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J. Biol. Chem., Vol. 277, Issue 10, 7799-7807, March 8, 2002

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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

Received for publication, August 6, 2001, and in revised form, December 13, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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; min¹) 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 H3_proS as catalyzed by NAD(P) isocitrate dehydrogenase (0.1, 0.05), and (iii) the irreversible exchanges of glutamate H3_proR and H3_proS 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 H3_proR (0.3, 0.2) or the H2 and H3_proS 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Hydrogen turnover in hepatic metabolites begins with the transport of water across the plasma membrane (1, 2). Cytosolic water is then distributed rapidly among different subcellular organelles (3, 4) experiencing a variety of enzyme catalyzed exchange reactions which replace stereospecifically, the pre-existing metabolite hydrogens with those from subcellular water (5, 6). The turnover completes when replaced metabolite hydrogens return to intracellular water through the same exchange reactions, eventually leaving the organelle and the cell. Classical studies analyzed the exchange between specific hydrogens of metabolites and those of the solvent, providing well established tools to investigate the kinetic mechanisms of enzymes in vitro (6, 7). Similarly, tritiated or deuterated water were used to probe metabolic turnover of fatty acids and carbohydrates (8, 9). However, the exchange of specific hydrogens from hepatic amino acids, like glutamate or aspartate, with hydrogens from intracellular water or its potential metabolic implications remained less explored.

¹³C NMR techniques have been used routinely in the last decades to investigate the turnover of individual metabolite carbons in vivo or in vitro in animals and humans by administering precursors selectively enriched in ¹³C (10, 11). Notably, most metabolite carbons are attached to one or more hydrogen atoms, a circumstance which should allow to extend the use of ¹³C NMR to analyze hydrogen turnover. To this end, we proposed earlier a double isotope labeling strategy which combined the administration of a ¹³C-labeled substrate and ²H₂O, monitoring hydrogen-deuterium exchange at steady state through the analysis of the geminal and vicinal¹ isotopic shifts or ²H-¹³C couplings detected in observable ¹³C resonances by high resolution ¹³C NMR (5, 12).

The present study reports on the kinetics of enzyme-catalyzed exchange of metabolite hydrogens with deuterons from intracellular heavy water with an emphasis on the exchanges occurring in the H2 and H3 hydrogens of hepatic [2-¹³C]glutamate and [3-¹³C]aspartate. The kinetics of ¹H-²H exchange in the H2 and H3 hydrogens of glutamate and aspartate depicted very rapid components, occurring significantly faster than previously reported time courses of ¹³C labeling in the neighboring carbons (13-17). The quicker time scale allowed to resolve in time, faster processes than previously accessible to conventional experiments of ¹³C turnover. In particular it became possible to observe directly -ketoglurate/glutamate exchange and oxalacetate/aspartate exchange as well as to follow in a time resolved manner, the sequence of events involved in the traffic of these metabolites through mitochondria and cytosol ex vivo. Preliminary accounts of this work have been reported (18, 19).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Perfusion Techniques and Experimental Design-- Male Swiss albino mice (30 ± 5 g) starved for 24 h but having access to drinking water ad libitum were used as donors. Mouse livers (2.5 ± 0.5 g) were isolated and perfused outside the magnet as described previously (12, 13, 20). Briefly, animals were anesthetized with Nembutal (50 mg kg¹ body weight), the abdomen opened and the portal vein cannulated. During cannulation and subsequent surgery, livers were pre-perfused in the flow-through mode with Krebs-Ringer bicarbonate (KRB) buffer (119 mM NaCl, 24 mM NaHCO₃, 4,7 mM KCl, 1,2 mM MgSO₄, 1,2 mM KHPO₄, 1,3 mM Ca₂Cl, 95% O₂, 5% CO₂, pH 7.4, ±0.04; 37 °C; 6 ml/min). After isolation of the liver from the animal carcass, the perfusion medium was changed to 6 mM [3-¹³C]alanine (99.9% ¹³C, Isotec Inc., Miamisburgh, OH) in KRB under recirculating conditions for 30 min. This time was sufficient to achieve steady state ¹³C labeling in glutamate, aspartate, and alanine carbons (12, 13, 21). After this equilibration period the perfusion medium was changed to 6 mM [3-¹³C]alanine in KRB containing 50% (v/v) ²H₂O (99.9% ²H, Appollo Scientific Ltd., Stockport, UK) maintaining the recirculation for 1, 3, 5, 7, 10, 15, or 30 min. During this perfusion period with 50% ²H₂O mouse livers maintained appropriate bioenergetics and metabolism (21). At the end of the perfusion, livers were freeze-clamped with aluminum tongs precooled in liquid nitrogen and stored at 70 °C until extraction. Perchloric acid extracts from these livers were prepared, neutralized with KOH, lyophilized, and resuspended in ²H₂O (99.9% ²H) prior to high resolution ¹³C NMR analysis (12). Total contents of glutamate, aspartate, and alanine of liver extracts obtained at the end of the perfusions were determined by automatic ionic exchange chromatography after ninhidrin derivatization (22). Hepatic oxygen consumption due to alanine oxidation was measured polarographicaly using Clark oxygen electrodes located before and after the liver in the perfusion circuit, correcting for the basal rate of endogenous oxygen consumption (23).

