Characterization of progesterone membrane binding sites from porcine liver probed with a novel azido-progesterone radioligand.
A new derivative of progesterone was synthesized for photoaffinity labelling of specific binding sites in porcine liver microsomes. Using progesterone-3-(O-carboxymethyl)-oximino-(3-125I-iodo-4-azidosa licylamidobutylamine) as a photoactivatable radioligand, selective covalent labelling of proteins was detected in porcine liver microsomes at apparent molecular weights of 90-100 kDa and 60-65 kDa by SDS-PAGE and subsequent radioautography. These proteins showed different ligand specifity for various steroids tested. On blue native polyacrylamide-gels three selectively labelled proteins were found corresponding to apparent molecular weights of approximately 310 kDa, approximately 215 kDa and approximately 75 kDa, respectively. Using two-dimensional electrophoresis for the analysis of these protein complexes, the 215 kDa-band could be correlated to the 90-100 kDa-band, while the 75 kDa-band may correspond to the 60-65 kDa-band at one-dimensional SDS-PAGE, respectively. One or more of these proteins may be involved in the rapid progesterone-induced increase of intracellular Ca2+ described previously in cultured hepatic cells.
Eisen
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C
Division of Clinical Pharmacology, Medizinische Klinik, Klinikum Innenstadt, University of Munich, Germany.
Meyer
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C
Wehling
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M
D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D018929:Cell Culture Techniques; D007457:Iodine Radioisotopes / Q000378:metabolism; D008024:Ligands; D008565:Membrane Proteins / Q000378:metabolism*; D008862:Microsomes, Liver / Q000378:metabolism*; D011374:Progesterone / Q000031:analogs & derivatives / Q000378:metabolism*; D013256:Steroids / Q000378:metabolism; D013552:Swine
D000345:Affinity Labels; D007457:Iodine Radioisotopes; D008024:Ligands; D008565:Membrane Proteins; D013256:Steroids; D011374:Progesterone