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Evaluation of the importance of transamination versus deamination in astrocytic metabolism of [U‐ 13 C] glutamate
Author(s) -
Westergaard Niels,
Drejer Joan,
Schousboe Arne,
Sonnewald Ursula
Publication year - 1996
Publication title -
glia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.954
H-Index - 164
eISSN - 1098-1136
pISSN - 0894-1491
DOI - 10.1002/(sici)1098-1136(199606)17:2<160::aid-glia7>3.0.co;2-6
Subject(s) - transamination , oxidative deamination , glutamine , aminooxyacetic acid , biochemistry , glutamate receptor , citric acid cycle , biology , metabolism , transaminase , glutamine synthetase , glutamate dehydrogenase , glutamate aspartate transporter , deamination , alanine , amino acid , metabotropic glutamate receptor , enzyme , receptor
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‐ 13 C]glutamate (0.5 mM) in the presence and absence of the transaminase inhibitor aminooxyacetic acid (AOAA) and in some cases with methionine sulfoximine, an 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 13 C‐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 due to 12 C incorporation into the C‐4 and C‐5 positions. Incorporation of 12 C 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‐ 13 C]glutamate 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. © 1996 Wiley‐Liss, Inc.