The Effect Of The Redox On Phosphotransferase Actixity And Apoptosis Induction In A549Cell Of NDPK-A And Its Mutants

Objective: To express and purify nucleoside diphosphate kinase A(NDPK-A) and its cysteinemutants。To measure their phosphotransferase activity under the redox and normal conditionsand study the influence of the redox and disulfide bond on phosphotransferase activity ofNDPK-A and its mutants. Nm23-H1，its mutators were amplified and recombinated vectorspEGFP-nm23-H1C145S, pEGFP-nm23-H1C109S were prepared. In order to investigate theeffects of NDPK-A and its Cys mutants in apopotsis，we transiently transfected recombitantplamids pEGFP-nm23-H1, pEGFP-nm23-H1C145S,pEGFP-nm23-H1C109S into A549cells.Methods: Wide type NDPK-A and its mutants proteins were expressed in E. coli and purified byDEAE-sepharose Fast Flow and Cibacron Blue3GA Sepharose CL-4B,their phosphotransferaseactivity under the redox and normal conditions were detected by reverse high performance liquidchromatography (HPLC). Taking pBV220-nm23-H1C145S, pBV220-nm23-H1C109S astemplates, the mutators were amplified using corresponding primers respectively and insertedinto the vector pEGFP after digested with BamH I and EcoR I. After sequencing, pEGFP-C1、pEGFP-nm23-H1、pEGFP-nm23-H1C145S、pEGFP-nm23-H1C109S were transientlytransfected into A549cells. Western blot was used to identify whether NDPK-A and its mutantswere expressed after the recombinated vectors were transfected into A549cells. In addition, thecells with green fluroence were sorted using flow cytometry to dectect the apoptosis of A549cells affected by NDPK-A and its mutants.Results: NDPK-A and its mutants were expressed in E.coli efficiently, the homogeneousrecombination proteins were obtained with the purity of98%；the phosphotransferase activity ofNDPK-A and its mutants under reducing conditions was higher than that under normalconditions; but lower than that under oxidizing conditions, obviously. We had succeeded inconstructing recombinated vectors, and the NDPK-A and its mutants were expressed after therecombinated vectors were transfected into A549cells. Flow cytometry showed that NDPK-Aand its mutants could urge the apoptosis of cancer cell A549.Conclusions: To a certain extent, redox affected the structure and phosphotransferaseactivity of NDPK-A and its mutants. And NDPK-A contains more complex mechanism of redoxregulation activity by the analysis of enzyme activities between mutants and wild type NDPK-A.We had succeeded in constructing recombinated vectors, its mutants were expressed and could urge the apoptosis of cancer cell A549.