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

点击翻译按钮获取中文摘要

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.