Journal of Experimental Botany, Vol. 52, No. 354, pp. 37-45,
January 2001
© 2001 Oxford University Press
Original Papers |
Contribution of glutamate dehydrogenase to mitochondrial glutamate metabolism studied by 13C and 31P nuclear magnetic resonance
1 Laboratoire de Physiologie Végétale, Département de Biologie Moléculaire et Structurale, CEA-Grenoble, 17 rue des Martyrs, F-38054 Grenoble Cédex 9, France
Received 31 May 2000; Accepted 29 August 2000
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Abstract |
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The relative contribution of glutamate dehydrogenase (GDH) and the aminotransferase activity to mitochondrial glutamate metabolism was investigated in dilute suspensions of purified mitochondria from potato (Solanum tuberosum) tubers. Measurements of glutamate-dependent oxygen consumption by mitochondria in different metabolic states were complemented by novel in situ NMR assays of specific enzymes that metabolize glutamate. First, a new assay for aminotransferase activity, based on the exchange of deuterium between deuterated water and glutamate, provided a method for establishing the effectiveness of the aminotransferase inhibitor amino-oxyacetate in situ, and thus allowed the contribution of the aminotransferase activity to glutamate oxidation to be assessed unambiguously. Secondly, the activity of GDH in the mitochondria was monitored in a coupled assay in which glutamine synthetase was used to trap the ammonium released by the oxidative deamination of glutamate. Thirdly, the reversibility of the GDH reaction was investigated by monitoring the isotopic exchange between glutamate and [15N]ammonium. These novel approaches show that the oxidative deamination of glutamate can make a significant contribution to mitochondrial glutamate metabolism and that GDH can support the aminotransferases in funnelling carbon from glutamate into the TCA cycle.
Key words: Carbon starvation, glutamate dehydrogenase, NMR spectroscopy, plant mitochondria, respiration.
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Introduction |
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Glutamate occurs at a metabolic crossroads, and its metabolism plays a pivotal role in the integration of carbon and nitrogen metabolism in higher plants. The enzymes for which glutamate is a substrate include glutamine synthetase (GS; EC 6.3.1.2), glutamate synthase (GOGAT; EC 1.4.1.13), glutamate dehydrogenase (GDH; EC 1.4.1.2), several aminotransferases, notably aspartate aminotransferase (AAT; EC 2.6.1.1), and glutamate decarboxylase (GDC; EC 4.1.1.15). These enzymes are distributed among several cellular compartments, including the cytosol (GS, AAT, GDC), the mitochondria (GDH, AAT) and the plastids (GS, GOGAT), and in some cases this subcellular distribution is tissue specific (Ireland and Lea, 1999
). While these factors complicate the analysis of glutamate metabolism, the functions of most of the enzymes have been established under a variety of physiological conditions. In particular, it is clear that ammonium, whether generated by nitrogen fixation, nitrate reduction, protein turnover or photorespiration, is generally assimilated by the GS/GOGAT cycle (Lea et al., 1990).
Ammonium assimilation was originally thought to be catalysed by GDH, but assigning a function to the enzyme in higher plants became more difficult after the discovery of the GS/GOGAT pathway. Interpretations in the literature have generally polarized between the view that GDH provides an assimilatory pathway for ammonium under some circumstances, for example, various stress conditions (Srivastava and Singh, 1987), and the contrary view that GDH has a catabolic role, catalysing the oxidative deamination of glutamate to facilitate the recycling of carbon and nitrogen (Robinson et al., 1991, 1992). Numerous experimental approaches have been deployed in the study of plant GDH, including the purification and in vitro characterization of the enzyme (Yamaya et al., 1984; Schlee et al., 1994; Loulakakis and Roubelakis-Angelakis, 1996; Turano et al., 1996; Athwal et al., 1997; Turano, 1998), metabolic studies on isolated mitochondria (Yamaya and Matsumoto, 1985; Yamaya et al., 1986), intact cells (Robinson et al., 1991, 1992), and mutant phenotypes (Magalhães et al., 1990; Stewart et al., 1995), and more recently cloning and expression studies (Sakakibara et al., 1995; Melo-Oliveira et al., 1996; Purnell et al., 1997; Turano et al., 1997). While these investigations have sometimes been consistent with an assimilatory role for GDH, they have rarely produced evidence for a quantitatively important contribution to ammonium assimilation. By contrast, metabolic studies have provided strong evidence for a catabolic role in germinating seedlings (Stewart et al., 1995) and carbon-starved cells (Robinson et al., 1991, 1992). However, the conclusion that GDH has a catabolic role has generated controversy in the literature (Oaks, 1994, 1995; Fox et al., 1995; Pahlich, 1996) and the function of GDH in higher plants still merits investigation.
