TY - JOUR
T1 - Oxidatively induced structural alteration of glutamine synthetase assessed by analysis of spin label incorporation kinetics
T2 - Relevance to Alzheimer's disease
AU - Butterfield, D. Allan
AU - Hensley, Kenneth
AU - Cole, Pamela
AU - Subramaniam, Ramachandran
AU - Aksenov, Michael
AU - Aksenova, Marina
AU - Bummer, Paul M.
AU - Haley, Boyd E.
AU - Carney, John M.
PY - 1997/6
Y1 - 1997/6
N2 - 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-tetramethyl-pyrroline-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 second-order 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 β-amyloid 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.
AB - 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-tetramethyl-pyrroline-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 second-order 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 β-amyloid 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.
KW - Alzheimer's disease
KW - Glutamine synthetase
KW - Protein oxidation
KW - Spin labeling
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U2 - 10.1046/j.1471-4159.1997.68062451.x
DO - 10.1046/j.1471-4159.1997.68062451.x
M3 - Article
C2 - 9166739
AN - SCOPUS:0030921729
SN - 0022-3042
VL - 68
SP - 2451
EP - 2457
JO - Journal of Neurochemistry
JF - Journal of Neurochemistry
IS - 6
ER -