High Resolution ¹³C NMR-- Extracts from livers were analyzed (pH 7.2, 22 °C) by ¹³C NMR at 14.09 T (150.9 MHz) using a Bruker AMX-600 NMR spectrometer (Bruker Analystische Messtechnik, Rheinsteten, Germany). Acquisition conditions (150.9 MHz) were: /3 pulses, 33.3 KHz sweep width, 128 K data table (1.97 acquisition time), 7.97-s recycle time, and ~600 scans. To minimize nuclear Overhauser effects in quantitation, broad band proton decoupling was applied only during the acquisition. In addition, ¹H-decoupled and ²H-decoupled ¹³C NMR spectra were obtained from the same samples at 11.9 Tesla with a Bruker AVANCE 500 NMR Spectrometer operating at 125.76 MHz for ¹³C, equipped with a commercial crioprobe refrigerated with liquid nitrogen. Conditions were: /3 pulses, 25.06 KHz, 64 K data table, 6-s total cycle time, and 4096 scans. ²H decoupling was performed simultaneously with ¹H decoupling only during the acquisition, using the lock channel and a software driven lock switch. Chemical shifts were referred to that of a 10% dioxane solution (67.4 ppm) placed in a concentric coaxial capillary. At least four extracts from different livers were analyzed for every time point.

Determination of Relative (¹H, ²H, ¹³C) Isotopomer Populations-- Covalent binding of one or more ²H atoms to ¹³C results in the formation of ²H-¹³C couplings and ²H-induced isotopic shifts which split and shift the perprotonated ¹³C resonance into characteristic ²H-¹³C multiplets (5, 12, 24). Replacement of one geminal hydrogen by one deuterium splits the original ¹³C singlet into a 1:1:1 triplet (19.21 < ¹J_CH < 22 Hz) inducing a geminal isotopic shift (0.25 < ₁ < 0.33 ppm). Multiple replacements of geminal hydrogens with two or three deuterons result in additive isotopic shifts and ²H-¹³C coupling patterns of five or seven line multiplets, respectively. Smaller and additive isotopic shifts are also observed upon deuterium substitution of one or more vicinal hydrogens (0.03 < ₂ <  0.11 ppm). Vicinally shifted resonances maintain the multiplicity caused by the geminal couplings since vicinal couplings to ²H are too small to be resolved. Thus high resolution ¹³C NMR allows to determine the number of deuterium replacements, their relative contributions, and their geminal or vicinal location with respect to the observed ¹³C carbon of specific ¹³C isotopomers through the analysis of the shifted and unshifted (¹H,²H)¹³C multiplets (5, 12). This is conveniently accomplished using the simulation program WINDAISY (Bruker Analystische Messtechnik, Rheinstetten, Germany) which allows the quantitative determination of the relative contributions of the individual (¹H,²H)¹³C multiplets to the analyzed resonance.

Model of ¹H-²H Exchange in [2-¹³C]Glutamate and [3-¹³C]Aspartate Isotopomers-- Fig. 1 depicts the model used for the analysis of the exchange of the H2 and H3 hydrogens from [2-¹³C]glutamate or [3-¹³C]aspartate with intracellular heavy water deuterons.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Model of 1H-2H exchange in the H2 and H3 hydrogens of [2-13C]glutamate (A) and [3-13C]aspartate (B). Exchange processes are characterized by rate constants (k) and input-ouput fluxes (f). Multiplicities for the individual (1H,2H)13C isotopomers detected by 13C NMR in the C2 carbon resonance of [2-13C]glutamate or the C3 carbon resonance of [3-13C]aspartate are abbreviated as: s, singlet; t, triplet; ss, shifted singlet; st, shifted triplet; dss, doubly shifted singlet (see Figs. 3 and 4).

In the case of [2-¹³C]glutamate the model considers, observing the C2 resonance (cf. Figs. 3 and 4); (i) the reversible exchange of H2 originating [2-¹³C, 2-²H]glutamate with rate constants k₁, k₁, catalyzed mainly by aspartate aminotransferases and revealed by a 1:1:1 triplet (t); (ii) the reversible exchange of the H3_proS hydrogen originating [2-¹³C, 3-²H_S]glutamate with rate constants k₂, k₂, catalyzed by cytosolic isocitrate dehydrogenase (NADP) revealed by the shifted singlet (ss); (iii) the reversible exchange of hydrogens H3_proR and H2 originating [2-¹³C, 2-²H, 3-²H_R]glutamate with rate constants k₃, k₃, caused by the combined activities of aconitase and aspartate aminotransferase, revealed by shifted triplet (st); and (iv) the irreversible deuterations of both H3 hydrogens originating [2-¹³C, 3-²H₂]glutamate with rate constant k₄ caused by the sequential activities of mitochondrial aconitase and isocitrate dehydrogenase in the tricarboxylic acid cycle, revealed by the doubly shifted singlet (dss).

