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Characterization of the purified molybdate-stabilized glucocorticoid receptor from rat liver. An in vitro transformable complex
| Content Provider | Scilit |
|---|---|
| Author | Idziorek, Thierry Formstecher, Pierre Danze, Pierre-Marie Sablonniere, Bernard Lustenberger, Patrick Richard, Claude Dumur, Viviane Dautrevaux, Michel |
| Copyright Year | 1985 |
| Description | Journal: Journal of Biological Inorganic Chemistry Rat liver glucocorticoid receptor was purified in the presence of molybdate by a three-step procedure comprising protamine sulfate precipitation, affinity chromatography on a dexamethasone matrix and high-performance size-exclusion chromatography (HPSEC) on a TSK G 3000 SW column. The [3H]triamcinolone-acetonide-receptor complex was obtained in 20% yield with an overall 11 800-fold purification. The dissociation rate constant of this complex was 1.6 X 10(-4) min-1. The purified receptor sedimented at 8.3 S in high-salt and 9.4 S in low-salt sucrose gradients containing molybdate. A 7.0-nm Stokes radius was determined by HPSEC on a TSK G 4000 column in high-salt buffer. The calculated Mr was 278000. Dodecyl sulfate/polyacrylamide gel electrophoresis revealed an almost homogeneous 90 000-Mr band. Three minor bands with Mr of 78 000, 72 000 and 48 000 were also inconstantly seen. An apparent pI = 5.1 was observed for the [3H]steroid complex by isoelectric focusing in agarose gel. Furthermore high-performance ion-exchange chromatography of the purified complex on a DEAE 545 LKB column (DEAE HPLC) yielded a sharp peak eluted at a 315 mM potassium ion concentration. This peak was shown to contain almost all the 90 000-Mr protein. Moreover the purified receptor complex appeared to be transformable to a DNA-binding state after molybdate removal followed by warming 30 min at 25 degrees C in presence of 0.2% bovine serum albumin: 50-78% transformation yield could be demonstrated by DNA-cellulose chromatography. Partial transformation could also be obtained at 0 degrees C in the absence of any added protein and was followed by DEAE HPLC. The transformed complex was eluted by 180 mM potassium. |
| Related Links | http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1985.tb09267.x/pdf |
| Ending Page | 74 |
| Page Count | 10 |
| Starting Page | 65 |
| ISSN | 09498257 |
| e-ISSN | 14321327 |
| DOI | 10.1111/j.1432-1033.1985.tb09267.x |
| Journal | Journal of Biological Inorganic Chemistry |
| Issue Number | 1 |
| Volume Number | 153 |
| Language | English |
| Publisher | Wiley-Blackwell |
| Publisher Date | 1985-11-01 |
| Access Restriction | Open |
| Subject Keyword | Journal: Journal of Biological Inorganic Chemistry Analytical Chemistry Purified Receptor |
| Content Type | Text |
| Resource Type | Article |
| Subject | Biochemistry Inorganic Chemistry |
