Evidence for the Glutamine Synthetase/Glutamate Synthase Pathway during the Photorespiratory Nitrogen Cycle in Spinach Leaves

Spinach leaf (Spinacia oleracea L.) discs infiltrated with [¹⁵N]glycine were incubated at 25°C in the light and in darkness for 0, 30, 60 and 90 minutes. The kinetics of ¹⁵N-incorporation into glutamine, glutamate, asparagine, aspartate, and serine from [¹⁵N]glycine was determined. At the beginning of the experiment, just after infiltration (0 min incubation) serine, and the amido-N of glutamine and asparagine were the only compounds significantly labeled in both light- and dark-treated leaf discs. Incorporation of ¹⁵N-label into the other amino acids was observed at longer incubation time. The per cent ¹⁵N-enrichment in all amino acids was found to increase with incubation. However, serine and the amido-N of glutamine remained the most highly labeled products in all treatments. The above pattern of ¹⁵N-labeling suggests that glutamine synthetase was involved in the initial refixation of ¹⁵NH₃ derived from [¹⁵N]glycine oxidation in spinach leaf discs.

The ¹⁵N-enrichment of the amino-N of glutamine was found to increase rapidly from 0 to 19% during incubation in the light. There was a comparatively smaller increase (4-9%) in the ¹⁵N-label of the amino-N of glutamine in tissue incubated in darkness. Furthermore the total flux of ¹⁵N-label into each of the amino acids examined was found to be greater in tissue incubated in the light than those in the dark. The above evidence indicates the involvement of the glutamine synthetase/glutamate synthase pathway in the recycling of photorespiratory NH₃ during glycine oxidation in spinach leaves.

     

Glutamic Acid metabolism and the photorespiratory nitrogen cycle in wheat leaves: metabolic consequences of elevated ammonia concentrations and of blocking ammonia assimilation

The effects of methionine sulfoximine and ammonium chloride on [¹⁴C] glutamate metabolism in excised leaves of Triticum aestivum were investigated. Glutamine was the principal product derived from [U¹⁴C]glutamate in the light and in the absence of inhibitor or NH₄Cl. Other amino acids, organic acids, sugars, sugar phosphates, and CO₂ became slightly radioactive. Ammonium chloride (10 mm) increased formation of [¹⁴C] glutamine, aspartate, citrate, and malate but decreased incorporation into 2-oxoglutarate, alanine, and ¹⁴CO₂. Methionine sulfoximine (1 mm) suppressed glutamine synthesis, caused NH₃ to accumulate, increased metabolism of the added radioactive glutamate, decreased tissue levels of glutamate, and decreased incorporation of radioactivity into other amino acids. Methionine sulfoximine also caused most of the ¹⁴C from [U-¹⁴C]glutamate to be incorporated into malate and succinate, whereas most of the ¹⁴C from [1-¹⁴C]glutamate was metabolized to CO₂ and sugar phosphates. Thus, formation of radioactive organic acids in the presence of methionine sulfoximine does not take place indirectly through “dark” fixation of CO₂ released by degradation of glutamate when ammonia assimilation is blocked. When illuminated leaves supplied with [U-¹⁴C] glutamate without inhibitor or NH₄Cl were transferred to darkness, there was increased metabolism of the glutamate to glutamine, aspartate, succinate, malate, and ¹⁴CO₂. Darkening had little effect on the labeling pattern in leaves treated with methionine sulfoximine.     

A Two-Translocator Model for the Transport of 2-Oxoglutarate and Glutamate in Chloroplasts during Ammonia Assimilation in the Light

This study examines the transport of 2-oxoglutarate (2-OG) and other dicarboxylates during ammonia assimilation in illuminated spinach chloroplasts. The transport of all dicarboxylates examined was strongly inhibited by NH₄Cl preincubation in the light. Treatment with NH₄Cl caused a rapid depletion of the endogenous glutamate pool and a corresponding increase in endogenous glutamine content. The inhibition of transport activity by NH₄Cl was apparently linked to its metabolism in the light because inhibition of glutamine synthetase activity by the addition of l-methionine sulfoximine or carbonylcyanide-m-chlorophenylhydrazone abolished this affect. Measurements of endogenous metabolite pools showed that malate was most rapidly exchanged during the uptake of all exogenous dicarboxylates examined. Depending on the exogenous substrates used, the apparent half-times of efflux measured for endogenous malate, aspartate and glutamate were 10, 10 to 30, and 15 to 240 seconds, respectively. The transport of 2-OG was also inhibited by malate. But chloroplasts preincubated with malate in the presence or absence of NH₄Cl were found to have high transport activity similar to untreated chloroplasts. A two-translocator model is proposed to explain the stimulation of 2-OG transport as well as the stimulation of (NH₃, 2-OG)-dependent O₂ evolution by malate (KC Woo, CB Osmond 1982 Plant Physiol 69: 591-596) in isolated chloroplasts. In this model the transport of 2-OG on the 2-OG translocator and glutamate on the dicarboxylate translocator is coupled to malate counter-exchange in a cascade-like manner. This results in a net 2-OG/glutamate exchange with no net malate transport. Thus, during NH₃ assimilation the transport of 2-OG into and the export of glutamate out of the chloroplast occurs via the 2-OG and the dicarboxylate translocators, respectively.     