It has been argued that the conclusion that GDH has a catabolic role ignores the results of earlier experiments on isolated mitochondria (Oaks, 1995). These experiments showed: (i) that the concentrations of the relevant substrates in the mitochondria are such that the GDH reaction is likely to act in the aminating direction (Yamaya et al., 1984); (ii) that a low level of 15N is incorporated into glutamate when mitochondria are supplied with [15N]ammonium and [15N]glycine (Yamaya et al., 1986); and (iii) that the utilization of glutamate as a substrate for respiration is reduced in the presence of the aminotransferase inhibitor amino-oxyacetate (AOA) (Yamaya and Matsumoto, 1985). This last observation highlights the potential competition between GDH and AAT in directing carbon from glutamate into the TCA cycle (Fig. 1). In fact, glutamate, added as a single substrate, is oxidized very poorly by intact mitochondria (Hanson and Day, 1980), and substantial utilization of glutamate is only triggered in the presence of oxaloacetate (OAA) (Journet et al., 1982). The effect of OAA emphasizes the potentially dominant role of AAT in glutamate-dependent oxygen consumption; and even in the absence of added OAA, the aminotransferase activity appears to deliver more carbon for respiration than GDH, judging from measurements of oxygen consumption in the presence and absence of AOA (Yamaya and Matsumoto, 1985). Thus, given the generally high level of aminotransferase activity in the plant cell cytoplasm and mitochrondria, together with potential uncertainty about the extent of its inhibition by AOA, it is perhaps difficult to accept that GDH could have a catabolic role in mitochondrial metabolism. At the same time there is good metabolic evidence for such a role in germinating maize seedlings (Stewart et al., 1995) and carbon-starved cells (Robinson et al., 1991, 1992).
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In the experiments described here, the contradiction between the apparent dominance of the aminotransferase activity and the catabolic role for GDH has been addressed by using nuclear magnetic resonance (NMR) spectroscopy to investigate glutamate metabolism in suspensions of highly purified mitochondria from potato tubers. Adopting the same strategy as that used in a recent 31P NMR investigation of adenylate kinase (Roberts et al., 1997
), multiple NMR measurements were used to correlate the metabolic events observed during glutamate metabolism (Fig. 1
) with oxygen electrode measurements of the respiratory state of the mitochondria. This approach provided new insights into mitochondrial glutamate metabolism, including a non-metabolic criterion for establishing the effectiveness of the inhibitor AOA, and it demonstrated a significant role for GDH in maintaining glutamate-dependent oxygen consumption by isolated mitochondria.
