Abstract
Two rapid and high yield purification methods for the rat liver glucocorticoid receptor based on differential DNA affinity (method A) and ligand affinity (method B) chromatography are described. In method A, the amount of receptor in rat liver cytosol that can be activated and subsequently eluted from a DNA-cellulose column has been increased to 80% by introducing a second heat activation step. Using this method, 1.5 nmol of 25% pure glucocorticoid receptor can be routinely obtained per day from 15-20 rat livers. Method B yields about 2.2 nmol of 60% pure receptor with an overall yield of congruent to 60%. The quality of these purifications has been controlled by affinity labeling. In each case, more than 95% of purified binding activity represented the intact 92,000 +/- 400-Da glucocorticoid receptor polypeptide as shown by sodium dodecyl sulfate-gel electrophoresis and fluorography. No difference in the labeling pattern was observed using either [3H]triamcinolone acetonide (photoaffinity labeling) or [3H]dexamethasone 21-mesylate (electrophilic labeling). The electrophilic labeling step was performed in the cytosol prior to purification by method A to compare the labeled components thus purified with those obtained when the photoaffinity labeling was performed after the purification. Using this approach, distinct breakdown products of the glucocorticoid receptor were revealed, co-purifying during DNA affinity chromatography. Cross-linked receptor obtained by method A has been further purified to homogeneity by preparative sodium dodecyl sulfate-gel electrophoresis and successfully used as immunogen to raise glucocorticoid receptor antibodies in rabbits. These antibodies raised against glucocorticoid receptor, as well as those previously obtained using affinity chromatography-purified receptor, react with the receptor molecules irrespective of their method of purification. Glucocorticoid receptors purified by methods A and B have been analyzed for specific DNA-binding properties by the nitrocellulose filter binding assay.
Highlights
(photoaffinity labeling)or [3H]dexamethasone 21-me- to explain some of the discrepancies in reported molecular sylate
Purification of Rat Liver Glucocorticoid Receptor by DNA Affinity Column (Method A)-A schematic description of this method is presented in Fig. 1, which gives inadditiona detailed time schedule which is designed for the purification of glucocorticoid receptor from 15 to 20 rat livers/day
When the same experiment was performed with 0.5 pg of affinity eluate (Fig. 2g, lune I) or with 0.5 pg of affinity eluate further purified by DNA-cellulose chromatography (Fig. 2h) and antibodies raised against glucocorticoid receptor purified by method A, immunoreaction could be detected with the intact glucocorticoid receptor band (Fig. 40, lanes 1 and 2).Western blotting analysis with the degradation products purified by preparative SDS-gel electrophoresis (36,45, and 64kDa; Fig. 3D, lunes 2 and 3 ) demonstrated that only 45- and 64-kDa receptor fragments reacted with the antibodies
Summary
MATERIALS ANDMETHODS [3H]Triamcinolone acetonide (31.3 Ci/mmol) and [3H]dexamethasone (25 and 49 Ci/mmol) were purchased from New England Nuclear, Dreieich, Federal Republic of Germany. The remaining eluate was diluted 3-fold with buffer A, adjusted to pH7.8, activated for 30 min at 20 "C, and chromatographed on a 30-ml DNA-cellulose column. This column was washed and eluted, and theeluates were crosslinked or precipitated with 50% ammonium sulfate as described in method A. Cross-linked and noncross-linked preparations were precipitated with ammonium sulfate, centrifuged, and stored as described above after adjusting the receptor stock solution to 1 X lo-' M. Preparative SDS-Gel Electrophoresis of Receptor Purified by Method A-The ammonium sulfate-precipitated cross-linked receptor preparation from each DNA-cellulosecolumn eluate was dissolved separately in 1ml of SDS sample buffer containing bromphenol blue as marker, by heating 2-5 min at 80 "C. The separation was performed on a 4-ml 7.5% SDS-polyacrylamide gel with a 2-ml 4% stacking gel
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