Characterization of dicarboxylate stimulation of ammonia, glutamine, and 2-oxoglutarate-dependent o(2) evolution in isolated pea chloroplasts

Intact isolated chloroplasts from pea (Pisum sativum) leaves carried out light-dependent (NH₃, 2-oxoglutarate) and (glutamine, 2-oxoglutarate)-dependent O₂ evolution at rates of 3.3 ± 0.7 (n = 7) and 6.0 ± 0.4 (n = 5) micromoles per milligram chlorophyll per hour, respectively. Malate stimulated the rate of (NH₃, 2-oxoglutarate)-dependent O₂ evolution 2.1 ± 0.5 (n = 7)-fold in the absence of glutamine, and 3.3 ± 0.4 (n = 11)-fold in the presence of glutamine. Malate also stimulated (glutamine, 2-oxoglutarate)-dependent O₂ evolution in the presence of high concentrations of glutamine. The affinity (K_1/2) of (NH₃, glutamine, 2-oxoglutarate)-dependent O₂ evolution for 2-oxoglutarate was estimated at 200 to 250 micromolar in the absence of malate and 50 to 80 micromolar when malate (0.5 millimolar) was present. In contrast to malate and various other dicarboxylates, aspartate, glutarate, and glutamate did not stimulate (NH₃, glutamine, 2-oxoglutarate)-dependent O₂ evolution in isolated pea chloroplasts. Using both in vitro assays and reconstituted chloroplast systems, malate was shown to have no effect on the activities of either glutamine synthetase or glutamate synthase.

The concentration of malate required for maximal stimulation of O₂ evolution was dependent on the concentration of 2-oxoglutarate present. However, the small extent of the competition between malate and 2-oxoglutarate for uptake was not consistent with that predicted by the current `single carrier' model proposed for the uptake of dicarboxylates into chloroplasts.

     

Nitrogen Metabolism in Senescent Flag Leaves of Wheat (Triticum aestivum L.) in the Light

Nitrogen metabolism was examined in senescent flag leaves of 90- to 93-day-old wheat (Triticum aestivum L. cv Yecora 70) plants. CO₂ assimilation and the levels of protein, chlorophyll, and nitrogen in the leaves decreased with age. Glutamine synthetase activity decreased to one-eighth of the level in young flag leaves. Detached leaves were incubated (with the cut base) in ¹⁵N-labeled NH₃, glutamate, or glycine in the light (1.8 millieinstein per square meter per second) at 25°C in an open gas exchange system under normal atmospheric conditions for up to 135 minutes. The ¹⁵N-enrichment of various amino acids derived from these ¹⁵N-substrates were examined. The amido-N of glutamine was the first ¹⁵N-labeled product in leaves incubated with ¹⁵NH₄Cl whereas serine, closely followed by the amido- and amino-N of glutamine, were the most highly ¹⁵N-labeled products during incubation with [¹⁵N]glycine. In contrast, aspartate and alanine were the first ¹⁵N-labeled products when [¹⁵N] glutamate was used. These results indicate that NH₃ was assimilated via glutamine synthetase and glutamate synthase activities and the photorespiratory nitrogen cycle remained functional in these senescent wheat flag leaves. In contrast, an involvement of glutamate dehydrogenase in the assimilation of ammonia could not be detected in these tissues.     

