Determination of binding constant of transcription factor AP-1 and DNA - Application of inhibitors

The equilibrium binding and association kinetics of the foe-jun dimer (basi c and leucine zipper domain) to the AP-1 DNA were studied using a quantitat ive assay. The basic-region and leucine zipper (bZip) domain of c-fos was e xpressed as a fusion protein with glutathione S-transferase, and it was bou nd to glutathione-agarose. The GST-fused fos bZip region was allowed to for m a heterodimer with the bZip domain of c-jun, to which radiolabeled AP-1 n ucleotides were added. After thorough washing, the gel bound radioactivity was counted. The binding and dissociation rate constants (k(1) and k(-1)) o f the fos-jun dimer and DNA could be obtained from a time-course experiment . The association binding constant (K-1) was determined using both a thermo dynamic equation and kinetic parameters. Nordihydroguaiaretic acid (NDGA), momordin I, natural product inhibitors of the fos-jun/DNA complex formation , was applied to this jun-GST-fused fos system and it was found to decrease the apparent equilibrium binding of dimer and DNA. The thermodynamic const ant of dimer and inhibitor binding was also determined.