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Materials and methods |
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Intact mitochondria were isolated from 10–20 kg of potato tubers (Neuburger et al., 1982
), with two successive Percoll gradients. The mitochondria were stored on ice as a thick slurry (180–220 µg protein µl-1) in a pH 7.2 buffer containing 5 mM MgCl2, 10 mM inorganic phosphate, 0.1% (w/v) BSA, and 300 mM mannitol. For the NMR experiments, 15 or 30 µl of the mitochondrial slurry was suspended in 3 ml of the same buffer with the addition of further components, including glutamate, according to the nature of the experiment. Typically, glutamate-dependent respiration was observed in a medium containing 0.3 mM NAD+, 0.2 mM ADP and 0.1 mM TPP; and in some cases glutamate utilization was stimulated by adding 10 mM glucose to the incubation medium. Phosphorylation of the glucose, either by the mitochondrial hexokinase (Kesseler et al., 1992) or by a commercial hexokinase (1 mg lyophilizate, corresponding to 70 U) added to the incubation medium, provided a convenient method for recycling ATP and thus preventing the switch to state 4 respiration. Each NMR sample included 5% 2H2O for the spectrometer lock signal, and this concentration was increased to 45% in some cases for the observation of deuterium exchange effects. The medium was bubbled with pure oxygen immediately before and after the addition of the mitochondria, and in some cases the sample was removed from the magnet and reoxygenated for varying lengths of time during the course of the experiment. In particular, reoxygenation was necessary after 1 h during experiments in which state 3 respiration on glutamate was maintained by recycling the ATP. For the oxygen electrode experiments, 3 µl of the mitochondrial slurry was suspended in 1 ml of the buffer and the oxygen consumption of the sample was measured using the method described previously (Neuburger et al., 1982). Proton decoupled 13C NMR spectra of the mitochondrial suspensions were recorded at 100.62 MHz using a Bruker AMX400 spectrometer equipped with a 10 mm broadband probehead. Spectra were accumulated in 5, 7.5, 15 or 60 min blocks using a 90° pulse angle, a 6 s recycle time and a spectral width of 20000 Hz. The Waltz sequence was used for 1H-decoupling, with a power level of 0.33 W during data acquisition and 0.15 W during the delay period. The 13C NMR signal was digitized using 32K data points and the spectrum was zero-filled to 64K before processing with a line broadening of either 0.2 or 1 Hz. Chemical shifts were referenced by using a coaxial capillary tube containing hexamethyldisiloxane to generate a signal at 2.7 ppm.
Proton decoupled 31P NMR spectra of the mitochondrial suspensions were recorded at 161.98 MHz using the same spectrometer and probehead. Spectra were accumulated in 5 min blocks using a 70° pulse angle, a 2.23 s recycle time, and a spectral width of 9090 Hz. The Waltz sequence was used for proton decoupling with the same parameters as above. The 31P NMR signal was digitized using 8K data points and the spectrum was zero-filled to 16K before processing with a line broadening of 1 Hz. Chemical shifts were referenced to phosphoric acid at 0 ppm by using a coaxial capillary tube containing methylene diphosphonate as an external reference.
Labelled glutamate was obtained from the following sources: 20% [U-13C]-L-glutamate and 99% [4-13C]-L-glutamate (Cambridge Isotopes); 99% [2-13C]-L-glutamate, 99% [2-13C]-DL-glutamate, 99% [3-13C]-L-glutamate, and 99% [3-13C]-DL-glutamate (Leman); 98% [15N]-L-glutamate (Sigma). Purified enzymes were used as follows: GDH (Boehringer, product No. 127 710); AAT (Sigma, product No. G2751); GS (Sigma, product No. G1270); and hexokinase (Boehringer, product No. 131 7466).
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Results |
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Dependence of glutamate metabolism on aminotransferase activity
Potato mitochondria oxidized glutamate slowly in comparison with the oxidation of succinate or
-ketoglutarate, and the rate of oxygen consumption in state 3 with glutamate as substrate fell to approximately 40% of its original value in the presence of freshly prepared AOA (Fig. 2). The effect of the inhibitor, which confirms the involvement of the aminotransferase activity in glutamate oxidation, was comparable to that observed in experiments on mitochondria derived from shoot tissue of maize (Zea mays) and pea (Pisum sativum) seedlings (Yamaya and Matsumoto, 1985), where 1 mM AOA reduced the rate of oxygen uptake to between 25% and 45% of the value observed in the absence of the inhibitor. Thus AOA did not abolish glutamate-dependent oxygen consumption completely, indicating either incomplete inhibition of the aminotransferase activity or a significant contribution to glutamate oxidation from GDH. These two possibilities were investigated in a series of NMR experiments.
