摘要
利用PCR定点突变技术构建人GSTp三种半胱氨酸突变体C^(47/101)、C^(14/47/101)和C^(14/47/101/169)。将CSTp 野生型和突变体表达质粒转染293细胞,以CDNB 为底物测定胞内GST 的转移酶活性,结果显示各类突变体均明显抑制了细胞内源性CST 的催化活性,具有显著的负显性(dominant nega-tive)突变体的功能;将CSTp 野生型和突变体与c-Jun、NF-kB 和p53的报告基因载体共转染,通过萤光素酶活性测定发现突变体C^(14/47/101)和C^(14/47/101/169)能明显激活c-Jun 和p21的转录活性;Western印迹分析显示突变体均能上调细胞内p21蛋白的水平,细胞存活率的测定表明GSTp 突变体能增强293细胞对H_2O_2刺激的敏感性;实验结果表明半胱氨酸残基对于维持GSTp 在对抗细胞氧化应激过程中的保护作用至关重要。
Site-directed mutagenesis was used to generate three cysteine mutants of GSTp,C^(47/101),C^(14/47/101) and C^(14/47/101/169).GSTp,C^(47/101.C14/47/101) and C^(14/47/101/169) were transfected into 293 cellsseparately and GST activity was deterrmined by using CDNB as substrate.Data showed that eachcysteine mutant inhibited endogenous GST catalyzatic activity and had remarkable dominant nega-tive effect.The expression vectors of wide type GSTp and its cysteine mutants were co-transfectedwith c-Jun,NF-kB,or p21 luciferase reporting vector,into 293 cells separately,luciferase activi-ty showed that C^(14/47/101) and C^(14/47/101/169) can dramatically activate c-Jnn and p21 transcriptional ac-tivity.Each cysteine mutant can increase endogenous p21 level,and also increased mortality rateof 293 cells when exposed to H_2O_2.These results suggest that cysteine residues of GSTp play animportant role in protecting cells against oxitative stress.
出处
《实验生物学报》
SCIE
CAS
CSCD
北大核心
2004年第3期176-182,共7页
Acta Biologiae Experimentalis Sinica
基金
国家自然科学基金(30270527)
国家教委留学回国人员主启动基金(2002SWXSBJBC22)~~