In the case of [3-¹³C]aspartate the model considers, observing the C3 resonance (cf. Figs. 3 and 4); (v) the reversible exchange of H2 originating [3-¹³C, 2-²H]aspartate as catalyzed by aspartate aminotransferase (k₅, k₅) and detected in the shifted singlet (ss); (vi) the reversible exchange of H3_proR originating [3-¹³C, 3-²H_R]aspartate as catalyzed by fumarase (k₆, k₆), and detected in the triplet (t); and (vii) the occurrence of both H2 and H3_proS exchanges on the same [3-¹³C]aspartate isotopomer originating [3-¹³C, 2-²H, 3-²H_S]aspartate as catalyzed by aspartate aminotransferase and the tricarboxylic acid cycle enzymes succinate dehydrogenase, fumarase, and malate dehydrogenase (k₇, k₇), detected in the corresponding shifted triplet (st). The following differential equations apply,

(Eq. 1)

(Eq. 2)

(Eq. 3)

(Eq. 4)

(Eq. 5)

(Eq. 6)

(Eq. 7)

(Eq. 8)

(Eq. 9)

where the concentration of the different (¹H, ²H, ¹³C) isotopomers is given as a relative contribution to the total C2 glutamate or C3 aspartate resonances observed as indicated in Fig. 3; k₁₍₁₎  k₇₍₇₎ refer to the apparent pseudo-first order rate constants of the reactions depicted in Fig. 1 and f₁-f₇ denote the relative input-output fluxes required to maintain steady state conditions in the system, respectively. Optimal values for these parameters were estimated by nonlinear fitting of the kinetics of deuteration in the different (¹H, ²H) isotopomers of [2-¹³C]glutamate and [3-¹³C]aspartate (cf. Fig. 5). Briefly, the system of differential equations was provided with an initial set of estimated parameters which was successively refined iteratively by minimizing the squared difference between calculated and mean experimental values of relative deuteration for every investigated isotopomer in each time point. For this purpose, we used the Stella package for simulations of system dynamics (High Performance Systems, Hanover, NH) as implemented in a desktop Pentium III platform and the BMDP package (BMDP Statistical Software Inc., Los Angeles, CA) as implemented on a Alpha 2100 mainframe computer (Digital Corp., San Diego, CA).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

¹H-²H Exchange in Hepatic Metabolites as Detected by ¹³C NMR-- Fig. 2 shows illustrative examples of the time courses of ¹H-²H exchange as detected in the (¹H,²H)¹³C multiplets of the C2 carbon of [2-¹³C]glutamate (top) and the C3 carbon of [3-¹³C]aspartate (bottom) from extracts obtained at increasing times of perfusion with [3-¹³C]alanine in KRB containing 50% ²H₂O.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Time course of 1H-2H exchange in the C2 carbon resonance from [2-13C]glutamate (A) and the C3 carbon resonance of [3-13C]aspartate (B) as observed by 13C NMR. Livers were perfused with 6 mM [3-13C]alanine in Krebs-Ringer buffer containing 50% 2H2O for increasing periods of time. Extracts were prepared at the end of the perfusions and analyzed by 13C NMR (150.90 MHz, 22 °C, pH 7.2). Results show representative spectra obtained after the combination of extracts from three livers perfused under identical conditions.

Similar spectra were obtained for the C2 carbon of [2-¹³C]aspartate, the C3 carbon of [3-¹³C]lactate, the C3 carbon of [3-¹³C]glutamate and, with significantly smaller intensity, for the C4 carbon of [4-¹³C]glutamate (not shown). All these resonances depicted evident (¹H,²H)¹³C multiplet patterns revealing characteristic and time-dependent increases in deuteration. These spectra were obtained at 14.09 Tesla, a field which improved considerably earlier results both in terms of sensitivity and resolution (12). However, despite the high field used, more quantitative determinations of the kinetics of ¹H-²H exchange required deconvolution of the observed (¹H,²H)¹³C multiplets into their individual components.

Fig. 3 shows representative deconvolutions of the C2 carbon resonance of [2-¹³C]glutamate and C3 carbon resonance from [3-¹³C]aspartate, respectively. Deconvolution of the C2 carbon resonance revealed contributions from five different [¹H, ²H, ¹³C]glutamate isotopomers: (i) [2-¹³C]glutamate as the unshifted singlet (55.50 ppm); (ii) [2-¹³C, 2-²H]glutamate as a triplet (¹J_¹³C²H = 19,2 Hz) shifted 0.32 ppm; (iii) [2-¹³C, 3-²H]glutamate as an upfield shifted singlet (₁= 0.07 ppm); (iv) [2-¹³C, 2-²H, 3-²H']glutamate as an upfield shifted triplet (₁= 0.39 ppm, ¹J_¹³C²H = 19.2 Hz); and (v) [2-¹³C, 3,3'-²H₂]glutamate as a doubly shifted singlet (₁ = 0.14 ppm). Similarly, deconvolution of the C3 resonance from [3-¹³C]aspartate revealed the relative contributions of four different (¹³C, ¹H, ²H) isotopomers: (i) [3-¹³C]aspartate, as an unshifted singlet (37.30 ppm); (ii) [3-¹³C, 2-²H]aspartate, as a singlet isotopically shifted 0.08 ppm; (iii) [3-¹³C, 3-²H]aspartate, as a 1:1:1 triplet (¹J_¹³C²H = 19,21 Hz) shifted 0.28 ppm; and (iv) [3-¹³C, 2,3-²H₂]aspartate, as a 1:1:1 triplet shifted 0.36 ppm. These assignments were confirmed in independent experiments, in which simultaneous ¹H and ²H decoupling were applied to the same samples during ¹³C NMR acquisition.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Representative deconvolutions of the C2 carbon resonances from [2-13C]glutamate (left panels) and the C3 carbon resonances from [3-13C]aspartate (right panels) into the contributions of individual (1H,2H)13C multiplets. 13C NMR spectra (150.90 MHz, 22 °C, pH 7.2) were obtained from the extracts of single livers perfused with 6 mM [3-13C]alanine in 50% 2H2O for 15 min (C2 glutamate) or 10 min (C3 aspartate). Relative contributions of specific isotopomers are given as the fractional contribution of the corresponding multiplet to the total area of the analyzed resonance taken arbitrarily as one. Simulations were performed with the WINDAISY program. Multiplicities of individual components are abbreviated as described in the legend to Fig. 1.

