Progesterone receptor from chick oviduct: purification of molybdate-stabilized form and preliminary characterization.
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-17 beta-carboxamide-substituted Sepharose gel, purified the receptor 1500-2700-fold with approximately equal to 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 greater than 6700-fold. The third step was filtration through Ultrogel AcA 34, resulting in overall purification approximately equal to 7400-fold with overall recovery approximately equal to 25% of pure receptor on the basis of 1 binding site/molecule of Mr 85000. The purified molybdate-stabilized receptor had a sedimentation coefficient approximately equal to 7.9S +/- 0.1 (n = 4) in 0.15 M or 0.4 M KCl containing sucrose 5-20% gradient and approximately equal to 8.9S +/- 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 approximately equal to 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 approximately equal to 85000-Mr polypeptide chains.
D000818:Animals; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002621:Chemistry; D002645:Chickens; D002845:Chromatography / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D008982:Molybdenum; D010057:Oviducts / Q000032:analysis*; D011980:Receptors, Progesterone / Q000302:isolation & purification*
D011980:Receptors, Progesterone; D008982:Molybdenum