Hydrogen Turnover and Subcellular Compartmentation of Hepatic
[2-13C]Glutamate and [3-13C]Aspartate as
Detected by 13C NMR*
María L.
García-Martín
,
María A.
García-Espinosa§,
Paloma
Ballesteros¶,
Marta
Bruix
, and
Sebastián
Cerdán**
From the Instituto de Investigaciones Biomédicas C.S.I.C.,
c/Arturo Duperier 4, E-28029 Madrid, Spain, the
¶ Department of Organic Chemistry and Biology UNED,
c/Senda del Rey 9 E-28040 Madrid, Spain, and the
Instituto de Estructura de la Materia C.S.I.C.,
c/Serrano 119, E-28006 Madrid, Spain
13C 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
-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 and
H3proS 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.
*
This work was supported in part by Grants SAF 2001-2245
from the Spanish Ministry of Science and Technology (MCT), Grants 08.1/0023/97 and 08.1/0046/98 from the Community of Madrid (to S. C. and P. B.), and strategic group Grant 2000-3 from the
Community of Madrid (to P. B.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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