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

新型O_2^--发生装置的研制及其应用

基本信息

批准号：
13480297
负责人：
TAMURA Minoru
金额：
$ 7.94万
依托单位：
Ehime University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13480297/
关键词：
Superoxide Reactive oxygen NADPH oxidase Cytochrome b_<558>Noxl Duox family Neutrophils Fusion Proteins Small GTPase 低分子量Gタンパク Superoxide Reactive oxygen Cytochrome b_<558>duox family Neutrophilts Fusion protein Small GTPas

项目摘要

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.

1)新型产氧器的建立我们通过改进中性粒细胞NADPH氧化酶的调节成分，创造了一种新型的产氧器。对p47N和p67N之间的融合蛋白进行了基因工程，并将其与RAC的组成活性形式racQ61L一起用于无细胞重组，发现这种方法产生了非常稳定的酶。此外，我们通过优化细胞色素b_&lt；558&gt；的脂类组成，在没有刺激剂十二烷基硫酸钠的情况下成功地激活了该酶。当用高浓度的蛋白质组分激活酶并快速冷冻时，形成的复合体保持储存并在解冻后显示出全部活性。2)将新设备应用于细胞实验当该设备加入HEK细胞时，该设备经常用于淀粉样蛋白形成的实验，这是阿尔茨海默病的特征，细胞的增殖在设备溶液的1：5000稀释时停止，细胞死亡在设备的1：500稀释时发生。加入超氧化物歧化酶后未观察到这种现象，表明它们是由O2^-引起的。另一方面，神经元细胞系对O_2^-的抵抗力更强，但当加入较高浓度的设备时，细胞开始死亡。