Purification and preliminary characterization of a macromolecular inhibitor of glucocorticoid receptor binding to DNA.
Rat liver cytosol contains a heat-labile macromolecule that inhibits the binding of the transformed glucocorticoid-receptor complex to nuclei or DNA-cellulose (Milgrom, E., and Atger, M. (1975) J. Steroid Biochem. 6, 487-492; Simons, S. S., Jr., Martinez, H. M., Garcea, R. L., Baxter, J. D., and Tomkins, G. M. (1976) J. Biol. Chem. 251, 334-343. We have developed a quantitative assay for the inhibitor and have purified it 600-700-fold by ammonium sulfate precipitation, ethanol precipitation, and phosphocellulose and Sephacryl S-300 chromatography. The inhibitory activity copurifies with a Mr = 37,000 protein doublet. Under low salt conditions, both the inhibitory activity and the 37-kDa protein doublet behave as high Mr aggregates that subsequently dissociate in the presence of salt. The inhibitor is positively charged at physiological pH, and it is not affected by digestion with several serine proteases or RNase. The inhibitor does not affect the transformation process, and it does not cause the release of steroid-receptor complexes that have been prebound to DNA-cellulose. The inhibitor preparation does not cleave receptors in L-cell cytosol that are covalently labeled with the site-specific affinity steroid [3H]dexamethasone 21-mesylate. If the steroid-receptor complex is first separated from the great majority of cytosol protein by transforming it and binding it to DNA-cellulose, addition of the inhibitor preparation results in receptor cleavage. Under these conditions, cleavage can be blocked with 1-chloro-3-tosylamido-7-amino-L-2-heptanone and antipain, but protease inhibitors do not affect the inhibition of DNA binding that occurs in whole cytosol. The inhibitor acts through an interaction with the receptor, not with DNA. We suggest that the inhibitor may prove to be a useful tool for studying the interaction of the steroid-receptor complex with DNA or nuclei and speculate that it may be important in determining normal events of the receptor cycle as they occur in the intact cell.
D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D002482:Cellulose / Q000031:analogs & derivatives; D002846:Chromatography, Affinity; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000031:analogs & derivatives / Q000378:metabolism*; D003907:Dexamethasone / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D007739:L Cells / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D008297:Male; D051379:Mice; D008970:Molecular Weight; D011506:Proteins / Q000302:isolation & purification*; D051381:Rats; D011919:Rats, Inbred Strains; D011965:Receptors, Glucocorticoid / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D011987:Receptors, Steroid / Q000378:metabolism*; D014222:Triamcinolone Acetonide / Q000378:metabolism
D000345:Affinity Labels; C033190:DNA-cellulose; D011506:Proteins; D011965:Receptors, Glucocorticoid; D011987:Receptors, Steroid; D003907:Dexamethasone; D002482:Cellulose; D004247:DNA; D014222:Triamcinolone Acetonide; C031710:dexamethasone 21-methanesulfonate