A new type of O(2)(-)-generating tool for oxidative stress studies by remodeling neutrophil NADPH oxidase.
The effects of reactive oxygen species on cells have attracted much attention in relation to redox regulation and oxidative stress-related diseases. Superoxide (O(2)(-)) is the reactive oxygen species primarily formed in biological systems. However, no convenient O(2)(-)-generating device has been available for use in cell or tissue culture. The neutrophil NADPH oxidase, a professional enzyme for killing bacteria, has a high ability to produce O(2)(-). However, the cell-free activation process requires several protein factors and an anionic amphiphile, and moreover, the activation is transient. To utilize the enzyme as an O(2)(-) generator, we improved the cell-free activation method by remodeling regulatory components, optimizing lipid composition, and modifying the mixing conditions. We established a new method to produce an active enzyme that is stable, efficient, and preservable. As an application, we examined the effect of the device on cultured HEK293 cells and observed that it caused cell death. This system has several advantages over the xanthine oxidase system often used. The new device will be useful for studies of oxidative stress and related diseases.
Tamura
Minoru
M
Department of Applied Chemistry, Faculty of Engineering, Ehime University, Matsuyama, Ehime 790-8577, Japan. tamura@eng.ehime-u.ac.jp
Nagasawa
Teruaki
T
Tange
Tomoyuki
T
Miyano
Kei
K
Kobayashi
Shin-Ichi
S
Nakai
Toshiki
T
Miura
Satoshi
S
D000818:Animals; D002460:Cell Line; D002474:Cell-Free System / Q000201:enzymology; D006801:Humans; D019255:NADPH Oxidases / Q000737:chemistry* / Q000235:genetics; D009504:Neutrophils / Q000201:enzymology*; D018384:Oxidative Stress; D013481:Superoxides / Q000378:metabolism*; D013552:Swine; D014969:Xanthine Oxidase / Q000737:chemistry
D013481:Superoxides; D014969:Xanthine Oxidase; D019255:NADPH Oxidases