氧化还原对NDPK-A及突变体磷酸转移酶活性的影响  

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

作　　者：张志红[1] 吕芬[1] 高雪[1] 杨依丽[1] 罗勇[1] 熊盛[1] 

机构地区：[1]暨南大学生命科学技术学院生物医药研究开发基地,广州510632

出　　处：《生物技术通报》2012年第6期129-135,共7页Biotechnology Bulletin

基　　金：国家自然科学基金项目(30873082);国家科技支撑计划项目(2008BAI63B05);教育部新世纪优秀人才支持计划(NCET-07-0376);暨南大学211计划

摘　　要：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。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.

关 键 词：核苷二磷酸激酶A 氧化还原 磷酸转移酶活性 

分 类 号：R730.2[医药卫生—肿瘤]