Abstract
One of the forms of phosphate activated glutaminase (PAG) is associated with the inner mitochondrial membrane. It has been debated whether glutamate formed from glutamine in the reaction catalyzed by PAG has direct access to mitochondrial or cytosolic metabolism. In this study, metabolism of [U-13C]glutamine (3 mM) or [U-13C]glutamate (10 mM) was investigated in isolated rat brain mitochondria. The presence of a functional tricarboxylic (TCA) cycle in the mitochondria was tested using [U-13C]succinate as substrate and extensive labeling in aspartate was seen. Accumulation of glutamine into the mitochondrial matrix was inhibited by histidine (15 mM). Extracts of mitochondria were analyzed for labeling in glutamine, glutamate and aspartate using liquid chromatography-mass spectrometry. Formation of [U-13C]glutamate from exogenous [U-13C]glutamine was decreased about 50% (P < 0.001) in the presence of histidine. In addition, the 13C-labeled skeleton of [U-13C]glutamine was metabolized more vividly in the tricarboxylic acid (TCA) cycle than that from [U-13C]glutamate, even though glutamate was labeled to a higher extent in the latter condition. Collectively the results show that transport of glutamine into the mitochondrial matrix may be a prerequisite for deamidation by PAG.
Similar content being viewed by others
Abbreviations
- LC-MS:
-
Liquid chromatography-mass spectrometry
- PAG:
-
Phosphate-activated glutaminase
- TCA:
-
Tricarboxylic acid
References
Kvamme E, Torgner IA, Roberg B (2001) Kinetics and localization of brain phosphate activated glutaminase. J Neurosci Res 66:951–958
Roberg B, Torgner IA, Laake J, Takumi Y, Ottersen OP, Kvamme E (2000) Properties and submitochondrial localization of pig and rat renal phosphate-activated glutaminase. Am J Physiol Cell Physiol 279:C648–C657
Albrecht J, Dolinska M, Hilgier W, Lipkowski AW, Nowacki J (2000) Modulation of glutamine uptake and phosphate-activated glutaminase activity in rat brain mitochondria by amino acids and their synthetic analogues. Neurochem Int 36:341–347
Laake JH, Takumi Y, Eidet J, Torgner IA, Roberg B, Kvamme E, Ottersen OP (1999) Postembedding immunogold labelling reveals subcellular localization and pathway-specific enrichment of phosphate activated glutaminase in rat cerebellum. Neuroscience 88:1137–1151
Schousboe A, Hertz L, Svenneby G, Kvamme E (1979) Phosphate activated glutaminase activity and glutamine uptake in primary cultures of astrocytes. J Neurochem 32:943–950
McKenna MC, Waagepetersen HS, Schousboe A, Sonnewald U (2006) Neuronal and astrocytic shuttle mechanisms for cytosolic-mitochondrial transfer of reducing equivalents: current evidence and pharmacological tools. Biochem Pharmacol 71:399–407
Palaiologos G, Hertz L, Schousboe A (1988) Evidence that aspartate aminotransferase activity and ketodicarboxylate carrier function are essential for biosynthesis of transmitter glutamate. J Neurochem 51:317–320
Waagepetersen HS, Sonnewald U, Gegelashvili G, Larsson OM, Schousboe A (2001) Metabolic distinction between vesicular and cytosolic GABA in cultured GABAergic neurons using 13C magnetic resonance spectroscopy. J Neurosci Res 63:347–355
Roberg B, Torgner IA, Kvamme E (1995) The orientation of phosphate activated glutaminase in the inner mitochondrial membrane of synaptic and non-synaptic rat brain mitochondria. Neurochem Int 27:367–376
Zieminska E, Hilgier W, Waagepetersen HS, Hertz L, Sonnewald U, Schousboe A, Albrecht J (2004) Analysis of glutamine accumulation in rat brain mitochondria in the presence of a glutamine uptake inhibitor, histidine, reveals glutamine pools with a distinct access to deamidation. Neurochem Res 29:2121–2123
Lai JC, Clark JB (1976) Preparation and properties of mitochondria derived from synaptosomes. Biochem J 154:423–432
Lai JC, Walsh JM, Dennis SC, Clark JB (1977) Synaptic and non-synaptic mitochondria from rat brain: isolation and characterization. J Neurochem 28:625–631
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
Biemann K (1962) The mass spectra of isotopically labeled molecules. In: Mass spectrometry; organic chemical applications. McGraw-Hill, New York, pp 223–227
Bak LK, Schousboe A, Sonnewald U, Waagepetersen HS (2006) Glucose is necessary to maintain neurotransmitter homeostasis during synaptic activity in cultured glutamatergic neurons. J Cereb Blood Flow Metab 26:1285–1297
Drejer J, Larsson OM, Kvamme E, Svenneby G, Hertz L, Schousboe A (1985) Ontogenetic development of glutamate metabolizing enzymes in cultured cerebellar granule cells and in cerebellum in vivo. Neurochem Res 10:49–62
Mason GF, Rothman DL, Behar KL, Shulman RG (1992) NMR determination of the TCA cycle rate and alpha-ketoglutarate/glutamate exchange rate in rat brain. J Cereb Blood Flow Metab 12:434–447
Berkich DA, Xu Y, LaNoue KF, Gruetter R, Hutson SM (2005) Evaluation of brain mitochondrial glutamate and alpha-ketoglutarate transport under physiologic conditions. J Neurosci Res 79:106–113
Bak LK, Waagepetersen HS, Melo TM, Schousboe A, Sonnewald U (2007) Complex Glutamate Labeling from [U-13C]glucose or [U-13C]lactate in Co-cultures of Cerebellar Neurons and Astrocytes. Neurochem Res 32:671–680
Chatziioannou A, Palaiologos G, Kolisis FN (2003) Metabolic flux analysis as a tool for the elucidation of the metabolism of neurotransmitter glutamate. Metab Eng 5:201–210
Waagepetersen HS, Qu H, Sonnewald U, Shimamoto K, Schousboe A (2005) Role of glutamine and neuronal glutamate uptake in glutamate homeostasis and synthesis during vesicular release in cultured glutamatergic neurons. Neurochem Int 47:92–102
Dolinska M, Albrecht J (1997) Glutamate uptake is inhibited by L-arginine in mitochondria isolated from rat cerebrum. Neuroreport 8:2365–2368
Haussinger D, Soboll S, Meijer AJ, Gerok W, Tager JM, Sies H (1985) Role of plasma membrane transport in hepatic glutamine metabolism. Eur J Biochem 152:597–603
Acknowledgments
The following granting agencies, The Danish State Medical Research Council (22-03-0250; 22-04-0314), the Polish Ministry of Science and Information (Grant 2P05A 066 28) and the Hørslev, Lundbeck and Alfred Benzon Foundations are cordially acknowledged for providing financial support. Ms Lene Vigh is cordially acknowledged for excellent technical assistance.
Author information
Authors and Affiliations
Corresponding author
Additional information
Special issue article in honor of Dr. Frode Fonnum.
Lasse K. Bak and Elżbieta Ziemińska contributed equally to the experimental work described in this paper.
Rights and permissions
About this article
Cite this article
Bak, L.K., Ziemińska, E., Waagepetersen, H.S. et al. Metabolism of [U-13C]Glutamine and [U-13C]Glutamate in Isolated Rat Brain Mitochondria Suggests Functional Phosphate-Activated Glutaminase Activity in Matrix. Neurochem Res 33, 273–278 (2008). https://doi.org/10.1007/s11064-007-9471-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11064-007-9471-1