Degradation without apparent change in size of molybdate-stabilized nonactivated glucocorticoid-receptor complexes in rat thymus cytosol.
We have investigated the stability of the [3H]dexamethasone 21-mesylate-labeled nonactivated glucocorticoid-receptor complex in rat thymus cytosol containing 20 mM sodium molybdate. Cytosol complexes were analyzed under nondenaturing conditions by gel filtration chromatography in the presence of molybdate and under denaturing conditions by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. When analyzed under nondenaturing conditions, complexes from fresh cytosol and from cytosol left for 2 h at 3 degrees C eluted from gel filtration as a single peak of radioactivity with a Stokes radius of approximately 7.7 nm, suggesting that no proteolysis of the complexes had occurred in either cytosol. When analyzed under denaturing conditions, however, whereas the fresh cytosol gave a receptor band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis at Mr approximately 90,000 (corresponding to the intact complex), the cytosol that had been left for 2 h at 3 degrees C gave only a fragment (Mr approximately 50,000). This fragment, just as the intact complex, could be thermally activated to a DNA-binding form. Proteolysis of the receptor could be blocked by preparing the cytosol in the presence of EGTA, leupeptin, or a heat-stable factor present in the cytosol of rat liver and WEHI-7 mouse thymoma cells. From these results we conclude: (i) 20 mM molybdate does not protect the nonactivated glucocorticoid-receptor complex present in rat thymus cytosol against proteolysis under conditions which are commonly used for cell-free labeling of the receptor, and (ii) the demonstration of a Stokes radius of approximately 8 nm for the nonactivated glucocorticoid-receptor complex is not sufficient to indicate that the receptor complex is present in its intact form.
Mendel
D B
DB
Holbrook
N J
NJ
Bodwell
J E
JE
D000818:Animals; D002154:Calpain; D002850:Chromatography, Gel; D003600:Cytosol / Q000378:metabolism; D003907:Dexamethasone / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000494:pharmacology; D008297:Male; D008970:Molecular Weight; D008982:Molybdenum / Q000494:pharmacology*; D011489:Protein Denaturation; D051381:Rats; D011919:Rats, Inbred Strains; D011965:Receptors, Glucocorticoid / Q000187:drug effects / Q000378:metabolism*; D011987:Receptors, Steroid / Q000378:metabolism*; D013950:Thymus Gland / Q000378:metabolism*
D011965:Receptors, Glucocorticoid; D011987:Receptors, Steroid; D004533:Egtazic Acid; D003907:Dexamethasone; D008982:Molybdenum; C024687:sodium molybdate(VI); D010450:Endopeptidases; D002154:Calpain