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 (7 α- N-(4-azido-2-hydroxy-5-iodo[ 125I]benzyl)-aminomethyl-5-androstene-3 β-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 μM, 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.