NADH-细胞色素b5还原酶突变型蛋白的结构与功能研究

目的 探讨NADH-细胞色素b5还原酶基因突变引起遗传性高铁血红蛋白血症的分子病理机制。研究突变型（b5R）蛋白结构和功能的关系。方法 用基因重组技术将野生型和突变型（L72P）b5R cDNA克隆于pGEX-2T载体，在大肠杆菌BL21中诱导表达。Western印迹鉴定表达的蛋白为CST-b5R融合蛋白。应用谷胱甘肽-Sepharose 4B亲扫层析，还原型谷胱甘肽洗脱得到纯化的GST-b5R和GST-b5R L72P融合蛋白，比较GST-b5R和GST-b5R L72P酶活性。结果 突变型酶活性低，为野生型的3％。结论 L72P突变可引起蛋白质二级结构改变从而导致酶的活性下降。

Study on structure and function of mutant type NADH-cytochrome b5 reductase

Objective To characterize the effect of L72P missense mutation on the enzymic function of NADH-cytochrome b5 reductase. Methods L72P mutant bSR cDNA was isolated from a patient with mutant type NADH-cytochrome b5 reductase by RT-PCR. The wild type and mutant bSR cDNAs were cloned into the expression vector pGEX-2T. The expressions of GST-fused wild type and L72P bSR mutant protein were analyzed by SDS-PAGE and Western blotting after inducing with IPTG for 4 hours. GST-fused wild type bSR and GST-fused L72P bSR were then purified with single-step affinity chromatography column of glutathione Sepharose 4B. The purity of both bSR proteins was analyzed by electrophoresis on PAGE. Results A single band with molecular mass of 58 kD on PAGE gel was obtained. By comparing with GST-fused b5 R and GST-fused b5 R L72P expressed in E. coli, the mutant enzyme activity was only 3% of the wild type enzyme. Conclusion The findings suggest that the L72P missense mutation may influence the secondary structure of NADH- cytochromc b5 reductase,which consequently results in decrease of enzymic activity.