OXIDATIVELY INDUCED STRUCTURAL ALTERATION OF GLUTAMINE-SYNTHETASE ASSESSED BY ANALYSIS OF SPIN-LABEL INCORPORATION KINETICS - RELEVANCE TO ALZHEIMERS-DISEASE

The activity of the astrocytic enzyme glutamine synthetase (GS) is dec reased in the Alzheimer's disease brain, which may have relevance to m echanisms of chronic excitotoxicity. The molecular perturbation(s) tha t results in GS inactivation is not known, although oxidative lesionin g 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/hy drogen peroxide and subsequently labeled with the thiol-specific nitro xide probe MTS [(1-oxyl-2,2,5,5-tetramethyl-pyrroline-3-methyl) methan ethiosulfonate]. The reaction of MTS with cysteine residues within GS was monitored in real time by electron paramagnetic resonance spectrom etry. Structural perturbation of GS, manifested as decreased thiol acc essibility, was inferred from an apparent decrease in the rate constan t for the second-order reaction of MTS with protein thiols. A subseque nt spin-labeling study was undertaken to compare the structural integr ity of GS purified and isolated from Alzheimer's disease-afflicted bra in (AD-GS) with that of GS isolated from nondemented, age-matched cont rol 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 wi th C-GS. The kinetic data were partially corroborated by spectroscopic data obtained from circular dichroism analysis of control and peroxid e-treated ovine GS. In an adjunct experiment, the interaction of GS wi th a synthetic analogue of the Alzheimer's-associated beta-amyloid pep tide, 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.