The use of simultaneous ¹H and ²H decoupling caused the multiplets derived from ²H-¹³C couplings to collapse into the corresponding ²H-decoupled singlets, thus reducing the complexity of the (¹H,²H)¹³C spectrum and increasing the sensitivity for ²H-¹³C isotopomer detection. This triple resonance experiment became particularly useful in the determination of the relative contributions of ²H-¹³C triplets or higher order multiplets appearing many times with relatively low intensity in ¹³C specta decoupled only for ¹H. Fig. 4 illustrates this aspect by showing a representative comparison of ¹H decoupled only (upper panels) and simultaneously ¹H- and ²H-decoupled ¹³C NMR spectra (lower panels) of the C2 glutamate and C3 aspartate resonances. The ²H- and ¹H-decoupled spectra depict more clearly resolved and with increased intensity, the singlet resonances corresponding to the ²H-¹³C triplets observed the proton decoupled spectrum of [2-¹³C, 2-²H]- and [2-¹³C, 3-²H, 2-²H]glutamate (t and st, left panel) or [3-¹³C, 3-²H]- and [3-¹³C, 3-²H, 2-²H]aspartate (t and st, right panel), respectively.

View larger version (11K): [in this window] [in a new window]   Fig. 4.   Effects of simultaneous 1H and 2H decoupling on the multiplet structure of the C2 resonance from [2-13C]glutamate (left) and the C3 resonance from [3-13C]aspartate (right). 13C NMR spectra (125.76 MHz, 22 °C, pH 7.2) were acquired from the extract of a liver perfused with [3-13C]alanine in KRB containing 50% 2H2O during 15 min as indicated under "Experimental Procedures." Upper panels, broad band 1H decoupling only. Lower panels, broad band 1H and 2H decoupling. Increased sensitivity and resolution in the lower panels allows detection of multiplets difficult to observe with 1H decoupling only (q and sq). Multiplicities of individual components are abbreviated as indicated in Fig. 1. q, quintet; sq, shifted quintet.

The concentrations of glutamate and aspartate measured in the extracts were 24.7 ± 4.5 µmol g¹ dry weight and 8.85 ± 2.3 µmol g¹ dry weight (n = 6), respectively, and did not vary appreciably during the entire ²H₂O perfusion period. Oxygen consumption due to alanine oxidation under these conditions was 2.3 ± 0.3 µmol min¹ g¹ dry weight (n = 4).

Turnover of Specific Hydrogens from [2-¹³C]Glutamate and [3-¹³C]Aspartate-- The relative contributions of specific (¹³C, ²H, ¹H) isotopomers of [2-¹³C]glutamate and [3-¹³C]aspartate was determined as indicated in Figs. 3 and 4, in liver extracts prepared at increasing times of perfusion with 50% deuterated KRB and [3-¹³C]alanine (Fig. 5).

View larger version (27K): [in this window] [in a new window]   Fig. 5.   Kinetics of deuteration of the H2 and H3 hydrogens from [2-13C]glutamate (A) or [3-13C]aspartate (B) during perfusions with [3-13C]alanine in KRB containing 50% 2H2O. Fractional contributions of individual isotopomers in each time point were determined as described in the legend to Fig. 3 and are indicated by specific symbols (insets). Lines represent the best fit to the model of Fig. 1 obtained with the rate constants and fluxes depicted in Table I. Multiplicities are abbreviated as indicated in Fig. 1 corresponding to the following (13C,2H,1H) isotopomers A; s, [2-13C]glutamate; t, [2-13C, 2-2H]glutamate; ss, [2-13C, 3-2H]glutamate; st, 2-13C, 2-2H, 3-2H']glutamate; dss, [2-13C, 3,3'-2H2]glutamate. B, s, [3-13C]aspartate; ss, [3-13C, 2-2H]aspartate; t, [3-13C, 3-2H]aspartate; st, [3-13C, 3-2H', 2-2H]aspartate.

In the case of glutamate, [2-¹³C, 2-²H]glutamate production (t) was the fastest process, [2-¹³C, 3-²H]glutamate was produced slower (ss) and [2-¹³C; 3-²H', 2-²H]glutamate or [2-¹³C, 3-²H₂]glutamates (st or dss) were produced even slower but at similar rates. Concerning deuteration of [3-¹³C]aspartate isotopomers, the fastest process was the deuteration of H2 to produce [3-¹³C, 2-²H]aspartate (ss), while the formation of [3-¹³C, 3-²H]- and [3-¹³C, 3-²H', 2-²H]aspartates (t and st) was slower. Taken together these results indicate that [2-¹³C]glutamate and [3-¹³C]aspartate are deuterated first to their (2-²H) isotopomers, the corresponding (3-²H), (3-²H', 2-²H), or (3-²H₂) isotopomers being produced successively later.

