title: Photoaffinity labeling of peroxisome proliferator binding proteins in rat hepatocytes; dehydroepiandrosterone sulfate- and bezafibrate-binding proteins.
abstract: To detect the cellular sites which directly interact with peroxisome proliferators (PPs) and mediate their inducing effect on peroxisomal enzymes in rat hepatocytes, two kinds of radiolabeled ligands, AD12 (7alpha-N-(4-azido-2-hydroxy-5-iodo[125I]benzyl)-aminomethyl-5-and rostene-3beta-ol-17-one-O-3-sulfate) and BZ5 (2-[p-[2-(4'-azido-3',5'-diiodo[125I]benzamido-2'-hydroxy)ethyl]phenoxy] -2-methylpropionic acid), were developed for photoaffinity labeling. These compounds were derivatives of dehydroepiandrosterone sulfate (DHEAS) and bezafibrate, respectively, with an azido group as the photoreactive functional group. Upon UV-irradiation following incubation with rat liver cytosol and nuclei, both the ligands effectively radiolabeled several proteins analyzed by SDS-polyacrylamide gel electrophoresis/radioluminography. When [125I]AD12 was used at a concentration of 0.2 microM, two cytosolic proteins with molecular masses of 55 and 28 kDa and a nuclear protein of 40 kDa were specifically labeled, as coincubation with a 1000-fold excess of DHEAS inhibited labeling. Photoaffinity labeling of the cytosolic 28-kDa protein was also affected by Wy-14,643, but not by unsulfated dehydroepiandrosterone or androsterone sulfate, consistent with our previous findings obtained in competitive binding studies of [3H]DHEAS-binding detected in rat liver cytosol (Yamada et al. (1994) Biochim. Biophys. Acta 1224, 139-146). On the other hand, [125I]BZ5 specifically labeled a cytosolic protein of 31 kDa, which was inhibited by coincubation with bezafibrate, clofibric acid and Wy-14,643, but not with DHEAS. Thus, [125I]AD12 and [125I]BZ5 labeled several proteins which recognized DHEAS and bezafibrate, respectively, in rat liver cytosol and nuclei, providing a useful means to investigate PP-binding proteins.
authors.affiliation: Department of Clinical Biochemistry, Tokyo University of Pharmacy and Life Science, Hachioji, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Sugiyama
authors.forename: J
authors.initials: J
authors.lastname: Yamada
authors.forename: H
authors.initials: H
authors.lastname: Takama
authors.forename: Y
authors.initials: Y
authors.lastname: Kodama
authors.forename: T
authors.initials: T
authors.lastname: Watanabe
authors.forename: T
authors.initials: T
authors.lastname: Taguchi
authors.forename: T
authors.initials: T
authors.lastname: Suga
mesh_terms: D000345:Affinity Labels; D000818:Animals; D001629:Bezafibrate / Q000031:analogs & derivatives / Q000138:chemical synthesis; D002352:Carrier Proteins / Q000032:analysis*; D003687:Dehydroepiandrosterone / Q000031:analogs & derivatives / Q000138:chemical synthesis; D004790:Enzyme Induction; D047908:Intracellular Signaling Peptides and Proteins; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008830:Microbodies / Q000201:enzymology; D010777:Photochemistry; D051381:Rats
chemical_list: D000345:Affinity Labels; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; C052998:peroxisome proliferator-binding protein, rat; D003687:Dehydroepiandrosterone; D001629:Bezafibrate