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Establishing the effectiveness of AOA as an aminotransferase inhibitor is crucial to assessing the relative contribution of transamination and oxidative deamination to mitochondrial glutamate metabolism, because incomplete inhibition of the aminotransferases could still leave a residual activity that exceeds the activity of GDH. However, this question can be answered definitively with a sensitive NMR assay that depends on monitoring the hydrogen exchange reaction that occurs between the solvent and the glutamate during transamination. Schiff base formation during transamination leads to solvent exchange of the hydrogen attached to glutamate C2 (Hilton et al., 1954
; Mitra et al., 1976), and this provides a mechanism for the deuteration of the glutamate if the solvent is enriched in deuterium (Fig. 3). This process can be observed by 13C NMR, because the replacement of hydrogen with deuterium at C2 causes deuterium isotope shifts (Simpson, 1986) in the glutamate spectrum. These shifts are readily observed and they provide the basis for an experiment that proves that glutamate-dependent oxygen consumption in the presence of AOA is not the result of incomplete suppression of the aminotransferase activity.
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Figure 4
shows the effect of the deuteration of glutamate C2 on the 13C NMR signals from glutamate C2, C3 and C4. Uniformly labelled glutamate, with 20% enrichment, was used in this experiment and the signals from the C2, C3 and C4 carbons showed the expected fine structure from the scalar coupling to the adjacent 13C nuclei (Fig. 4A). The suspending medium also contained 45% 2H2O, and adding mitochondria to the solution reduced the intensity of the glutamate signals in parallel with the appearance of a new set of signals with lower chemical shift values (Fig. 4B). For example, the original glutamate C3 triplet (Fig. 4A) was transformed into two overlapping triplets separated by 0.1 ppm (Fig. 4B), with the new signals arising from glutamate that had been deuterated at C2. The isotopic equilibration of the glutamate with the solvent could be monitored by following the peak intensities of these C3 triplets over time (Fig. 5) and it was found that the intensity changes occurred in the presence or absence of respiration, at rates that depended on the mitochondrial density, the concentration of glutamate and the availability of oxaloacetate (data not shown). Exactly the same changes were observed when glutamate solutions were incubated with commercial AAT (data not shown), and it can be concluded that the changes in Figs 4 and 5 were caused by the deuteration of glutamate C2, following binding to the pyridoxal phosphate prosthetic group of the mitochondrial aminotransferases.
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Deuteration had the greatest effect on the signal from the directly bonded C2 carbon, in keeping with the expected result for isotope induced shifts (Simpson, 1986
). Thus the C2 signal showed an upfield -shift of approximately 0.4 ppm (Fig. 4B), but the shifted signals were broadened through the interaction with the directly bonded 2H nucleus, and they were also difficult to detect because the absence of a directly bonded hydrogen atom increased the relaxation time and reduced the nuclear Overhauser effect. The isotope shifts on the other glutamate signals were easier to detect: the C3 signal showed an upfield ß-shift of 0.1 ppm, giving rise to two overlapping triplet signals, as discussed above; and the C4 signal showed an upfield 
-shift of 0.03 ppm, again giving rise to overlapping multiplets from the protonated and deuterated isotopomers (Fig. 4B). Thus it was easier to observe the aminotransferase-mediated deuterium exchange effects on C3 and C4, than on the directly affected carbon C2.
This deuterium exchange assay allowed the inhibitory effect of AOA on the aminotransferase activity in a mitochondrial suspension to be assessed directly. In fact, AOA abolished the deuterium exchange effect on the glutamate signals from a mitochondrial suspension (Fig. 6), indicating that AOA inhibited the aminotransferase activity completely. However, AOA did not prevent the formation of -ketoglutarate by the mitochondria (Fig. 6) and the only alternative pathway to this respiratory substrate is via the oxidative deamination of GDH. Most significantly, these measurements of deuterium exchange provide an unambiguous demonstration that mitochondrial respiration fuelled by glutamate in the presence of AOA (Fig. 2) contains no contribution from aminotransferase activity, and can therefore be attributed entirely to GDH.