We performed a more quantitative interpretation of the kinetics of ²H incorporation into [2-¹³C]glutamate and [3-¹³C]aspartate by simulating the experimental data of Fig. 5 with the model shown in Fig. 1. This model describes the dynamics of exchange of the H2 and H3 hydrogens of [2-¹³C]glutamate or [3-¹³C]aspartate by intracellular heavy water deuterons, starting from the perprotonated molecules. The best fits for the kinetics of individual (¹³C, ¹H, ²H) isotopomers, obtained as described under "Experimental Procedures," are illustrated by the different lines in Fig. 5. A good agreement between calculated and experimentally measured values can be observed in every case. The apparent rate constants and input-output fluxes optimized in this way are summarized in Table I.

Notably, the kinetics for the exchange of some of the hydrogens shown in Table I are significantly faster than previously measured values of turnover from the neighboring ¹³C carbon, indicating that these hydrogens turn over several times during a single ¹³C turnover (13, 15, 25, 26). This is the case of the H2 hydrogens of glutamate and aspartate. In previous experiments of mouse liver perfusion performed under the same conditions, [3-¹³C]alanine-labeled the C2 and C3 carbons of glutamate with apparent rate constants of 0.07 ± 0.02 and 0.06 ± 0.01 min¹ and aspartate C2 and C3 with apparent rate constants of 0.15 ± 0.02 and 0.033 ± 0.003 min¹, respectively (13).² Steady state ¹³C labeling of the glutamate carbons under these conditions is achieved after approximately 30 min of perfusion. However, the steady-state deuteration of these carbons is reached after 10-15 min of perfusion only (Fig. 5). As indicated in Table I, the exchange of H2 glutamate occurs with a rate constant of the order of 0.3 min¹, approximately five times faster than the turnover of the geminally attached ¹³C2 carbon. Moreover, in addition to the fast exchange of glutamate H2, it is possible to measure also a slower exchange process of the H2 glutamate hydrogen, occurring only in those glutamate molecules deuterated in H3' (see [2-¹³C, 2-²H, 3-²H']glutamate, st in Figs. 3-5). This slower H2 exchange occurs essentially at the same rate than the exchange of both H3 hydrogens by deuterium (see [2-¹³C, 3,3'-²H₂]glutamate, dss in Figs. 3-5). A similar situation occurs with the H2 hydrogen of [3-¹³C]aspartate which exchanges approximately five times faster than the attached ¹³C3 carbon and presents additionally a slower exchange, occurring after one H3' deuteration (see the production of [2-¹³C, 2-²H]- and [2-¹³C, 2-²H, 3-²H']aspartate, ss and st in Figs. 3-5).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

During the course of metabolism hydrogen atoms from hepatic glutamate and aspartate are exchanged with those of intracellular water by distinct enzymes (5, 12, 27, 28). The H2 hydrogens of aspartate or glutamate are exchanged mainly by aspartate and alanine aminotransferases and glutamate dehydrogenase (28-31). The H3_proS hydrogen of glutamate (in the -ketoglutarate skeleton) may be exchanged either reversibly by cytosolic isocitrate dehydrogenase (NADP) or irreversibly by mitochondrial isocitrate dehydrogenase (NAD) (32). The glutamate H3_proR hydrogen is exchanged by cytosolic and mitochondrial aconitases (6, 33-35) and glutamate H4 hydrogens are exchanged by citrate synthase (36). In the case of aspartate, the H3_proR hydrogen is exchanged by both cytosolic and mitochondrial fumarases while the H3_proS hydrogen can be traced with equal probablity to the H2 or H3 hydrogens of fumarate and succinate or to the H3 and H4 hydrogens of -ketoglutarate (34, 37). Present results show that the kinetics of the ¹H-²H exchange of the H2 and H3 hydrogens of glutamate and aspartate can be conveniently followed by high field ¹³C NMR and occur in some cases significantly faster than the previously measured time courses of ¹³C enrichment (13, 25, 26). The next sections show that the faster timescale and ubiquitous location of ¹H-²H exchange enzymes, together with the exclusive mitochondrial location the tricarboxylic acid cycle, allows to investigate the exchange of -ketoglutarate/glutamate or oxalacetate/aspartate between mitochondrial and cytosolic compartments with more detail than previously possible with classical experiments of ¹³C turnover.

¹H-²H Exchange and Subcellular Compartmentation during [3-¹³C]Alanine Metabolism-- The hepatic metabolism of alanine and [3-¹³C]alanine has been covered in several classical studies (38-41). [3-¹³C]Alanine is transported to the cytosol and transaminated to [3-¹³C]pyruvate. In the fasted liver, [3-¹³C]pyruvate enters the mitochondrial tricarboxylic acid cycle mainly through pyruvate carboxylase, originating [3-¹³C]oxalacetate and [2-¹³C]-ketoglutarate in the first turn. Further metabolism of [2-¹³C]-ketoglutarate in the cycle yields an approximately equimolar mixture of [1-¹³C]- and [4-¹³C]succinate and oxalacetate molecules, which loose subsequently the ¹³C label by decarboxylation. Equilibration of mitochondrial [3-¹³C]oxalacetate by malate dehydrogenase and fumarase, originates an equimolar mixture of [2-¹³C]- or [3-¹³C]oxalacetate and, after condensation with new acetyl-CoA molecules in the tricarboxylic acid cycle, a mixture of [3-¹³C]- or [2-¹³C]-ketoglutarate. Mitochondrial [¹³C]oxalacetate and [¹³C]-ketoglutarate are then rapidly transaminated to the corresponding [¹³C]aspartate and [¹³C]glutamate.

