Construction Of Nucleoside Diphosphate Kinase A Isomers And Preliminary Study On Its Bio-activity

Objective: In order to study the difference of enzymatic activity and anticancer activity among these rhNDPK-A redox isomers. NDPK-A was selected as the model molecule, and four rhNDPK-A redox isomers are prepared.Methods: We used the technique of PCR-based method for site-directed Mutagenesis with a synthetic oligonucleotide primer, where the codon for cysteine-4 of wild type nm23-H1 gene was replaced with serine. After sequencing, the mutant gene was inserted into the expression vector pBV220 to form the recombinant expression plasmid pBV-Nm23-H1 C4S;pBV-Nm23-H1 C109S;pBV-Nm23-H1 C145S;pBV-Nm23-H1 CallS. E.coli DH5αstrain transformed by the recombinant plasmid over expressed the product of the gene when it was induced at 42℃. A feasible method for obtaining the purified NDPK-A isomers was established by using DEAE-cellulose anion exchange chromatography and Cibaeron Blue affinity chromatography. The peptide maps of NDPK-A isomers were analyzed by matrix assisted laser desorption ionization time-of-flight MS (MALDI-TOF MS) to validate the site-mutation in protein level. The electrophoresis character of NDPK-A isomers were analyzed by Reduce and non-reduce SDS-PAGE; The NDP kinase activity of the purified NDPK-A isomers were measured by reverse-HPLC; The DNA cleavage (DNase) activity was analyzed; Effect of NDPK-A isomers on cell proliferation of K562 cells were assayed by MTT method.Results: We had constructed four NDPK-A isomers recombinant plasmid and prepared NDPK-A isomers by purification methods, the purity of purified NDPK-A isomers were over 97%; The peptide maps of NDPK-A isomers analyzed by MALDI-TOF MS demonstrated that the 3 cysteines of rhNDPK-A were mutated into serine respectively; The electrophoresis results suggested that 145-cysteine is the key residue to disulfide bond isomerism of NDPK-A; The enzymatic activity of NDPK-A isomers was measured by reverse-HPLC: NDPK-A:3558U/mg; C4S:3769U/mg; C109S:4191U/mg; C145S:3938U/mg; Ca11S:3359 U/mg. And the difference among NDPK-A isomers are follow:C109S＞C145S＞C4S＞NDPK-A wild type＞CallS; DNA cleavage activity analysis suggested that the DNase activity of NDPK-A C4S is higher than NDPK-A wildtype and other NDPK-A ismoers distinctly; MTT assay showed that NDPK-A isomers have no inhibitory action to cell proliferation of K562 cells.Conclusions: Four purified NDPK-A isomers were preparated. The difference of NDP kinase activity and DNase activity among NDPK-A wild type and NDPK-A isomers were analyzed. We also found that the NDP kinase activity and DNase activity of NDPK-A C109S and NDPK-A C4S is higher than other isomers respectively. Investigations in this paper provided no only materials for study on the correlation between structure and bioactivities; but also materials for studies on the activites regulation and mechanism of NDPK-A. Besides, A reasonable method to evaluate the DNase activity of NDPK-A were established.