PHOTOAFFINITY-LABELING OF PEROXISOME PROLIFERATOR BINDING-PROTEINS INRAT HEPATOCYTES - DEHYDROEPIANDROSTERONE SULFATE-BINDING AND BEZAFIBRATE-BINDING PROTEINS

To detect the cellular sites which directly interact with peroxisome p roliferators (PPs) and mediate their inducing effect on peroxisomal en zymes in rat hepatocytes, two kinds of radiolabeled ligands, AD12 (7 x y-5-iodo[I-125]benzyl)-aminomethyl-5-androstene-3 beta-ol-17-one-O-3-s ulfate) and BZ5 zamido-2'-hydroxy)ethyl]phenoxy]-2-methylpropionic aci d), were developed for photoaffinity labeling. These compounds were de rivatives of dehydroepiandrosterone sulfate (DHEAS) and bezafibrate, r espectively, 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 ana lyzed by SDS-polyacrylamide gel electrophoresis/radioluminography. Whe n [I-125]AD12 was used at a concentration of 0.2 mu M, two cytosolic p roteins with molecular masses of 55 and 28 kDa and a nuclear protein o f 40 kDa were specifically labeled, as coincubation with a 1000-fold e xcess of DHEAS inhibited labeling. Photoaffinity labeling of the cytos olic 28-kDa protein was also affected by Wy-14,643, but not by unsulfa ted dehydroepiandrosterone or androsterone sulfate, consistent with ou r previous findings obtained in competitive binding studies of [H-3]DH EAS-binding detected in rat liver cytosol (Yamada et al. (1994) Biochi m. Biophys, Acta 1224, 139-146). On the other hand, [I-125]BZ5 specifi cally labeled a cytosolic protein of 31 kDa, which was inhibited by co incubation with bezafibrate, clofibric acid and Wy-14,643, but not wit h DHEAS. Thus, [I-125]AD12 and [I-125]BZ5 labeled several proteins whi ch recognized DHEAS and bezafibrate, respectively, in rat liver cytoso l and nuclei, providing a useful means to investigate PP-binding prote ins.