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Ammonium release and exchange catalysed by GDH
Further evidence for the involvement of GDH in the utilization of glutamate was obtained by using 13C NMR to follow the metabolism of the glutamate amino group. First, a coupled NMR assay for the deamination of glutamate was developed using glutamine synthetase (GS) to trap the ammonium released by the oxidative deamination of glutamate. No ammonium is formed when -ketoglutarate is produced by the aminotransferase reaction (Fig. 1), and so this assay could be used to test the validity of the conclusions drawn from the experiments with AOA. Adding GS to an oxygenated incubation medium containing the nucleotides and cofactors required for respiration led to the detection of a strong 13C NMR signal from glutamine, as well as signals from aspartate, succinate, and malate (Fig. 7). The observation of the TCA cycle intermediates was consistent with earlier work (Journet et al., 1982), and, by providing a method for recycling ATP, the addition of GS prevented the attainment of state 4 in the NMR tube and sustained the utilization of glutamate. The GS also provided a trap for ammonium. Since the incubation medium itself contained no ammonium, and since endogenous ammonium was negligible, the detection of labelled glutamine in Fig. 7 indicates that the ammonium must have been generated by the metabolism of the added glutamate. The direct deamination of glutamate by GDH is the simplest pathway that can be envisaged for generating the ammonium, and since AOA prevented the labelling of aspartate while still allowing the detection of glutamine (data not shown), deamination of the glutamate by an aminotransferase as the first step in a multistep pathway to the release of ammonium from some other metabolite can be ruled out. Accordingly, the spectra in Fig. 7 provide further strong evidence for a GDH-dependent contribution to the utilization of glutamate as a respiratory substrate by the mitochondria. This coupled assay of GDH using GS permits GDH activity to be monitored in the presence of aminotransferase activity, a distinct advantage over the experiments using the aminotransferase inhibitor AOA.
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Secondly, the reversibility of the GDH reaction was investigated by conducting 14N/15N exchange experiments in the presence of [15N]ammonium. Incubating mitochondria with NAD+, [2-13C]-DL-glutamate and [15N]ammonium for many hours caused a splitting of the glutamate C2 signal (Fig. 8
), but had no effect on C3 and C4 (data not shown). The same effect was observed when the mitochondria in the incubation medium were replaced with commercial GDH, and it was unaffected by the presence of deuterium in the water (data not shown). These experiments show that GDH, in agreement with the proposed mechanism for the enzyme (Stillman et al., 1993) and in contrast to the aminotransferases, does not mediate deuterium exchange at carbon 2, but that it does catalyse the isotopic exchange of the nitrogen atom in the glutamate amino group. Exchange of the normally abundant 14N nucleus for 15N caused a 6 Hz splitting of the C2 signal and an upfield isotope shift of 0.02 ppm. Thus the glutamate C2 signal in Fig. 8 is a superposition of a singlet from [14N, 2-13C]glutamate and a doublet from [15N, 2-13C]glutamate, and this interpretation was confirmed by recording natural abundance 13C NMR spectra of [14N]- and [15N]glutamate (data not shown). An important metabolic feature of the experiment in Fig. 8 is that conditions were chosen that would favour isotopic equilibration, with a long incubation time, a high ammonium concentration, and no respiration to compete for -ketoglutarate. However, despite these considerations, the results show that the exchange process occurred at a rate that was significantly slower than any of the other reactions involving glutamate described above.
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Discussion |
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The results presented in this paper provide a further demonstration of the practicality of using NMR to probe the metabolic events that occur in plant mitochondria during oxygen consumption in defined respiratory states (Roberts et al., 1997
). In the first application of this approach, 31P NMR was used to monitor changes in nucleotide levels in potato tuber mitochondria before and after the onset of state 3 respiration, and the results highlighted the importance of adenylate kinase in nucleotide metabolism. Evidence was also obtained for the existence of a small pool of tightly sequestered ATP in the purified mitochondria, and it was shown that plant mitochondria can readily regenerate nucleoside triphosphates from the AMP and nucleoside diphosphates that arise during cellular biosynthesis (Roberts et al., 1997). As reported here, a similar approach, using 13C-labelled substrates and 13C NMR, can be used to investigate the metabolism of respiratory substrates, but allowance has to be made for the fact that each substrate molecule is capable of generating more than one ATP molecule.
