Metabolism of [U-13C]Glutamine and [U-13C]Glutamate in Isolated Rat Brain Mitochondria Suggests Functional Phosphate-Activated Glutaminase Activity in Matrix
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.
KeywordsMass spectrometry Glutaminase PAG Heterogeneous
Liquid chromatography-mass spectrometry
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.
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