Four biomarkers of neuronal protein oxidation [W/S ratio of MAL‐6 spin‐labeled synaptosomes, phenylhydrazine‐reactive protein carbonyl content, glutamine synthetase (GS) activity, creatine kinase (CK) activity] in three brain regions [cerebellum, inferior parietal lobule (IPL), and hippocampus (HIP)] of Alzheimer's disease (AD)‐demented and age‐matched control subjects were assessed. These endpoints indicate that AD brain protein may be more oxidized than that of control subjects. The W/S ratios of AD hippocampal and inferior parietal synaptosomes are 30 and 46% lower, respectively, than corresponding values of tissue isolated from control brain; however, the difference between the W/S ratios of AD and control cerebellar synaptosomes is not significant. Protein carbonyl content is increased 42 and 37% in the Alzheimer's HIP and IPL regions, respectively, relative to AD cerebellum, whereas carbonyl content in control HIP and IPL is similar to that of control cerebellum. GS activity decreases an average of 27% in the AD brain; CK activity declines by 80%. The brain regional variation of these oxidation‐sensitive biomarkers corresponds to established histopathological features of AD (senile plaque and neurofibrillary tangle densities) and is paralleled by an increase in immunoreactive microglia. These data indicate that senile plaque‐dense regions of the AD brain may represent environments of elevated oxidative stress.

The activity of the astrocytic enzyme glutamine synthetase (GS) is decreased in the Alzheimer's disease brain, which may have relevance to mechanisms of chronic excitotoxicity. The molecular perturbation(s) that results in GS inactivation is not known, although oxidative lesioning of the enzyme is one likely cause. To assess structural perturbation induced in GS by metal-catalyzed oxidation, a series of spin-labeling studies were undertaken. Ovine GS was oxidized by exposure to iron/hydrogen peroxide and subsequently labeled with the thiol-specific nitroxide probe MTS [(1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)methanethiosulfonate]. The reaction of MTS with cysteine residues within GS was monitored in real time by electron paramagnetic resonance spectrometry. Structural perturbation of GS, manifested as decreased thiol accessibility, was inferred from an apparent decrease in the rate constant for the secondorder reaction of MTS with protein thiols. A subsequent spin-labeling study was undertaken to compare the structural integrity of GS purified and isolated from Alzheimer's disease-afflicted brain (AD-GS) with that of GS isolated from nondemented, age-matched control brain (C-GS). The rate constant for reaction of MTS with AD-GS was markedly decreased relative to that for the reaction of spin label with C-GS. The kinetic data were partially corroborated by spectroscopic data obtained from circular dichroism analysis of control and peroxide-treated ovine GS. In an adjunct experiment, the interaction of GS with a synthetic analogue of the Alzheimer's-associated ßamyloid peptide, known to induce free radical oxidative stress, indicated strong interaction of the enzyme with the peptide as reflected by a decrease in the rate constant for MTS binding to reactive protein thiols.

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The aim of this study was to investigate the role of mitochondrial ionic homeostasis in promoting reoxygenation-induced hypercontracture in cardiac muscle. Mitochondrial membrane potential and intramitochondrial Ca2+ concentration ([Ca2+]) were measured using confocal imaging in guinea pig ventricular myocytes exposed to anoxia and reoxygenation. Anoxia produced a variable, but often profound, mitochondrial depolarization. Some cells mounted a recovery of their mitochondrial membrane potential during reoxygenation; the depolarization was sustained in other cells. Recovery of the mitochondrial membrane potential seemed essential to avoid reoxygenation-induced hypercontracture. Reoxygenation also caused a sizable elevation in intramitochondrial [Ca2+], the amplitude of which was correlated with the likelihood of a cell undergoing hypercontracture. A sustained Ca2+load analogous to that seen during reoxygenation was imposed on cardiac mitochondria through permeabilization of the plasma membrane. Elevation of intracellular [Ca2+] to 800 nM caused a substantial mitochondrial depolarization. We propose that the conditions seen in guinea pig ventricular myocytes during reoxygenation are well suited to produce Ca2+-dependent mitochondrial depolarization, which may play a significant role in promoting irreversible cell injury.