Glutamine transport and the role of the glutamine translocator in chloroplasts

The transport of l-[¹⁴C]glutamine in oat (Avena sativa L.) and spinach (Spinacia oleracea L.) chloroplasts was studied by a conventional single-layer and a newly developed stable double-layer silicone oil filtering system. [¹⁴C]Glutamine was actively transported into oat chloroplasts against a concentration gradient. Metabolite uptake was greatly affected by the endogenous dicarboxylate pools, which could be easily changed by preloading the chloroplast with specific exogenous substrate. Glutamine uptake was decreased by 44 to 75% in oat chloroplasts preloaded with malate, 2-oxoglutarate (2-OG), and aspartate, but increased by 52% in chloroplasts preloaded with l-glutamate. On the other hand, the uptake of the other four dicarboxylates was decreased by 47 to 79% in chloroplasts preloaded with glutamine. In glutamine-preloaded chloroplasts the uptake of glutamine was inhibited only by l-glutamate. The observed inhibition by l-glutamate was competitive with an apparent K_i value of 32.1 millimolar in oat and 6.7 millimolar in spinach chloroplasts. This study indicates that there are two components involved in glutamine transport in chloroplasts. The major component was mediated via a specific glutamine translocator. It was specific for glutamine and did not transport other dicarboxylates except l-glutamate. A K_0.5 value of 1.25 millimolar and V_max of 45.5 micromoles per milligram of chlorophyll per hour were determined for the glutamine translocator in oat chloroplasts. The respective values were 1.0 millimolar and 16.7 micromoles per milligram of chlorophyll per hour in spinach chloroplasts. A three translocator model, involving the glutamine, dicarboxylate, and 2-OG translocators, is proposed for the reassimilation of photorespiratory NH₃ in chloroplasts of C₃ species. In this three-translocator model the additional transport of glutamine into the chloroplast is coupled to the export of glutamate via the glutamine translocator. This is an extension of the two-translocator model, involving the dicarboxylate and 2-OG translocators, proposed for spinach chloroplasts, (KC Woo, UI Flügge, HW Heldt 1987 Plant Physiol 84: 624-632).     

Light-dependent reduction of pyruvate by pea chloroplasts in the presence of glutamate dehydrogenase and C5-dicarboxylic acids

Illuminated pea chloroplasts supported (glutamine plus α-oxoglutarate (α-OG)) and (NH₃ plus α-OG)-dependent O₂ evolution. The properties of these reactions were consistent with light-coupled glutamate synthase and glutamine synthetase activities. In the presence of a glutamate-oxidizing system (component C) comprised of NAD-specific glutamate dehydrogenase (NAD-GDH), lactate dehydrogenase (LDH), 4 mM pyruvate and 0.2 mM NAD, illuminated chloroplasts supported O₂ evolution in the presence of glutamine. The reaction did not proceed in the absence of any one of the constituents of component C and the properties of O₂ evolution were consistent with light-coupled glutamate synthase activity. In the presence of component C, chloroplasts also catalysed O₂ evolution in the presence of catalytic concentrations of glutamate. Studies of O₂ evolution and metabolism of [¹⁴C]-glutamate in the presence of the inhibitors methionine sulphoximine (MSO) and azaserine suggest that O₂ evolution was dependent on the synthesis of glutamine from the products of glutamate oxidation. This was supported by polarographic studies using α-OG and NH₃ instead of glutamate.The results are consistent with a C₅-dicarboxylic acid shuttlemechanism for the export of reducing equivalents from illuminated chloroplasts (glutamate) and recycling of the oxidation products (α-OG and NH₃).     

The role of malate in ammonia assimilation in cotyledons of radish (Raphanus sativus L.)

The relationships between the metabolism of malate, nitrogen assimilation and biosynthesis of amino acids in response to different nitrogen sources (nitrate and ammonium) have been examined in cotyledons of radish (Raphanus sativus L.). Measurements of the activities of some key enzymes and pulse-chase experiments with [¹⁴C]malate indicate the operation of an anaplerotic pathway for malate, which is involved in the synthesis of glutamine during increased ammonia assimilation. It is most likely that the tricarboxylicacid cycle is supplied with carbon through entry of malate, formed via the phosphoenolpyruvate (PEP)-carboxylation pathway, when 2-oxoglutarate leaves the cycle to serve as precursor for an increased synthesis of glutamine via glutamate. This might occur predominantly in the cytosol via the activity of the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle, the NADH-dependent GOGAT being the rate-limiting activity.Abbreviations DTT dithiothreitol - EDTA ethylenediamine-tetraacetic acid - GDH glutamate dehydrogenase - GOGAT glutamate synthase (glutamine: 2-oxoglutarate aminotransferase) - GOT aspartate aminotransferase (glutamate: oxaloacetate transaminase) - GS glutamine synthetase - HPLC high-performance liquid chromatography - MCF extraction medium of methanol: chloroform: 7M formic acid, 12:5:3, by vol. - MDH malate dehydrogenase - MSO L-methionine, sulfoximine - PEPCase phosphoenolpyruvate carboxylase - TLC thin-layer chromatography     