Under steady-state conditions, a continuous cycling of [¹³C]aspartate and [¹³C]glutamate occurs between mitochondria and cytosol, associated mainly to the malate aspartate shuttle as described in Fig. 6. Briefly, [¹³C]glutamate enters the mitochondrial matrix through the aspartate/glutamate exchanger, returning later to the cytosol as [¹³C]-ketoglutarate through the dicarboxylate carrier after tricarboxylic acid cycle metabolism (40, 42, 43). Coupled to the exit of [¹³C]-ketoglutarate, [¹³C]malate enters the matrix though the dicarboxylate carrier, leaving it later as [¹³C]aspartate through aspartate/glutamate exchanger (42). If under steady-state conditions of ¹³C metabolite cycling, intracellular water is substituted partially by ²H₂O, deuterons from heavy water will become almost instantly available for exchange with the H2 and H3 hydrogens from both mitochondrial and cytosolic [¹³C]glutamates and aspartates or the corresponding ketoacid precursors. This is so because ²H₂O transport to cytosol or mitochondria proceeds in the millisecond or microsecond range, a much faster rate than that measured in this work for the ¹H-²H exchange reactions (cf. Fig. 5) (1, 3, 5). However, the exclusive mitochondrial location of pyruvate carboxylase, succinate dehydrogenase, and citrate synthase will make those irreversible ¹H-²H exchanges and ¹³C exchanges associated to the operation of the tricarboxylic acid cycle to occur only in the mitochondria.

View larger version (38K): [in this window] [in a new window]   Fig. 6.   Subcellular compartmentation of -ketoglutarate/glutamate and oxalacetate/aspartate exchange as reflected in the kinetics of deuteration of the H2 and H3 hydrogens from [2-13C]glutamate and [3-13C]aspartate. [2-2H]- or [3-2HS]glutamate (t and ss in Fig. 5A) are produced fast (f) in the cytosol and transported to the mitochondria (a). In the mitochondrial space, glutamate is transaminated to -ketoglutarate loosing the cytosolic H2 deuteron (b). The cytosolic [3-2H3S]deuteron is lost later in the tricarboxylic acic cycle where [2-13C]- and [2-13C, 3-2HS]-ketoglutarate produce a mixture of undeuterated and deuterated [1-13C]- and [4-13C]succinate, fumarate, malate (c), and oxalacetate molecules which eventually condense with incoming acetyl-CoA. Mitochondrial H3R () and H3S () deuterons are incorporated successively slower (s) into newly formed [2-13C]- or [3-13C]-ketoglutarate molecules produced from [3-13C]alanine entering the cycle through pyruvate carboxylase (d). Mitochondrial [2-13C]-ketoglutarate molecules deuterated in H3 can finally receive the slowest deuteron (or hydrogen) in H2(s), with transamination kinetics limited by mitochondrial transport and/or the tricarboxylic acid cycle activity (e, st, and dss in Fig. 5A). Further metabolism of H3 deuterated [2-13C]- or [3-13C]-ketoglurate in the cycle originates a mixture of deuterated [2-13C]- and [3-13C]succinate, fumarate, and malate (g, scrambled 13C molecules of succinate and fumarate are illustrated over the same carbon skeleton). Cytosolic [3-13C]oxalacetate may be deuterated fast (f) in the cytosol to [3-13C, 2-2H]aspartate through AAT (h) or to [3-13C, 3-2HR]aspartate through fumarase (ss and t in Fig. 5B), a process which appears to inhibit H2 deuteration (i). However, [3-13C, 3-2HS]oxalacetate or [2-13C, 3-2HS]oxalacetate molecules produced during tricarboxylic acid cycle metabolism of [13C]-ketoglutarate molecules deuterated in H3 or H4 (d), may become deuterated in H2 originating the slowest (3-13C, 3-2HS, 2-2H) (or 2-13C, 3-2HS, 2-2H) aspartate components (j and st in Fig. 5B). Acetyl-CoA entering the cycle is assumed to be perprotonated. A, deuterated [2-13C]glutamate isotopomers observed in Fig. 5A. B, deuterated [3-13C]aspartate isotopomers observed in Fig. 5B. Pre, before TCA cycle metabolism; Post, after TCA cycle metabolism; C, aspartate/glutamate exchanger; D, dicarboxylate carrier; AAT, aspartate aminotransferase; CS, citrate synthase; AC, aconitase (NAD or NADP); ICDH, isocitrate dehydrogenase; MDH, malate dehydrogenase; FM, fumarase; -KG, -ketoglutarate; Glu, glutamate; Asp, aspartate; Mal, malate; Fum, fumarate. , 13C; , scrambled 13C; , 12C, 2H (,   , ). Black or gray symbols indicate cytosolic or mitochondrial deuterations, respectively. Left or right location of deuterons in the carbon skeleton indicates S or R configuration of the attached carbon. Subscripts c or m indicate cytosolic or mitochondrial. f, fast; s, slow. 13C multiplicities are indicated in parentheses and abbreviated as in Fig. 5.

