Purification of "nontransformed" avian progesterone receptor and preliminary characterization.
The nontransformed (molybdate-stabilized) avian oviduct progesterone receptor has been purified to near homogeneity by a simple four-step procedure: ammonium sulfate fractionation, affinity chromatography, gel filtration, and DEAE-Sephadex A-25 chromatography. The affinity resin (deoxycorticosterone-agarose) is very resistant to chemical breakdown and enzymatic destruction and can be used repeatedly. The material obtained after gel filtration was separated into two receptor components by DEAE-chromatography. The two components, termed I and II, are obtained in about 18% yield and are purified by about 6000-fold (I) and 4000-fold (II). They are highly purified as only a single major Coomassie blue-stained polypeptide of about Mr = 90,000 in both components I and II is seen on sodium dodecyl sulfate-gel electrophoresis plus a second band of Mr = 104,000 which is seen only in component II. Both purified receptor components have sedimentation coefficients of 8 S in glycerol gradients containing molybdate (10 mM). In the absence of molybdate, however, both receptor forms have sedimentation coefficients of about 4 S in gradients containing 300 mM KCl. Therefore, stabilization of the 8 S form by molybdate is still evident in highly purified receptor preparations. The Stokes radii, binding specificity, and steroid dissociation rate of purified receptor are similar to those found in crude cytosol. These results confirm our previous observation on the existence of two 8 S forms of the progesterone receptor and show that these molybdate-stabilized forms remain intact throughout purification.
D000818:Animals; D002645:Chickens; D003600:Cytosol / Q000378:metabolism; D004054:Diethylstilbestrol / Q000494:pharmacology; D005260:Female; D007700:Kinetics; D008970:Molecular Weight; D010057:Oviducts / Q000187:drug effects / Q000378:metabolism*; D011374:Progesterone / Q000378:metabolism; D011980:Receptors, Progesterone / Q000302:isolation & purification* / Q000378:metabolism
D011980:Receptors, Progesterone; D011374:Progesterone; D004054:Diethylstilbestrol