Glutamate dehydrogenase reaction as a source of glutamic acid in synaptosomes.
The role of the glutamate dehydrogenase reaction as a pathway of glutamate synthesis was studied by incubating synaptosomes with 5 mM 15NH4Cl and then utilizing gas chromatography-mass spectrometry to measure isotopic enrichment in glutamate and aspartate. The rate of formation of [15N]glutamate and [15N]aspartate from 5 mM 15NH4Cl was approximately 0.2 nmol/min/mg of protein, a value much less than flux through glutaminase (4.8 nmol/min/mg of protein) but greater than flux through glutamine synthetase (0.045 nmol/min/mg of protein). Addition of 1 mM 2-oxoglutarate to the medium did not affect the rate of [15N]glutamate formation. O2 consumption and lactate formation were increased in the presence of 5 mM NH3, whereas the intrasynaptosomal concentrations of glutamate and aspartate were unaffected. Treatment of synaptosomes with veratridine stimulated reductive amination of 2-oxoglutarate during the early time points. The production of ([15N]glutamate + [15N]aspartate) was enhanced about twofold in the presence of 5 mM beta-(+/-)-2-aminobicyclo [2.2.1]heptane-2-carboxylic acid, a known effector of glutamate dehydrogenase. Supplementation of the incubation medium with a mixture of unlabelled amino acids at concentrations similar to those present in the extracellular fluid of the brain had little effect on the intrasynaptosomal [glutamate] and [aspartate]. However, the enrichment in these amino acids was consistently greater in the presence of supplementary amino acids, which appeared to stimulate modestly the reductive amination of 2-oxoglutarate. It is concluded: (a) compared with the phosphate-dependent glutaminase reaction, reductive amination is a relatively minor pathway of synaptosomal glutamate synthesis in both the basal state and during depolarization; (b) NH3 toxicity, at least in synaptosomes, is not referable to energy failure caused by a depletion of 2-oxoglutarate in the glutamate dehydrogenase reaction; and (c) transamination is not a major mechanism of glutamate nitrogen production in nerve endings.
Division of Biochemical Development and Molecular Disease, Children's Hospital of Philadelphia, Pennsylvania.
M
M
Yudkoff
I
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Nissim
D
D
Nelson
Z P
ZP
Lin
M
M
Erecińska
D000596:Amino Acids / Q000378:metabolism / Q000494:pharmacology; D000598:Amino Acids, Cyclic; D000641:Ammonia / Q000378:metabolism; D000818:Animals; D001224:Aspartic Acid / Q000096:biosynthesis; D004734:Energy Metabolism; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism; D005971:Glutamates / Q000096:biosynthesis / Q000378:metabolism*; D018698:Glutamic Acid; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D013574:Synaptosomes / Q000378:metabolism*; D014701:Veratridine / Q000494:pharmacology
D000596:Amino Acids; D000598:Amino Acids, Cyclic; D005971:Glutamates; C006504:2-aminobicyclo(2,2,1)heptane-2-carboxylic acid; D001224:Aspartic Acid; D018698:Glutamic Acid; D014701:Veratridine; D000641:Ammonia; D005969:Glutamate Dehydrogenase; D005974:Glutamate-Ammonia Ligase