The Rat Liver Glutathione S-Transferase Ya Subunit Gene: Characterization of the Binding Properties of a Nuclear Protein from HepG2 Cells That Has High Affinity for the Antioxidant Response Ele
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- 作者：Suxing Liu and Cecil B. Pickett
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：September 3, 1996
- 年：1996
- 卷：35
- 期：35
- 页码：11517 - 11521
- 全文大小：384K
- 年卷期：v.35,no.35(September 3, 1996)
- ISSN：1520-4995
文摘
A nuclear protein from HepG2 cells (YABP) that binds theantioxidant response element (ARE),which is required for activation of the rat glutathioneS-transferase (GST) Ya subunit gene by planararomatic compounds and phenolic antioxidants, was further characterizedby quantitative competitionbinding experiments and DNA mutational analysis. The apparentdissociation constant of the YABP-ARE complex was estimated as 
0.77 nM, suggesting that the YABP hasvery high affinity for the ARE.There is no difference in the affinity of the YABP for the AREwhen HepG2 cells are treated with inducersthat transcriptionally activate the GST Ya subunit gene.Quantitative competition binding analyses inconjunction with mutagenesis of the ARE revealed that an 11-nucleotideregion in the 41-nucleotide ARE,5'-GGTGACAAAGC-3', is responsible for binding to the YABP. Eightnucleotides of this core sequenceare in close proximity to the YABP, indicating that there is a broaderspectrum of protein contact pointsthan those required for the transcriptional activation. van'tHoff analysis of effects of temperature onbinding has revealed that the binding reaction is governed mainly byentropy changes, which could resultfrom conformational changes in the YABP and/or the ARE upon theformation of the complex. In addition,the native molecular weight of the YABP was determined to be 74 300using gel filtration chromatography.These data together with previous UV cross-linking data suggestthat the YABP exists as a heterodimer.
0.77 nM, suggesting that the YABP hasvery high affinity for the ARE.There is no difference in the affinity of the YABP for the AREwhen HepG2 cells are treated with inducersthat transcriptionally activate the GST Ya subunit gene.Quantitative competition binding analyses inconjunction with mutagenesis of the ARE revealed that an 11-nucleotideregion in the 41-nucleotide ARE,5'-GGTGACAAAGC-3', is responsible for binding to the YABP. Eightnucleotides of this core sequenceare in close proximity to the YABP, indicating that there is a broaderspectrum of protein contact pointsthan those required for the transcriptional activation. van'tHoff analysis of effects of temperature onbinding has revealed that the binding reaction is governed mainly byentropy changes, which could resultfrom conformational changes in the YABP and/or the ARE upon theformation of the complex. In addition,the native molecular weight of the YABP was determined to be 74 300using gel filtration chromatography.These data together with previous UV cross-linking data suggestthat the YABP exists as a heterodimer.