Inactivation of astrocytic glutamine synthetase by hydrogen peroxide requires iron

Abstract

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 H 2O 2 directly inactivates GS or whether GS is primarily inactivated by hydroxyl radicals that are produced by the Fenton reaction when H 2O 2 reacts with ferrous iron. Exposure of purified sheep brain GS to supraphysiological concentrations of H 2O 2 (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 H 2O 2, 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 H 2O 2, with approximately 37% of activity remaining 3 h after incubation with 1 mM H 2O 2. 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 H 2O 2 indirectly via the Fenton reaction as it required the presence of chelatable intracellular iron.