Development of a new O_2^--generating device and its application

点击翻译按钮获取中文摘要

1) Establishment of a new O_2^- generating deviceWe created a new-type O_2^- generating nano-machine by improving the regulatory components of neutrophil NADPH oxidase. A fusion protein between p47N and p67N was genetically engineered and used in a cell-free reconstitution in combination with RacQ61L, a constitutively active form of Rac, and found that this method produced a extremely stabilized enzyme. Furthermore, we succeeded to activate the oxidase without a stimulator SDS by optimizing the lipid composition for relipidation of cytochrome b_<558>. When the enzyme was activated with high concentrations of protein components and frozen quickly, the formed complex was maintained in storage and showed a full activity after thawing.2) Application of the new device to cell experimentsWhen the device was added to HEK cells which has often been used for experiments of amyloid formation, a characteristic of Alzheimer's disease, proliferation of the cells stopped at 1 : 5000 dilution of the device solution and cell death took place at 1 : 500dilution of the device. Such phenomena was not observed when superoxide dismutase was added, indicating that they were caused by O_2^-. On the other hand, a neuron cell line was more resistant to O_2^-, but when added at a higher concentration of the device cells started to die.