Chemical cross-linking of chick oviduct progesterone-receptor subunits by using a reversible bifunctional cross-linking agent.
Chick oviduct progesterone-receptor proteins were treated in cytosol with the reversible cross-linking reagent methyl 4-mercaptobutyrimidate. The product of the reaction was a 7S complex that could be detected and recovered after sucrose-density-gradient centrifugation in 0.3M-KCl. The extent of the reaction was dependent on the concentration of methyl 4-mercaptobutyrimidate and independent of the presence of bound hormone, since unlabelled receptors could also be cross-linked. The cross-linking reaction required conditions in which the cytosol 6S complex was preserved. A Stokes radius of 7.3 nm was determined by gel filtration in Agarose A-1.5 m in 0.3 M-KCl. The sedimentation coefficient, which was also determined in 0.3 M-KCl, allowed us to calculate a mol. wt. of 228,000. We were also able to cross-link partially purified receptor forms isolated by using an Agarose A-15 m column. On reduction with beta-mercaptoethanol the complex broke down to 4S monomers that were identified by DEAE-cellulose and phosphocellulose chromatography, adsorption on DNA-cellulose and gel filtration in an Agarose A-1.5 m column. In most cases, A and B receptor proteins were released in equivalent amounts, implying that the cross-linked form was an A-B complex.
D000818:Animals; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002621:Chemistry; D002645:Chickens; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D046911:Macromolecular Substances; D010057:Oviducts / Q000032:analysis*; D011980:Receptors, Progesterone; D013438:Sulfhydryl Compounds; D014461:Ultracentrifugation
D046911:Macromolecular Substances; D011980:Receptors, Progesterone; D013438:Sulfhydryl Compounds