Characterization of the purified molybdate-stabilized glucocorticoid receptor from rat liver. An in vitro transformable complex
| Autor: | Claude Richard, Pierre Formstecher, Patrick Lustenberger, Pierre‐Marie Danze, M. Dautrevaux, Bernard Sablonnière, Viviane Dumur, Thierry Idziorek |
|---|---|
| Rok vydání: | 1985 |
| Předmět: |
Receptor complex
Molybdate In Vitro Techniques Biochemistry High-performance liquid chromatography Triamcinolone Acetonide Chromatography Affinity Dexamethasone chemistry.chemical_compound Receptors Glucocorticoid Affinity chromatography Centrifugation Density Gradient Animals Bovine serum albumin Cellulose Polyacrylamide gel electrophoresis Molybdenum Chromatography biology Isoelectric focusing DNA Chromatography Ion Exchange Rats chemistry Liver biology.protein Chromatography Gel Agarose Electrophoresis Polyacrylamide Gel Isoelectric Focusing |
| Zdroj: | European journal of biochemistry. 153(1) |
| ISSN: | 0014-2956 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Plný text