Determination of binding constant of transcription factor AP-1 and DNA. Application of inhibitors.
The equilibrium binding and association kinetics of the fos-jun dimer (basic and leucine zipper domain) to the AP-1 DNA were studied using a quantitative assay. The basic-region and leucine zipper (bZip) domain of c-fos was expressed as a fusion protein with glutathione S-transferase, and it was bound to glutathione-agarose. The GST-fused fos bZip region was allowed to form a heterodimer with the bZip domain of c-jun, to which radiolabeled AP-1 nucleotides were added. After thorough washing, the gel-bound radioactivity was counted. The binding and dissociation rate constants (k(1) and k-(1)) of 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 thermodynamic 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 constant of dimer and inhibitor binding was also determined.
Kwon
H
H
School of Chemistry and Molecular Engineering, Seoul National University, Korea.
Park
S
S
Lee
S
S
Lee
D K
DK
Yang
C H
CH
D003001:Cloning, Molecular; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D004247:DNA / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D020128:Inhibitory Concentration 50; D007700:Kinetics; D009637:Masoprocol / Q000494:pharmacology; D008954:Models, Biological; D009828:Oleanolic Acid / Q000031:analogs & derivatives; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D016760:Proto-Oncogene Proteins c-fos / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012503:Saponins / Q000494:pharmacology; D012685:Sepharose / Q000737:chemistry / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D013816:Thermodynamics; D013997:Time Factors; D018808:Transcription Factor AP-1 / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism
D016861:Cyclooxygenase Inhibitors; D016760:Proto-Oncogene Proteins c-fos; D011993:Recombinant Fusion Proteins; D012503:Saponins; D018808:Transcription Factor AP-1; C408362:momordin I (Oleanolic Acid); D012965:Sodium Chloride; D009828:Oleanolic Acid; D009637:Masoprocol; D004247:DNA; D012685:Sepharose; D005982:Glutathione Transferase; D005978:Glutathione