Progesterone Receptor from Chick Oviduct:. Purification of Molybdate-Stabilized Form and Preliminary Characterization

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Abstract
A molydate-stabilized, ‘non-activated’ form of the progesterone receptor from the cytosol of oestrogen-stimulated-chick oviduct has been purified to homogeneity by a three-step procedure. The first step, affinity chromatography using a N-(12-amino-dodecyl)-3-oxo-4-androsten-17β-carboxamide-substituted Sepharose gel, purified the receptor 1500–2700-fold with ∼ 50% recovery. In the second step, ion-exchange chromatography through a DEAE-cellulose column, progesterone receptor was eluted as a single peak at 0.1 M KCl. Purification after this step was > 6700-fold. The third step was filtration through Ultrogel AcA 34, resulting in overall purification ∼ 7400-fold with overall recovery ∼ 25% of pure receptor on the basis of 1 binding site/molecule of Mr, 85000. The purified molybdate-stabilized receptor had a sedimentation coefficient ∼ 7.9 S ± 0.1 (n= 4) in 0.15 M or 0.4 M KCl containing sucrose 5–20% gradient and ∼ 8.9 S ± 0.2 (n= 6) in 0.15 M KCl containing glycerol 10–35% gradient, and its Stokes radius was 7.05 ± 0.10 nm (n= 3) (calculated Mr between 240000 and 280000). Binding specificity of the purified receptor was the same as that found in crude cytosol. SDS-PAGE revealed a single band migrating as a polypeptide of Mr∼ 85000 ± 2300 (n= 9). PAGE under non-denaturing conditions at total acrylamide concentrations 5%, 7% and 9% showed a single [3H]ORG 2058-protein band (ORG 2058 is a high-affinity analogue more suitable than progesterone for electrophoretic studies). The data suggest that the high molecular weight molybdate-stabilized progesterone receptor purified from oestrogen-primed chick oviduct is composed of only ∼ 85000-Mr polypeptide chains.