The principal aim of the investigation was to explore the interplay between GDH and aminotransferase activity in the utilization of glutamate as a respiratory substrate and, in particular, to address the apparent contradiction (Oaks, 1995) between metabolic experiments on cell suspensions and maize seedlings that provide strong evidence for a catabolic role for GDH (Robinson et al., 1991, 1992; Fox et al., 1995; Stewart et al., 1995) and experiments on isolated mitochondria that have been interpreted in terms of a role for GDH in glutamate synthesis (Yamaya et al., 1984, 1986; Yamaya and Matsumoto, 1985). Three significant observations were made in the course of this work that have a bearing on this controversy.
First, although the aminotransferase inhibitor AOA abolished deuteration at glutamate C2 in the presence of 2H2O (Figs 4, 6), it did not eliminate glutamate-dependent oxygen consumption (Fig. 2), and it did not prevent the conversion of glutamate to -ketoglutarate (Fig. 6). Deuteration is an inevitable consequence of the aminotransferase mechanism (Hilton et al., 1954) and the absence of deuteration when AOA was present proves that the inhibitor was fully effective in the mitochondrial suspension. Accordingly, there is clear evidence that glutamate utilization by the mitochondria included a contribution from a pathway that did not involve aminotransferase activity, i.e. GDH.
Secondly, there was a substantial conversion of glutamate to glutamine when GS was included in the incubation medium with the respiring mitochondria (Fig. 7), and while AOA prevented the formation of aspartate it did not prevent the synthesis of glutamine. The oxidative deamination of glutamate is the most direct route for generating the ammonium required for glutamine synthesis, and the possibility that the amino group is first transferred to an oxo-acid as the first step in a sequence leading to the deamination of some other compound is ruled out by the effect of AOA. Accordingly, this result provides further strong evidence for a contribution to glutamate utilization from GDH.
Thirdly, the glutamate amino group became labelled with 15N when [15N]ammonium was included in the incubation medium with the mitochondria (Fig. 8), indicating slow exchange due to the reversibility of the GDH reaction. This observation supports the argument developed earlier (Pahlich, 1996), that the mitochondria should be capable of supporting a net flux towards glutamate if the metabolic conditions are suitable. Indeed, limited synthesis of glutamate in isolated plant mitochondria has been reported (Yamaya et al., 1986), but the observed rates of glutamate production were very slow in comparison with the glutamate utilization rates observed here. For example, in experiments such as the one in Fig. 7, the glutamate consumption rate over the first 30 min of the reaction was approximately 0.5 µmol h-1 mg-1 protein, while the intensity of the glutamine signal indicated that oxidative deamination of glutamate occurred at approximately 0.25 µmol h-1 mg-1 protein. In contrast, the synthesis of [15N]glutamate from [15N]ammonium occurred at approximately 3 nmol h-1 mg-1 protein with isolated maize mitochondria, and at 30–40 nmol h-1 mg-1 protein with isolated pea mitochondria (Yamaya et al., 1986). The very slow isotopic equilibration of the glutamate amino group in the presence of [15N] ammonium (Fig. 8) is consistent with these low rates of glutamate synthesis, and so, while it is clear that GDH can operate reversibly, it is the oxidative deamination of glutamate that is more important in the experiments reported here.