Hydrogen-Deuterium Exchange of H2 and H3 Glutamate-- The time courses of deuteration of the H2 and H3 hydrogens of [2-¹³C]glutamate, as detected by ¹³C NMR (Fig. 5) support the exchange of -ketoglutarate/glutamate through cytosol and mitochondria as depicted in Fig. 6. A crucial observation is that the H2 and one of the H3 hydrogens from [2-¹³C]glutamate are shown to be replaced successively at two different rates, fast and slow (Fig. 5). This indicates necessarily that H2 and H3 deuterons incorporated initially in the fast processes (noted with f in Fig. 6) must be removed before the slow deuteron from the solvent (noted with s in Fig. 6) occupies, at a later stage, the same position in the carbon skeleton of -ketoglutarate/glutamate. In the case of the H2 deuterations of [2-¹³C]-ketoglutarate, the formation of [2-¹³C, 2-²H]glutamate occurs first while the slower H2 deuteration occurs always after one (or both) H3 hydrogens of [2-¹³C]ketoglutarate have been replaced by deuterium (cf. Fig. 5). For H3 deuterations, the first deuteration originates [2-¹³C, 3-²H]glutamate fast, the subsequent H3 deuterations producing [2-¹³C, 3-²H', 2-²H]- and [2-¹³C, 3-²H₂]glutamate much slower and at similar rates.

This sequence of events agrees well with the dynamics of -ketoglutarate/glutamate exchange between cytosol and mitochondria and with the topology of the tricarboxylic acid cycle. First, [2-¹³C, 2-²H]- or [2-¹³C, 3-²H]glutamates are produced rapidly in the cytosol from -ketoglutarate by cytosolic aspartate aminotransferase, NADP isocitrate dehydrogenase, or aconitase (Fig. 6, f, Pre). These molecules are transferred to the mitochondria where the cytosolic ²H2 deuteron is lost by mitochondrial transamination originating mitochondrial [2-¹³C]- or [2-¹³C, 3-²H]-ketoglutarate which can enter then the tricarboxylic acid cycle. [2-¹³C]-Ketoglutarate will necessarily be decarboxylated during tricarboxylic acid metabolism to originate [1-¹³C]- or [4-¹³C]succinate, fumarate, malate, and oxalacetate and loosing later the ¹³C label by decarboxylation. [2-¹³C, 3-²H]-Ketoglutarates will produce [1-¹³C, 2-²H]succinate and fumarate, [1-¹³C, 2-²H]- or [1-¹³C, 3-²H]malate, and eventually [1-¹³C, 3-²H]oxalacetate, loosing both ¹³C and ²H labels in the next turn of the cycle. Therefore, both H3 hydrogens (or deuterons) from the original cytosolic -ketoglutarate are removed in the mitochondria during the first turn (and in every turn) of the tricarboxylic acid cycle. Simultaneously, new [3-¹³C]oxalacetate molecules are produced in the mitochondria each time [3-¹³C]pyruvate enters the cycle through pyruvate carboxylase, generating citrate, isocitrate, and newly formed molecules of [2-¹³C]-ketoglutarate. The new mitochondrial [2-¹³C]-ketoglutarate molecules synthesized by the cycle must incorporate the slower mitochondrial deuterium labeling (Fig. 6, s) in either or both of its H3 hydrogens through mitochondrial aconitase and NAD-isocitrate dehydrogenase, respectively. These newly synthesized [2-¹³C, 3-²H]- or [2-¹³C, 3-²H₂]-ketoglutarate molecules can receive finally the slow deuteron in H2 either by transamination or by reductive amination through glutamate dehydrogenase (Fig. 6, s, Post). The reductive amination process of -ketoglutarate requires previously the formation of mitochondrial [4-²H_S]NAD(P), resulting probably in a slower and smaller contribution to H2 deuteration of glutamate than transamination (28, 44).

Stereospecific Exchange of the H3,H3' Hydrogens of Glutamate-- ¹³C NMR is not able to resolve directly the stereochemistry of the single deuterations of the proR and proS H3 hydrogens of [2-¹³C]glutamate from the vicinal and geminal isotopic shifts observed in the C2 resonance (cf. Figs. 4 and 5). However, it is possible to infer the stereospecificity because the presence of substituents in the 3-R or 3-S configuration of the amino acid is known to have important consequences on the aminotransferase catalyzed exchange of H2 (28, 45). H3 substituents in the erythro-configuration with the amino group inhibit transamination probably because of a steric hinderance, while H3 substituents in the threo-configuration have no significant effect (45). This indicates that [3-²H_R]-ketoglutarate molecules produced by aconitase may incorporate deuterium in H2 normally, while the [3-²H_S]-ketoglutarate molecules deuterated by NAD- or NADP-isocitrate dehydrogenases will not incorporate appreciably ²H in H2 or will do it very slowly. This agrees well with the situation found experimentally in Fig. 5. In the case of [2-¹³C]glutamate, the slow H3' deuteration allows deuterium incorporation in H2 and produces [2-¹³C, 3-²H', 2-²H]glutamate (st in Figs. 4 and 5), suggesting that it corresponds to the 3-R configuration as incorporated by mitochondrial aconitase. In contrast, substitution of the other H3 hydrogen either fast, as in [2-¹³C, 3-²H]glutamate (ss in Figs. 4 and 5), or slow as in [2-¹³C, 3-²H₂]glutamate (dss in Figs. 4 and 5), appears to preclude ²H incorporation in H2, suggesting that it corresponds to the 3-S configuration as exchanged by isocitrate dehydrogenases. The fast ²H3_S exchange occurs most probably on pre-existing cytosolic [2-¹³C]-ketoglutarate while the slow one uses mitochondrial [2-¹³C]-ketoglutarate newly formed in the tricarboxylic acid cycle. In the latter case, the slow ²H3_S deuteration is limited by the previous incorporation of the ²H3_R through aconitase. In agreement with this, ²H3_R or ²H3_R and ²H3_S deuterations are found experimentally to proceed at the same rate, since both are limited by mitochondrial transport and the tricarboxylic acid cycle activity. In this respect it is worthwhile mentioning that the value of 1.15 ± 0.2 µmol min¹ g¹ for the tricarboxylic acid cycle flux calculated from oxygen consumption measurements,³ matches well the value for the tricarboxylic acid cycle flux of 0.9 ± 0.2 µmol min¹ g¹ calculated as the product of the glutamate concentration times the slow rate constant for double deuteration in H3 (k₄).

