We have developed a method using novel latex beads for rapid identification of drug receptors using affinity purification. Composed of a glycidylmethacrylate (GMA) and styrene copolymer core with a GMA polymer surface, the beads minimize nonspecific protein binding and maximize purification efficiency. We demonstrated their performance by efficiently purifying FK506-binding protein using FK506-conjugated beads, and found that the amount of material needed was significantly reduced compared with previous methods. Using the latex beads, we identified a redox-related factor, Ref-1, as a target protein of an anti-NF-kappaB drug, E3330, demonstrating the existence of a new class of receptors of anti-NF-kappaB drugs. Our results suggest that the latex beads could provide a tool for the identification and analysis of drug receptors and should therefore be useful in drug development.

Reduction-oxidation (redox) regulation has been implicated in the activation of the transcription factor NF-B. However, the significance and mechanism of the redox regulation remain elusive, mainly due to the technical limitations caused by rapid proton transfer in redox reactions and by the presence of many redox molecules within cells. Here we establish versatile methods for measuring redox states of proteins and their individual cysteine residues in vitro and in vivo, involving thiolmodifying reagents and LC-MS analysis. Using these methods, we demonstrate that the redox state of NF-B is spatially regulated by its subcellular localization. While the p65 subunit and most cysteine residues of the p50 subunit are reduced similarly in the cytoplasm and in the nucleus, Cys-62 of p50 is highly oxidized in the cytoplasm and strongly reduced in the nucleus. The reduced form of Cys-62 is essential for the DNA binding activity of NF-B. Several lines of evidence suggest that the redox factor Ref-1 is involved in Cys-62 reduction in the nucleus. We propose that the Ref-1-dependent reduction of p50 in the nucleus is a necessary step for NF-B activation. This study also provides the first example of a drug that inhibits the redox reaction between two specific proteins.The redox states of cysteine residues, which can change reversibly within cells, often greatly influence the various properties of proteins, such as protein stability, chaperone activity, enzymatic activity, and protein structure (1-5). It has also been suggested that several transcription factors bind to their cognate sites in a redox-regulated manner. Well characterized cases include the prokaryotic transcription factors SoxR and OxyR, which function as oxidative stress sensors, their DNA binding activated through oxidation of critical cysteine residues (6 -7). In most cases, however, the roles and mechanisms of redox regulation are not fully defined because it is difficult to monitor the alteration of redox states of proteins mainly due to the rapid proton transfer in redox reactions. A few have directly quantified the redox state of cysteine clustered with iron or amounts of oxidized cysteines using physicochemical or biochemical techniques (3, 8 -9), but these methods cannot describe the whole picture of redox states of a protein and are not widely applicable to other proteins. Therefore, most researchers have chosen an indirect way of using cysteine-substitution mutant proteins (3-5, 7).NF-B 1 is a eukaryotic transcription factor that regulates a wide variety of genes involved in immune function and development (10). NF-B is composed of two subunits, p50 and p65, both of which are members of the Rel family of transcription factors. NF-B normally exists in the cytoplasm, forming an inactive ternary complex with the inhibitor protein IB␣. Following the application of appropriate stimuli, NF-B is released from IB␣ and translocates into the nucleus, where it binds DNA and activates transcription of target genes. Mechanisms of NF-B activation have been exten...

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Phthalate esters are commonly used plasticizers; however, some are suspected to cause reproductive toxicity. Administration of high doses of di-(2-ethylhexyl) phthalate (DEHP) induces germ cell death in male rodents. Mono-(2-ethylhexyl) phthalate (MEHP), a hydrolyzed metabolite of DEHP, appears to be responsible for this testicular toxicity; however, the underlying mechanism of this chemical's action remains unknown. Here, using a one-step affinity purification procedure, we identified glycogen debranching enzyme (GDE) as a phthalate-binding protein. GDE has oligo-1,4-1,4-glucanotransferase and amylo-1,6-glucosidase activities, which are responsible for the complete degradation of glycogen to glucose. Our findings demonstrate that MEHP inhibits the activity of oligo-1,4-1,4-glucanotransferase, but not of amylo-1,6-glucosidase. Among various phthalate esters tested, MEHP specifically binds to and inhibits GDE. We also show that DEHP administration affects glycogen metabolism in rat testis. Thus, inhibition of GDE by MEHP may play a role in germ cell apoptosis in the testis.