Purification of the molybdate-stabilized 9-10 S estradiol receptor from calf uterus
| Autor: | Roland Hähnel, Sahidan Senafi, Thomas Ratajczak, V. Atrache |
|---|---|
| Rok vydání: | 1985 |
| Předmět: |
Receptor complex
Protein Conformation Sodium chemistry.chemical_element Receptors Estradiol In Vitro Techniques Molybdate Biochemistry High-performance liquid chromatography Chromatography Affinity chemistry.chemical_compound Cytosol Animals Receptor Molecular Biology Chromatography High Pressure Liquid Stokes radius Molybdenum Gel electrophoresis Chromatography Estradiol Elution Uterus Cell Biology Molecular Weight Receptors Estrogen chemistry Cattle Female |
| Zdroj: | Journal of Biological Chemistry. 260:5936-5941 |
| ISSN: | 0021-9258 |
| DOI: | 10.1016/s0021-9258(18)88919-0 |
| Popis: | The molybdate-stabilized calf uterine estradiol receptor has been purified to near-homogeneity by a three-step procedure. Initial purification by heparin-Sepharose chromatography provides a concentrated receptor extract in 40% yield with a 5-10-fold increase in purity. The inclusion of molybdate in phosphate-buffered cytosol enhances 9-10 S receptor stability in high salt and allows elution of the oligomeric receptor complex from heparin-Sepharose with 0.4 M KCl. A second affinity step utilizing estrone carboxymethyloxime coupled to diaminoethyl bis(2-hydroxypropoxy)butane-Sepharose Cl-4B increases purification by a further 1600-fold. High performance liquid chromatography gives homogeneous receptor which migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a polypeptide of Mr approximately 89,000. The purified molybdate-stabilized receptor sediments at 9.3 +/- 0.2 S (n = 4) in glycerol gradients and has a Stokes radius of 74 +/- 3 A (n = 2) giving a calculated Mr approximately 290,000. These properties and the steroid-binding specificity of the purified receptor bear a close similarity to those found for the 9-10 S receptor in crude cytosol. |
| Databáze: | OpenAIRE |
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