Here we report and validate a simple method for measuring intracellular activities of glial glutamine synthetase (GS) and glutaminase (GLNase) in intact glial cells. These enzymes are responsible for glutamate and glutamine recycling in the brain, where glutamate and glutamine transport from the blood stream is strongly limited by the blood-brain barrier. The intracellular levels of glutamate and glutamine are dependent on activities of numerous enzymatic processes, including (i) cytosolic production of glutamine from glutamate by GS, (ii) production of glutamate from glutamine by GLNase that is primarily localized between mitochondrial membranes, and (iii) mitochondrial conversion of glutamate to the TCA intermediate α-ketoglutarate in the reactions of oxidative deamination and transamination. We measured intracellular activities of GS and GLNase by quantifying enzymatic interconversions of L-[3H]glutamate and L-[3H]glutamine in cultured rat astrocytes. The intracellular substrate and the products of enzymatic reactions were separated in one step using commercially available anion exchange columns and quantified using a scintillation counter. The involvement of GS and GLNase in the conversion of [3H]-labeled substrates was verified using irreversible pharmacological inhibitors for each of the enzymes, and additionally validated by measuring intracellular amino acid levels using an HPLC. Overall, this manuscript describes optimized conditions and pharmacological controls for measuring GS and GLNase activities in intact glial cells.
- glutamate-glutamine cycle
- glutamine synthetase
- Copyright © 2011, American Journal of Physiology - Cell Physiology