2-4-dinitrophenylhydrazine and peroxynitric-acid

Topics: Aldehydes; Alzheimer Disease; Analysis of Variance; Biomarkers; DNA Damage; Glycation End Products, Advanced; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Histocytochemistry; Humans; Iron; Membrane Proteins; Neurodegenerative Diseases; Neurofibrillary Tangles; Neurons; Nitrates; Oxidation-Reduction; Oxidative Stress; Oxygen; Phenylhydrazines; Plaque, Amyloid; Tyrosine

The regulation of free intracellular calcium [Ca2+]i is altered in neurons from the aged brain, possibly due to reductions in the activity of Ca2+ transporters. The plasma membrane Ca(2+)-ATPase (PMCA) plays a critical role in Ca2+ homeostasis, and its kinetic properties change in aged rat brain. These changes could be due to oxidative modification of PMCA as a result of age-related chronic oxidative stresses. The present studies were undertaken to determine the sensitivity of the neuronal PMCA to in vitro exposure of synaptic plasma membranes (SPMs) to reactive oxygen species (ROS). We examined the effects of three oxidants including peroxyl radicals generated by azo-initiators, 2,2'-Azobis 2-amidinopropane dihydrochloride (AAPH) and 4,4'-Azobis 14-cyanovaleric acid (ACVA), hydrogen peroxide (H2O2), and peroxynitrite (ONOO-). Synaptic plasma membranes briefly exposed to these oxidants were analyzed for functional and structural alterations in PMCA. Although all three oxidants led to significant loss of PMCA activity, the effect of ONOO- was the most potent, followed by peroxyl radicals and H2O2. Kinetic analysis of PMCA activity after oxidant treatment showed decreases in Vmax without significant changes in K(act). Immunoblots revealed oxidant-induced cross-linking of PMCA molecules that were partially reversed under reducing conditions and completely reversed with addition of urea. The PMCA appears to be very sensitive to inhibition by ROS and hence may be a target of oxidative stress in the aging brain. Reduction in its activity may contribute to age-related alterations in neuronal [Ca2+]i regulation.

Topics: Age Factors; Amidines; Animals; Azo Compounds; Brain; Calcium; Calcium-Transporting ATPases; Hydrogen Peroxide; Kinetics; Male; Nitrates; Oxidative Stress; Phenylhydrazines; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Synaptic Membranes; Tyrosine; Urea; Valerates

Proteins are targets of reactive species and detection of oxidatively modified proteins is often used as an index of oxidative stress. Peroxynitrite is a strong oxidant formed by reaction of nitric oxide with superoxide. Using fatty acid-free bovine serum albumin as a model we examined peroxynitrite-mediated protein modifications. The reaction of protein with peroxynitrite resulted in the oxidation of tryptophan and cysteine, in the nitration of tyrosine, in the formation of dityrosine, in the production of 2,4 dinitrophenylhydrazine-reactive carbonyls and in protein fragmentation. The formation of 3-nitrotyrosine represents a specific peroxynitrite-mediated protein modification that is different from modifications mediated by reactive oxygen species.

Topics: Animals; Cysteine; Electrophoresis, Polyacrylamide Gel; Fluorescence; Hydrogen-Ion Concentration; Male; Nitrates; Oxidation-Reduction; Oxidative Stress; Phenylhydrazines; Rats; Rats, Sprague-Dawley; Serum Albumin, Bovine; Tryptophan; Tyrosine