Hydrogen-Deuterium Exchange of H2 and H3 Aspartate-- The sequence of deuterations observed in the H2 and H3 hydrogens of aspartate follow a similar trend to those described for glutamate. The fast deuteration of H2 occurs first originating [3-¹³C, 2-²H]aspartate, followed by slower deuterations originating [3-¹³C, 3-²H]aspartate and [3-¹³C, 2-²H, 3-²H']aspartate, respectively. Similar reasonings to those described for glutamate allow to infer the stereospecificity of H3 deuterations in [3-¹³C]aspartate. The slow H3' deuteration, originating [3-¹³C, 3-²H']oxalacetate and malate should be assigned the ²H3_S configuration since it allows H2 deuteration to produce [2-¹³C, 2-²H, 3-²H_S]aspartate. The fast H3 deuteration originating [3-¹³C, 3-²H]aspartate may then correspond to the [²H3_R]aspartate diasteroisomer. Therefore the fast ²H3_R deuteration appears to be caused by cysolic fumarase while the slow ²H3_S should be incorporated through mitochondrial tricarboxylic acid cycle activity.

Concluding Remarks-- The results described in this study may present more general implications. Classical ¹³C NMR approaches to cerebral, cardiac, and hepatic metabolisms used ¹³C turnover of glutamate carbons to determine flux through the tricarboxylic acid cycle and associated anaplerotic reactions. Rate-limiting roles of citrate synthase (v_tca) and a fast and complete equilibration of all intracellular -ketoglutarate/glutamate pools by aspartate aminotransferases (v_x) and across the mitochondrial membrane were initially assumed in brain, heart, and liver (14-16, 26, 46). Model calculations provided in these cases values for v_x in the range from 1 to 72 times faster than v_tca. More recently, further modeling approaches proposed glutamate/oxoglutarate exchange and transport across the mitochondrial membrane as the rate-limiting step of [¹³C]glutamate turnover in perfused heart (47-51) with v_x values closer to unity or even smaller (17, 48). The hydrogen turnover experiments reported here provide direct information on the relative rates of aspartate aminotransferases and the tricarboxylic acid cycle through the specific deuteration reactions observed in the H2 and H3 hydrogens of [¹³C]glutamate. Our results show that intracellular -ketoglutarate/glutamate exchange can now be resolved at least in two kinetically different components. A fast component involves H2 exchange of the -ketoglutarate/glutamate couple in the cytosol, proceeding approximately five times faster than the tricarboxylic acid cycle rate (v_xc  5 v_tca). A slower component involves deuteration of the H3_proR or H3_proR and H3_proS hydrogens of glutamate requiring -ketoglutarate/glutamate transport through the mitochondrial membrane and the operation of the tricarboxylic acid cycle (v_xm  1). Some kinetic limitation precludes the equilibration of the mitochondrially produced ²H3_R and [3-²H₂]glutamates with the complete cellular glutamate pool. However, it is not possible at present to determine which process, tricarboxylic acid cycle, exchange through the mitochondrial membrane, or other, is limiting the formation of the slow components of [¹³C,²H]glutamate labeling.

In summary, this study shows that careful analysis of ²H-¹³C couplings and ²H-induced isotopic shifts observed by ¹³C NMR in the C2 glutamate and C3 aspartate carbon resonances reveals accurately the exchange by deuterium of the neighboring H2 and H3 hydrogens. This provides a wealth of information on hepatic -ketoglutarate/glutamate and oxalacetate/aspartate exchange and their subcellular compartmentation. The proposed (¹H, ²H, ¹³C) approach may be easily extended to alternative metabolites and additional carbons, other metabolic pathways or to different tissues.

	ACKNOWLEDGEMENTS

We are deeply indebted to Sylvain Meguellatni, Bruker Analytische Messtechnik, Rheinstetten (Germany), and Dr. Detlev Moskau, Bruker AG, Fallanden (Switzerland), for performing the triple resonance, simultaneous proton, and deuterium decoupling experiments.

	FOOTNOTES

^* 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.

Supported by a predoctoral fellowship from the MCT.

^§ Received a predoctoral fellowship from Consejo Superior de Investigaciones Científicas.

^** To whom correspondence and reprint requests should be addressed. Tel.: 34-91-585-4633; Fax: 34-91-585-4587; E-mail: scerdan@iib.uam.es.

Published, JBC Papers in Press, December 14, 2001, DOI 10.1074/jbc.M107501200

¹ Geminal or vicinal locations relative to an observed ¹³C refer to hydrogen or deuterium atoms located at one or two bonds distance from the observed ¹³C.

² M. García-Martín and S. Cerdán, unpublished observations.

³ Note that two oxygen molecules are used for every acetyl-CoA oxidized in the cycle.

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