4-maleimido-2-2-6-6-tetramethylpiperidinooxyl and peroxynitric-acid

Peroxynitrite (ONOO ) is a highly reactive, oxidizing anion with a half-life of <1 s that is formed by reaction of superoxide radical anion with nitric oxide. Several reports of ONOO--induced oxidation of lipids, proteins, DNA, sulfhydryls, and inactivation of key enzymes have appeared. ONOO- has also been implicated as playing a role in the pathology of several neurodegenerative disorders, such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis, among others. Continuing our laboratory's interest in free radical oxidative stress in brain cells in AD, the present study was designed to investigate the damage to brain neocortical synaptosomal membrane proteins and the oxidation-sensitive enzyme glutamine synthetase (GS) caused by exposure to ONOO-. These synaptosomal proteins and GS have previously been shown by us and others to have been oxidatively damaged in AD brain and also following treatment of synaptosomes with amyloid beta-peptide. The results of the current study showed that exposure to physiological levels of ONOO- induced significant protein conformational changes, demonstrated using electron paramagnetic resonance in conjunction with a protein-specific spin label, and caused oxidation of proteins, measured by the increase in protein carbonyls. ONOO- also caused inactivation of GS and led to neuronal cell death examined in a hippocampal cell culture system. All these detrimental effects of ONOO- were successfully attenuated by the thiol-containing antioxidant tripeptide glutathione. This research shows that ONOO- can oxidatively modify both membranous and cytosolic proteins, affecting both their physical and chemical nature. These findings are discussed with reference to the potential involvement of ONOO- in AD neurodegeneration.

Topics: Alzheimer Disease; Animals; Cell Survival; Cells, Cultured; Cyclic N-Oxides; Cytosol; Gerbillinae; Glutathione; Hippocampus; Male; Membrane Proteins; Neurons; Nitrates; Nitric Oxide; Oxidants; Oxidation-Reduction; Oxidative Stress; Spin Labels; Synaptosomes

The effect of several oxidative agents (hydrogen peroxide, tert-butyl hydroperoxide, menadione, AAPH, peroxynitrite and ionizing radiation) on the ratio of weakly to strongly immobilized residues of erythrocyte membrane-bound maleimide-tempo spin label (h(w)/h(s) ratio) was studied in order to test the hypothesis that a decrease in the h(w)/h(s) ratio may be a general index of oxidative damage to membrane proteins. Most of the agents studied decreased though H2O2 almost did not affect and ionizing radiation increased the h(w)/h(s) ratio. In parallel, the ratio of DTNB accessible/DTNB inaccessible membrane protein-SH groups was determined from membrane-SH group measurements with the Ellman reagent in the absence and in the presence of sodium dodecyl sulfate. This ratio decreased in all cases studied and seems to be a more universal and easy to measure parameter to describe the oxidative damage to membrane proteins.

Topics: Adult; Amidines; Cyclic N-Oxides; Dithionitrobenzoic Acid; Electron Spin Resonance Spectroscopy; Erythrocyte Membrane; Gamma Rays; Humans; Hydrogen Peroxide; Membrane Proteins; Nitrates; Oxidants; Oxidative Stress; Peroxides; Spin Labels; Sulfhydryl Compounds; tert-Butylhydroperoxide; Vitamin K