| 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. |
