氧化还原对NDPK-A及突变体磷酸转移酶活性的影响

对核苷二磷酸激酶A(NDPK-A)及其4种半胱氨酸突变体进行诱导表达及纯化,测定它们在氧化还原条件及正常条件下的磷酸转移酶活性,研究氧化还原及二硫键异构对NDPK-A及突变体活性的影响。将实验室之前构建成功的野生型NDPK-A(PBV-NDPK-A)及4种突变型NDPK-A基因(PBV-NDPK-A C4S,PBV-NDPK-A C109S,PBV-NDPK-A C145S,PBV-NDPK-A C4/109/145S)在大肠杆菌中高效表达;以DEAE-sepharose Fast Flow离子交换层析与Cibacron Blue 3GA Sepharose CL-4B亲和层析技术纯化目的蛋白;HPLC法测定比较野生型NDPK-A及突变体在氧化还原和正常环境下磷酸转移酶活性。结果显示,NDPK-A及突变体在大肠杆菌中高效表达;经纯化分别获得了均一的NDPK-A蛋白及突变体蛋白,纯度均达到98%;在还原环境下NDPK-A及突变体的磷酸转移酶活性均高于正常环境下的活性,但是在氧化环境下的磷酸转移酶活性明显低于正常环境下。氧化还原环境对NDPK-A结构异构及磷酸转移酶活性有一定的影响,提示氧化还原环境可能调控NDPK-A二硫键的形成,影响蛋白的聚集状态,从而影响蛋白的磷酸转移酶活性,并且NDPK-A结构中可能有更为复杂的氧化还原调控酶活性机制。

Effect of the Redox on Phosphotransferase Actixity of NDPK-A and Its Mutants

It was to express and purify nucleoside diphosphate kinase A(NDPK-A) and its cysteine mutants,measure their phosphotransferase activity under the redox and normal conditions,and study the influence of redox and disulfide bond on phosphotransferase activity of NDPK-A and its mutants.Wide type NDPK-A and its mutants proteins were expressed in E.coli and purified by DEAE-sepharose Fast Flow and Cibacron Blue 3GA Sepharose CL-4B,and their phosphotransferase activity under the redox and normal conditions were detected by reverse high performance liquid chromatography(HPLC).Results showed that NDPK-A and its mutants were expressed in E.coli efficiently,the homogeneous recombination proteins were obtained with the purity of 98%.The phosphotransferase activity of NDPK-A and its mutants under reducing conditions was higher than that under normal conditions,but lower than that under oxidizing conditions,obviously.To a certain extent,redox affected the structure and phosphotransferase activity of NDPK-A and its mutants.NDPK-A contains complex mechanism of redox regulation activity by the analysis of enzyme activities between mutants and wild type NDPK-A.