Electron paramagnetic resonance investigations of free radical-induced alterations in neocortical synaptosomal membrane protein infrastructure.
Evidence is presented that free radical stress can directly induce physico-chemical alterations in rodent neocortical synaptosomal membrane proteins. Synaptosomes were prepared from gerbil cortical brain tissue and incubated with 3 mM ascorbate and various concentrations of exogenous Fe2+ for 30-240 min at 37 degrees C. Synaptosomes were then lysed and covalently labeled with the protein thiol-selective spin label MAL-6 (2,2,6,6-tetramethyl-4-maleimidopiperdin-1-oxyl) and subjected to electron paramagnetic resonance (EPR) spectrometry. In separate experiments, synaptosomal membranes were labeled with the thiol-specific spin label MTS ((1-oxyl-2,2,5,5-tetramethyl-pyrroline-3-methyl)-methanethiosulfonate), or the lipid-specific spin probe 5-NS (5-nitroxide stearate). Free radical stress induced by iron/ascorbate treatment has a rigidizing effect on the protein infrastructure of these membranes, as appraised by EPR analysis of membrane protein-bound spin label, but no change was detected in the lipid component of the membrane. These results are discussed with reference to potential oxidative mechanisms in aging and neurological disorders.
Hensley
K
K
Department of Chemistry, University of Kentucky, Lexington 40506.
Carney
J
J
Hall
N
N
Shaw
W
W
Butterfield
D A
DA
D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology*; D002540:Cerebral Cortex / Q000378:metabolism*; D003497:Cyclic N-Oxides; D004228:Dithionitrobenzoic Acid; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D005609:Free Radicals; D005849:Gerbillinae; D007501:Iron / Q000494:pharmacology*; D007700:Kinetics; D008297:Male; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D013113:Spin Labels; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism*
D003497:Cyclic N-Oxides; D005609:Free Radicals; D009419:Nerve Tissue Proteins; D013113:Spin Labels; C017480:4-maleimido-2,2,6,6-tetramethylpiperidinooxyl; D004228:Dithionitrobenzoic Acid; D007501:Iron; D001205:Ascorbic Acid