Obesity and oxidative stress: involvement of SOD in the physiology of metabolic syndrome and atherosclerosis

纯化了重组细胞外超氧化物歧化酶，并对其肝素亲和力进行了研究。从473mL表达重组EC-SOD细胞株HECY6-7的培养基中，经层析、肝素亲和、羟基磷灰石、凝胶过滤三步纯化出野生型人酶104微克。去除n -聚糖降低了ConA的反应性，提高了酶的比活性和肝素亲和力。肝素结合域的两个突变研究（氨基酸替换，Glu237Lys和加倍碱性氨基酸簇，KERKKRRRESEC => KERKKRRRESECKEKKRRRESEC）导致肝素结合EC-SOD升高。另外两个改变n -聚糖位置的突变（N121Q/Q207N和N121Q/S234N）影响了酶对肝素的亲和力。这些结果表明，n -聚糖的结构和位置在EC-SOD的功能和定位中起着重要作用。

Recombinant extracellular superoxide dismutases were purified and the heparin affinity was investigated. 104 micrograms of the wild-type human enzyme was purified from 473mL of cultured medium of recombinant EC-SOD expressing cell line HECY6-7 through three steps of chromatography, heparin-affinity, hydroxyapatite, and gel-filtration. Removing the N-glycan reduced reactivity of ConA, and increased both of specific activity and heparin affinity of the enzyme. Two mutation studies at heparin binding domain (substitution of amino acid, Glu237Lys and doubling basic amino acid cluster, KERKKRRRESEC => KERKKRRRESECKEKKRRRESEC) gave rise to higher heparin binding EC-SOD. Another two mutation that moved the position of N-glycan (N121Q/Q207N and N121Q/S234N) affected the heparin affinity nature of the enzyme. These results suggest that the structure and position of N-glycan play an important role in the function and localization of EC-SOD.