Freeman B A, Rosen G M, Barber M J
J Biol Chem. 1986 May 15;261(14):6590-3.
Cultured porcine thoracic aorta endothelial cells were covalently labeled with 4-maleimido-2,2,6,6-tetramethylpiperidinooxyl. Electron paramagnetic resonance spectrometry revealed two major binding environments representing strongly and weakly immobilized species. The disorder parameter of weak/strong, determined from the respective peak amplitudes, was irreversibly elevated following incubation of endothelial cells with a superoxide-generating system, indicating increased membrane fluidity. The rate of increase in membrane disorder was dependent upon superoxide generation rates. Incorporation of the spin-label at concentrations less than 250 microM had no effect on cell viability. The cellular proteins reacting with the spin-label were predominantly membrane proteins, characterized by immunoblotting using a rabbit anti-4-maleimido-2,2,6,6-tetramethylpiperidinooxyl IgG, following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophorectic transfer to nitrocellulose.
将培养的猪胸主动脉内皮细胞用4-马来酰亚胺基-2,2,6,6-四甲基哌啶氮氧自由基进行共价标记。电子顺磁共振光谱显示出两种主要的结合环境，分别代表强固定化和弱固定化物种。根据各自的峰幅度确定的弱/强无序参数，在内皮细胞与超氧化物生成系统孵育后不可逆地升高，表明膜流动性增加。膜无序度的增加速率取决于超氧化物的生成速率。以低于250 microM的浓度掺入自旋标记对细胞活力没有影响。与自旋标记反应的细胞蛋白主要是膜蛋白，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和电泳转移至硝酸纤维素膜后，用兔抗4-马来酰亚胺基-2,2,6,6-四甲基哌啶氮氧自由基IgG进行免疫印迹分析来表征。