Effect of inhibitors on ammonia-, 2-oxoglutarate-, and oxaloacetate-dependent o(2) evolution in illuminated chloroplasts

The evolution of O₂ in spinach chloroplasts in the presence of oxaloacetate (OAA) was inhibited by a wide range of dicarboxylates. In contrast, (ammonia, 2-oxoglutarate)-dependent O₂ evolution was stimulated by malate, succinate, fumarate, glutarate, maleiate, and l-tartrate although OAA has little effect. This increase in O₂ evolution was accompanied by a similar increase in ¹⁴C incorporation from [5-¹⁴C]oxoglutarate into amino acids which was sensitive to azaserine inhibition. Glutamate and aspartate inhibited (ammonia, 2-oxoglutarate)-dependent O₂ evolution, but this inhibition was relieved by the addition of succinate, malate, or fumarate. OAA-dependent O₂ evolution also was inhibited by glutamate and aspartate, but succinate, malate, or fumarate had little effect on this inhibition. Phthalonate and n-butyl malonate inhibited (ammonia, 2-oxoglutarate)-dependent O₂ evolution competitively with respect to 2-oxoglutarate and uncompetitively with respect to malate. Both these inhibitors inhibited OAA-dependent O₂ evolution competitively. This evidence suggests that different mechanisms might be involved in the transport of OAA, 2-oxoglutarate, and malate into the chloroplasts.     

Characteristics of 2-oxoglutarate and glutamate transport in spinach chloroplasts

The transport of glutamate, 2-oxoglutarate and malate in intact spinach chloroplasts was determined using a double-silicone-layer centrifugation technique in which the silicone layers stayed separated at the end of centrifugation. Glutamate was found to be transported via the dicarboxylate but not the 2-oxoglutarate translocator. Hence the kinetic parameters (i.e.K _m,K _i andV _max) determined in glutamate-preloaded chloroplasts represent the kinetic constants of the dicarboxylate translocator. Measurements from malate- or succinate-preloaded chloroplasts represent the aggregate values of both the dicarboxylate and the 2-oxoglutarate translocators. Calculations showed that the 2-oxoglutarate and glutamate transport required to support the high fluxes of photorespiratory NH₃ recycling could be achieved if the transport of these two dicarboxylates occurred on separate translocators. It is proposed that during photorespiration the transport of 2-oxoglutarate into and glutamate out of the chloroplast occurred via the 2-oxoglutarate and the dicarboxylate translocators, respectively. These transports are coupled to malate counter-exchange in a cascade-like manner resulting in a net 2-oxoglutarate/glutamate exchange with no net malate uptake.Abbreviation 2-OG 2-oxoglutarate     

Carbon and nitrogen metabolism in a barley (Hordeum vulgare L.) mutant with impaired chloroplast dicarboxylate transport

A mutant line, RPr79/2, of barley (Hordeum vulgare L. cv. Maris Mink) has been isolated that has an apparent defect in photorespiratory nitrogen metabolism. The metabolism of ¹⁴C-labelled glutamine, glutamate and 2-oxoglutarate indicates that the mutant has a greatly reduced ability to synthesise glutamate, especially in air, although in-vitro enzyme analysis indicates the presence of wild-type activities of glutamine synthetase (EC 6.3.1.2) glutamate synthase (EC 1.4.7.1 and EC 1.4.1.14) and glutamate dehydrogenase (EC 1.4.1.2). Several characteristics of RPr79/2 are very similar to those described for glutamate-synthase-deficient barley and Arabidopsis thaliana mutants, including the pattern of labelling following fixation of ¹⁴CO₂, and the rapid rise in glutamine content and fall in glutamate in leaves on transfer to air. The CO₂-fixation rate in RPr79/2 declines much more slowly on transfer from 1% O₂ to air than do the rates in glutamate-synthase-deficient plants, and RPr79/2 plants do not die in air unless the temperature and irradiance are high. Analysis of (glutamine+NH₃+2-oxoglutarate)-dependent O₂ evolution by isolated chloroplasts shows that chloroplasts from RPr79/2 require a fivefold greater concentration of 2-oxoglutarate than does the wild-type for maximum activity. The levels of 2-oxoglutarate in illuminated leaves of RPr79/2 in air are sevenfold higher than in Maris Mink. It is suggested that RPr79/2 is defective in chloroplast dicarboxylate transport.     

