Inactivation of astrocytic glutamine synthetase by hydrogen peroxide requires iron

The specific activity of brain glutamine synthetase (GS) is lowered in several neurodegenerative diseases that involve iron-mediated oxidative stress. The present study has investigated whether H₂O₂ directly inactivates GS or whether GS is primarily inactivated by hydroxyl radicals that are produced by the Fenton reaction when H₂O₂ reacts with ferrous iron. Exposure of purified sheep brain GS to supraphysiological concentrations of H₂O₂ (1 mM for 30 min) reduced its specific activity by only 41%, indicating that the enzyme is fairly resistant to oxidation by peroxide. However, the enzyme was completely inactivated when co-incubated with H₂O₂, iron and ascorbate, indicating a vulnerability to oxidation by conditions that favour the production of hydroxyl radicals. Similarly, specific GS activity in cultured mouse astrocytes was resistant to supraphysiological concentrations of H₂O₂, with approximately 37% of activity remaining 3h after incubation with 1mM H₂O₂. This inactivation was prevented by the iron chelators 2,2'-dipyridyl or 1,10-phenanthroline, but not by their non-chelating analogues. These data suggest that inactivation of astrocytic GS is caused by H₂O₂ indirectly via the Fenton reaction as it required the presence of chelatable intracellular iron.