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Hydrogen turnover and subcellular compartmentation of hepatic [2-13C]glutamate and [3-13C]aspartate as detected by 13C NMR

AuthorsGarcía-Martín, María L. ; García-Espinosa, María A.; Ballesteros, Paloma ; Bruix, M. ; Cerdán, Sebastián
Issue Date2002
PublisherAmerican Society for Biochemistry and Molecular Biology
CitationJournal of Biological Chemistry 277(10): 7799-7807 (2002)
Abstract13C NMR monitored the dynamics of exchange from specific hydrogens of hepatic [2-13C]glutamate and [3-13C]aspartate with deuterons from intracellular heavy water providing information on α-ketoglutarate/glutamate exchange and subcellular compartmentation. Mouse livers were perfused with [3-13C]alanine in buffer containing or not 50% 2H2O for increasing periods of time (1 min < t < 30 min). Liver extracts prepared at the end of the perfusions were analyzed by high resolution 13C NMR (150.13 MHz) with 1H decoupling only and with simultaneous 1H and 2H decoupling. 13C-2H couplings and 2H-induced isotopic shifts observed in the glutamate C2 resonance, allowed to estimate the apparent rate constants (forward, reverse; min−1) for (i) the reversible exchange of [2-13C]glutamate H2 as catalyzed mainly by aspartate aminotransferase (0.32, 0.56), (ii) the reversible exchange of [2-13C]glutamate H3proS as catalyzed by NAD(P) isocitrate dehydrogenase (0.1, 0.05), and (iii) the irreversible exchanges of glutamate H3proR andH3proS as catalyzed by the sequential activities of mitochondrial aconitase and NAD isocitrate dehydrogenase of the tricarboxylic acid cycle (0.035), respectively. A similar approach allowed to determine the rates of 1H-2H exchange for the H2 (0.4, 0.5) or H3proR (0.3, 0.2) or the H2 and H3proS hydrogens (0.20, 0.23) of [3-13C]aspartate isotopomers. The ubiquitous subcellular localization of 1H-2H exchange enzymes and the exclusive mitochondrial localization of pyruvate carboxylase and the tricarboxylic acid cycle resulted in distinctive kinetics of deuteration in the H2 and either or both H3 hydrogens of [2-13C]glutamate and [3-13C]aspartate, allowing to follow glutamate and aspartate trafficking through cytosol and mitochondria
Identifiersdoi: 10.1074/jbc.M107501200
issn: 0021-9258
e-issn: 1083-351X
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