Stabilization and characterization of the dexamethasone-binding proteins in rat liver cytosol.
Conditions were worked out for maximal stabilization of dexamethasone binding activity of rat liver cytosol in the absence of the protective steroid ligand. Important stabilization factors are ionic strength, thiol-protecting agents, glycerol and pH. Maximal stability of the cytosol is observed in a buffer consisting of 20 mM Tris-HCl, pH 7.5, 50 mM KCl, 25 mM beta-mercaptoethanol and 20% glycerol. Chromatography of cytosol on DEAE-cellulose revealed the existence of three dexamethasone receptors, binder DE-1, present in the flow-through fraction and binders DE-2 and DE-3, eluting from the column with salt concentrations of 100 and 190 mM, respectively. Binders DE-2 and DE-3 are not adsorbed on phosphocellulose at pH 7.5, whereas binder DE-1 is. All three receptors are retained to varying degrees on DNA-cellulose columns: binder DE-1 is eluted with salt concentrations of 270 mM, whereas binders DE-2 and DE-3 are eluted between 180 and 200 mM NaCl. The dexamethasone receptors also bind natural glucocorticoids, but to varying degrees, the highest binding being observed to binder DE-2. The receptors obtained after chromatography on DEAE-cellulose, but not on phosphocellulose, cannot be to an appreciable extent charged with dexamethasone.
D000818:Animals; D002846:Chromatography, Affinity; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D003907:Dexamethasone / Q000378:metabolism*; D006207:Half-Life; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008297:Male; D009994:Osmolar Concentration; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011956:Receptors, Cell Surface / Q000187:drug effects; D013438:Sulfhydryl Compounds / Q000494:pharmacology
D011506:Proteins; D011956:Receptors, Cell Surface; D013438:Sulfhydryl Compounds; D003907:Dexamethasone