Superoxide perturbation of the organization of vascular endothelial cell membranes.
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.
D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D001013:Aorta, Thoracic / Q000166:cytology; D003497:Cyclic N-Oxides / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D004727:Endothelium / Q000166:cytology*; D007163:Immunosorbent Techniques; D008560:Membrane Fluidity; D008565:Membrane Proteins / Q000378:metabolism; D011817:Rabbits; D013481:Superoxides / Q000378:metabolism*; D013552:Swine
D000345:Affinity Labels; D003497:Cyclic N-Oxides; D008565:Membrane Proteins; D013481:Superoxides; C017480:4-maleimido-2,2,6,6-tetramethylpiperidinooxyl