The principal conclusion to be drawn from these three sets of observations is that GDH can make a significant contribution to glutamate-dependent oxygen consumption by isolated plant mitochondria, in agreement with the proposed catabolic role of the enzyme (Robinson et al., 1991, 1992). However, the significance of the flux through GDH into the TCA cycle in vivo is likely to be rather dependent on the needs of the cell and the availability of OAA. Thus while approximately one-third of the glutamate-dependent respiration rate in isolated mitochondria can be attributed to the activity of GDH (Fig. 2), in agreement with Yamaya and Matsumoto (Yamaya and Matsumoto, 1985), the observed rates were very small in comparison to the values obtained in the presence of an abundant supply of OAA (Journet et al., 1982). In vivo, significant pools of malate, together with high activities of malate dehydrogenase, may reasonably be expected to ensure no shortage of OAA under normal conditions, allowing delivery of -ketoglutarate to the mitochondrion via the aminotransferase. A further relevant point is that the two pathways are metabolically inequivalent: in principle, the GDH reaction can lead to the complete oxidation of glutamate by the TCA cycle, with the release of the fixed nitrogen as ammonium; whereas the aminotransferase reaction only allows the immediate oxidation of a single carbon atom, while retaining the nitrogen in a reduced organic form. These considerations suggest that the flux through the GDH reaction is likely to become more important under conditions of carbon limitation, when pools of metabolites such as malate are reduced. Moreover, if starvation limits the availability of OAA, then it may actually become necessary to use the GDH reaction to maintain a sufficient pool of OAA to allow transamination to occur. Thus the relative contribution of GDH and the aminotransferase activity to glutamate utilization can be predicted to change as starvation develops, and a situation can be envisaged in which the aminotransferase activity becomes dependent on the flux through GDH.
This analysis is supported by a number of observations on sugar-starved cell suspension cultures. For example, GDH activity increased in sucrose-starved carrot cells (Robinson et al., 1991, 1992) and this response correlated with the onset of protein catabolism and the excretion of ammonium into the growth medium (Robinson et al., 1992). Similarly, sucrose starvation of sycamore cells led to a rapid decline in the intracellular carbohydrate pools, triggering protein catabolism and a sharp increase in the levels of free amino acids in general and asparagine in particular (Genix et al., 1990). Although the two species differed in some of their responses to carbon limitation, for example, the carrot cells did not show a marked accumulation of asparagine and the sycamore cells did not release ammonium, the overall response of both cell cultures is consistent with protein breakdown being used to generate carbon skeletons for oxidation by the tricarboxylic acid cycle, and with GDH playing an increasingly important role as its activity increases.
Preliminary NMR and oxygen electrode experiments on mitochondria isolated from sycamore cells show that sucrose starvation increases both the utilization of glutamate and the GDH-dependent contribution to glutamate oxidation (data not shown). The pattern of metabolism observed by NMR is similar to that found with the potato tuber mitochondria, with slow utilization in the absence of a sink for ATP, detectable metabolism in the presence of AOA, complete abolition of deuterium exchange by AOA, and isotopic equilibration in the presence of [15N]ammonium. These observations suggest that the accumulation of asparagine that occurs in sucrose-starved sycamore cells (Genix et al., 1990) is the result of the combined action of the aminotransferase activity and GDH on the glutamate generated by protein catabolism. Thus AAT is likely to be important in the early stages of the starvation response, and this would explain the transient increase in the level of aspartate that precedes the accumulation of asparagine in carbon-starved maize root tips (Brouquisse et al., 1992); while the increase in the GDH activity as starvation continues would increase the availability of ammonium for the subsequent conversion of the aspartate to asparagine (Genix et al., 1990). So it would appear that the interaction between GDH and the aminotransferase activity is likely to be a critical feature in the outcome of protein catabolism during sucrose starvation, and it should now be possible to test this hypothesis quantitatively using the combination of NMR and oxygen electrode measurements described here. In particular, the methodology developed here should allow the changing balance between the two pathways to be quantified in mitochondria prepared from sycamore cells at different stages of sucrose starvation.
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Acknowledgments |
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We are indebted to Michelle Quemin for expert preparation of the mitochondria. RGR thanks the Novartis Fellowship Trust for a Novartis ACE Award; and JKMR was supported in part by a grant from the USDA (98351006146).
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Notes |
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2 To whom correspondence should be addressed at: Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK. Fax: +44 1865 275074. E-mail: george.ratcliffe{at}plants.ox.ac.ukPermanent
3 Permanent address: Department of Biochemistry, University of California, Riverside, CA 92521, USA.
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