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

Sepharose gel, purified the receptor 1500 - 2700-fold with z 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 KCI. Purification after this step was >6700-fold. The third step was filtration through Ultrogel AcA 34, resulting in overall purification z 7400-fold with overall recovery z 25 of pure receptor on the basis of 1 binding site/molecule of M, 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 KCI containing sucrose 5-20% gradient and z 8.9 S k 0.2 (n = 6) in 0.15 M KCI containing glycerol 10-35 ”/, gradient, and its Stokes radius was 7.05 k 0.10 nm (n = 3) (calculated M, 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 MI z 85000 -+ 2300 (n = 9). PAGE under nondenaturing 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-M, polypeptide chains.