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Evaluation of the importance of transamination versus deamination in astrocytic metabolism of [U- 13C] glutamate

✍ Scribed by Niels Westergaard; Joan Drejer; Arne Schousboe; Ursula Sonnewald

Publisher
John Wiley and Sons
Year
1996
Tongue
English
Weight
900 KB
Volume
17
Category
Article
ISSN
0894-1491

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✦ Synopsis

Glutamate metabolism was studied in primary cultures of cerebral cortical astrocytes to determine the significance of transamination for the oxidative metabolism of glutamate. Cultures were incubated with [U-13C]glutamate (0.5 mM) in the presence and absence of the transaminase inhibitor aminooxyacetic acid (AOAA) and in some cases with methionine sulfoximine, a n inhibitor of glutamine synthetase. Perchloric acid extracts of the cells as well as redissolved lyophilized incubation media were subjected to nuclear magnetic resonance spectroscopy to identify 13C-labeled metabolites. Additionally, biochemical analyses were performed to quantify amino acids, lactate, citrate, and ammonia. Glutamine released into the medium and intracellular glutamate were labeled uniformly to a large extent, but the C-3 position showed not only the expected apparent triplet but also a doublet clue to IZC incorporation into the C-4 and C-5 positions. Incorporation of IZC into the C-4 and C-5 positions of glutamate and glutamine as well as labeling of lactate, citrate, malate, and aspartate could only arise via metabolism of [U-13Clglutamate through the tricarboxylic acid (TCA) cycle. Entry of the carbon skeleton of glutamate into the TCA cycle must proceed via 2-oxoglutarate. This conversion can occur as a transamination or an oxidative deamination. After blocking transamination with AOAA, metabolism of glutamate through the TCA cycle was still taking place since lactate labeling was only slightly reduced. Glutamate and glutamine synthesis from 2-oxoglutarate could, however, not be detected under this condition. It therefore appears that while glutamate dehydrogenase is important for glutamate degradation, glutamate biosynthesis occurs mainly as a transamination.

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