedione markedly inhibited the binding of the activated receptor complex to DNA-cellulose. The chemical reagent, ethoxyformic anhydride, has been shown to preferentially ethoxyformylate histidyl residues. At a concentration of 34 mM, ethoxyformic anhydride virtually abolished steroid-receptor complex binding to DNA-cellulose. An additional probe for histidine residues, the photoactivated oxidant rose bengal at 80 μM, prevented more than 50% of the activated steroid-receptor complexes from binding to DNA-cellulose. The same inhibitor concentration of these inhibitors had little effect on specific steroid binding to receptor. The DNAbinding site of the activated glucocorticoid receptor may therefore require the activities of histidine, arginine, and lysine residues. Additional lysine and arginine residues influence the ligand- binding site activity of the receptor. Because pure preparations of receptor are unavailable and we had to use crude cytosol, the data presented in this paper are also compatible with interactions of chemical probes with some protein other than receptor which then can influence receptor to generate the behavior measure. Moreover, our measurements cannot discern primary interactions of chemical probes directly with active sites from interactions of these agents at distal sites, which exert their effects by allosteric mechanisms.
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