Photoaffinity labeling of the chick progesterone receptor proteins. Similar hormone binding domains detected after removal of proteolytic interference.
Chick progesterone receptor subunits A and B have been photoaffinity-labeled using [3H]R5020 ([17 alpha-methyl-3H]17,21-dimethyl-19-nor-pregn-4,9-diene-3,20-dione) by a modification of the procedures previously reported by our laboratory (Dure, L. S., IV, Schrader, W. T., and O'Malley, B. W. (1980) Nature (Lond.) 283, 784-786). [3H]R5020 binds to the same receptor sites as authentic progesterone, and has an apparent Kdiss of 2.0 nM. Use of a CuSO4 filter raised the coupling efficiency to 5% and labeled exclusively the receptor proteins. Smaller labeled macromolecules were found to be proteolytic fragments of receptors. The protease(s) could not be inhibited by any of the commonly used protease inhibitors. However, the proteolytic activity was completely removed by passage of crude receptor preparations through phosphocellulose columns. Receptor preparations, photoaffinity-labeled after this procedure, showed exclusively one radioactive band at Mr = 79,000 (subunit A) or Mr = 108,000 (subunit B) with no detectable side-reaction products. Labeled receptors A and B were digested with Staphylococcus aureus V8 protease to yield smaller [3H]R5020-protein fragments derived from both. Molecular weight estimates (Mr = 9,500) and apparent isoelectric points indicate similarities of these regions of both A and B. The photoaffinity protocol described here thus provides a method for study of the hormone-binding domain of progesterone receptors and of receptor proteolysis in crude extracts.
D000345:Affinity Labels; D000818:Animals; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007700:Kinetics; D008970:Molecular Weight; D009649:Norpregnadienes / Q000378:metabolism*; D010057:Oviducts / Q000378:metabolism*; D011397:Promegestone / Q000378:metabolism*; D011980:Receptors, Progesterone / Q000302:isolation & purification / Q000378:metabolism* / Q000528:radiation effects; D014316:Tritium; D014466:Ultraviolet Rays
D000345:Affinity Labels; D009649:Norpregnadienes; D011980:Receptors, Progesterone; D014316:Tritium; D011397:Promegestone