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核苷二磷酸激酶A(nucleoside diphosphate kinase A,NDPK-A)有广泛的生物学活性,在肿瘤转移和调控中起重要作用.单独抑制NDPK-A中任何1个Cys所形成的二硫键,不会降低NDPK-A的磷酸转移酶活性和DNase活性.本实验通过构建C4/109/145S突变体并研究其生物学效应,为NDPK-A结构与功能的研究提供参考.用定点突变法将NDPK-A的4位、109位和145位Cys突变为Ser,构建pBV220-NDPK-A C4/109/145S和pEGFP-NDPK-A C4/109/145S两种重组质粒.在大肠杆菌中高效表达NDPK-A C4/109/145S突变体,纯化后可获得均一的重组NDPK-A C4/109/145S突变体蛋白.HPLC法和DNA消化法测定发现,C4/109/145S突变体磷酸转移酶活性低于野生型NDPK-A,而DNase活性高于野生型NDPK-A.以A549细胞作为模式细胞的流式细胞仪周期检测表明,C4/109/145S突变体与野生型NDPK-A一致,均可将细胞周期延滞在S期和G2/M期.这些结果证实,NDPK-A结构异构与其磷酸转移酶活性密切相关,其酶活性至少需要1个Cys残基存在,NDPK-A结构中的二硫键也可能是其DNase活性的负调控机制之一,胞内NDPK-A的氧化还原异构可能对细胞周期无显著影响.
Nucleoside diphosphate kinase A (NDPK-A) has a wide range of biological activity and plays an important role in tumor metastasis and regulation.It inhibits the disulfide bond formed by any one Cys in NDPK-A alone, Will not reduce the phosphatase activity and DNase activity of NDPK-A.In this experiment, C4 / 109 / 145S mutants were constructed and their biological effects were studied to provide a reference for the study of the structure and function of NDPK-A.Using site-directed mutagenesis Two recombinant plasmids pBV220-NDPK-A C4 / 109 / 145S and pEGFP-NDPK-A C4 / 109 / 145S were constructed by mutation of Cys at position 4, 109 and 145 of NDPK-A into Ser. The recombinant plasmid was highly expressed in E.coli NDPK-A C4 / 109 / 145S mutants were obtained, and the recombinant NDPK-A C4 / 109 / 145S mutant protein was obtained after purification.It was found by HPLC and DNA digestion that the C4 / 109 / 145S mutant phosphotransferase activity Was lower than that of wild-type NDPK-A, while DNase activity was higher than that of wild-type NDPK-A. The flow cytometry cycle test using A549 cells as model cells showed that the C4 / 109 / 145S mutant was identical to wild-type NDPK- Can delay the cell cycle in the S phase and G2 / M phase.These results confirm that NDPK-A structural isoforms and its phosphotransferase activity is closely related, Its enzymatic activity needs at least one Cys residue exists. The disulfide bond in NDPK-A structure may also be one of the negative regulators of its DNase activity. The redox isoforms of intracellular NDPK-A may have no significant effect on cell cycle influences.