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KMID : 0380219990320060594
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Journal of Biochemistry and Molecular Biology
1999 Volume.32 No. 6 p.594 ~ p.598
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Quantitative Assay for the Binding of Jun-Fos Dimer and Activator Protein-1 Site
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Lee Sang-Kyou
Park Se-Yeon
Jun Gyo
Hahm Eun-Ryeong
Lee Dug-Keun
Yang Chul-Hak
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Abstract
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The Jun and Fos families of eukaryotic transcription factors form heterodimers capable of binding to their cognate DNA enhancer elements. We are interested in searching for inhibitors or antagonists of the binding of the Jun-Fos heterodimer to the activator protein-1 (AP-1) site. The basic-region leucine zipper (bZIP) domain of c-Fos was expressed as a fusion protein with glutathione S-transferase, and allowed to form a heterodimer with the bZIP domain of c-Jun. The heterodimer was bound to glutathione-agarose, to which were added radiolabeled AP-1 nucleotides. After thorough washing, the gel-bound radioactivity was counted. The assay is faster than the coventional electrophoretic mobility shift assay because the gel electrophoresis step and the autoradiography step are eliminated. Moreover, the assay is very sensitive, allowing the detection of picomolar quantities of nucleotides, and is not affected by up to 50% dimethylsulfoxide, a solvent for hydrophobic inhibitors. Curcumin and dihydroguaiaretic acid, recently known inhibitors of Jun-Fos-DNA complex formation, were applied to this Jun-GST-fused Fos system and revealed to decrease the dimer-DNA binding.
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KEYWORD
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AP-1, Curcumin, Dihydroguaiaretic acid, Glutathione agarose, GST-fused Fos
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