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Purification of the unactivated glucocorticoid receptor and its subsequent in vitro activation.

Open AccessPublished:March 10, 1984DOI:https://doi.org/10.1016/S0021-9258(17)43277-7
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      The unactivated, molybdate-stabilized rat hepatic glucocorticoid receptor has been purified approximately 4000-fold, as calculated by specific radioactivity, by affinity chromatography using a deoxycorticosterone-derivatized agarose, gel filtration on Bio-Gel A-1.5m agarose, and DEAE-cellulose chromatography. The final receptor sediments at 9-10 S in low salt (40 mM KCl) glycerol gradients containing molybdate. Elevated salt concentrations up to 1 M KCl reduce the sedimentation coefficient to 8-9 S. The final DEAE-cellulose eluted complexes exhibit a Stokes radius of 7.3 nm, a value similar to that reported for receptors in crude cytosol. From the hydrodynamic parameters an apparent Mr = 303,000 can be calculated for the steroid-receptor complex. Analysis of the receptor-containing fractions from DEAE-cellulose chromatography by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrates the occurrence of a major Mr = 90,000 protein band which closely followed the distribution of bound radioactivity. Two other proteins corresponding to Mr = 41,000 and 40,000 also exhibit the same distribution pattern. Saturation of cytosolic specific binding sites with unlabeled triamcinolone acetonide prior to receptor purification results in the disappearance of these three proteins from the DEAE-cellulose chromatogram. Furthermore, a Mr = 24,000 component, which is eluted from DEAE-cellulose at a salt concentration higher than that of the bound radioactivity peak itself, also disappears. These observations argue that the Mr = 90,000, 41,000, 40,000, and 24,000 components are related as components or degradation products of the unactivated, molybdate-stabilized rat hepatic glucocorticoid-receptor complex. Studies on the in vitro activation of purified steroid-receptor complexes have revealed that Sephadex G-25 gel filtration and warming (25 degrees C for 30 min) enables purified receptors to become activated judged by ability to bind to DNA-cellulose but to a lower extent than observed for receptors in crude tissue homogenates. A DNA-cellulose binding capacity, similar to that shown by crude liver cytosolic receptor under the same conditions, can be conferred on the purified complexes only in a reconstituted system in which crude cytosol has been added. Molybdate is shown to completely inhibit activation induced by gel-filtration and offers significant protection against heat-induced activation both in highly purified and reconstituted systems. The activation inhibitory effect of molybdate has also been confirmed by DEAE-cellulose chromatography.

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