Progesterone binding components of chick oviduct. X. Purification by affinity chromatography.
Cytoplasmic progesterone receptors of chick oviduct have been purified in 8% yield by steroid affinity and ion exchange chromatography. The affinity resin, deoxycorticosterone-bovine serum albumin-Sepharose, binds progesterone receptors with high affinity (KD equals 8 times 10-minus 10 M) and its use resulted in a greater than 2000-fold purification over the starting material in a single step. DEAE-Sephadex A-50 chromatography was then used to achieve final purification. NA dodecyl-SO4 gel electrophoresis and DEAE-cellulose chromatography showed that the purified receptors contained both of the previously described 4 S progesterone binding components in near equal amounts. Na dodocyl-SO4 gel electrophoresis also showed that these components consisted of single polypeptide chains with molecular weights of 110, 000 (A component) and 117, 000 (B component). There was no evidence for subunits of lower molecular weight. The purified materials have identical hormone-binding kinetics and steroid specificity to crude cytosol receptors. The isolated receptors retain the three biologically important properties exhibited by progesterone binding components present in cruder preparations: they bind specifically to (a) nuclei (KD equals 1.1 times 10-minus 9 M, 10, 000 sites per nucleus); (b) chromatin (KD equals 3 times 10-minus 9 M, 2000 sites per pg of DNA-);and (C) DNA.
D000818:Animals; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002645:Chickens; D002843:Chromatin / Q000378:metabolism; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000378:metabolism; D003900:Desoxycorticosterone; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007700:Kinetics; D008970:Molecular Weight; D009124:Muscle Proteins / Q000302:isolation & purification* / Q000378:metabolism; D010057:Oviducts / Q000378:metabolism*; D011374:Progesterone / Q000378:metabolism*; D011485:Protein Binding; D011956:Receptors, Cell Surface
D002843:Chromatin; D009124:Muscle Proteins; D011956:Receptors, Cell Surface; D003900:Desoxycorticosterone; D011374:Progesterone; D004247:DNA