Carbon and nitrogen metabolism in barley (Hordeum vulgare L.) mutants lacking ferredoxin-dependent glutamate synthase

Five mutant lines of barley (Hordeum vulgare L.), which are only able to grow at elevated levels of CO₂, contain less than 5% of the wild-type activity of ferredoxin-dependent glutamate synthase (EC 1.4.7.1). Two of these lines (RPr 82/1 and RPr 82/9) have been studied in detail. Leaves and roots of both lines contain normal activities of NADH-dependent glutamate synthase (EC 1.4.1.14) and the other enzymes of ammonia assimilation. Under conditions that minimise photorespiration, both mutants fix CO₂ at normal rates; on transfer to air, the rates drop rapidly to 15% of the wild-type. Incorporation of ¹⁴CO₂ into sugar phosphates and glycollate is increased under such conditions, whilst incorporation of radioactivity into serine, glycine, glycerate and sucrose is decreased; continuous exposure to air leads to an accumulation of ¹⁴C in malate. The concentrations of malate, glutamine, asparagine and ammonia are all high in air, whilst aspartate, alanine, glutamate, glycine and serine are low, by comparison with the wild-type parent line (cv. Maris Mink), under the same conditions. The metabolism of [¹⁴C]glutamate and [¹⁴C]glutamine by leaves of the mutants indicates a very much reduced ability to convert glutamine to glutamate. Genetic analysis has shown that the mutation in RPr 82/9 segregates as a single recessive nuclear gene.Abbreviations GDH glutamate dehydrogenase (EC 1.4.1.2) - GS glutamine synthetase (EC 6.3.1.2) - RuBP ribulose 1,5-bisphosphate     

Relationship between NH(4) Assimilation Rate and in Vivo Phosphoenolpyruvate Carboxylase Activity : Regulation of Anaplerotic Carbon Flow in the Green Alga Selenastrum minutum

The rate of NH₄⁺ assimilation by N-limited Selenastrum minutum (Naeg.) Collins cells in the dark was set as an independent variable and the relationship between NH₄⁺ assimilation rate and in vivo activity of phosphoenolpyruvate carboxylase (PEPC) was determined. In vivo activity of PEPC was measured by following the incorporation of H¹⁴CO⁻₃ into acid stable products. A linear relationship of 0.3 moles C fixed via PEPC per mole N assimilated was observed. This value agrees extremely well with the PEPC requirement for the synthesis of the amino acids found in total cellular protein. Determinations of metabolite levels in vivo at different rates of N assimilation indicated that the known metabolite effectors of S. minutum PEPC in vitro (KA Schuller, WC Plaxton, DH Turpin, [1990] Plant Physiol 93: 1303-1311) are important regulators of this enzyme during N assimilation. As PEPC activity increased in response to increasing rates of N assimilation, there was a corresponding decline in the level of PEPC inhibitors (2-oxoglutarate, malate), an increase in the level of PEPC activators (glutamine, dihydroxyacetone phosphate), and an increase in the Gln/Glu ratio. Treatment of N-limited cells with azaserine caused an increase in the Gln/Glu ratio resulting in increased PEPC activity in the absence of N assimilation. We suggest glutamate and glutamine play a key role in regulating the anaplerotic function of PEPC in this C₃ organism.     

Inhibition of Astrocyte Glutamine Production by α-Ketoisocaproic Acid

Abstract: We have evaluated the effect of α-ketoisocaproic acid (KIC), the ketoacid of leucine, on the production of glutamine by cultured astrocytes. We used ¹⁵NH₄Cl as a metabolic tracer to measure the production of both [5-¹⁵N]glutamine, reflecting amidation of glutamate via glutamine synthetase, and [2-¹⁵N]glutamine, representing the reductive amination of 2-oxoglutarate via glutamate dehydrogenase and subsequent conversion of [¹⁵N]-glutamate to [2-¹⁵N]glutamine. Addition of KIC (1 mM) to the medium diminished the production of [5-¹⁵N]glutamine and stimulated the formation of [2-¹⁵N]glutamine with the overall result being a significant inhibition of net glutamine synthesis. An external KIC concentration as low as 0.06 mM inhibited synthesis of [5-¹⁵N]glutamine and a level as low as 0.13 mM enhanced labeling (atom% excess) of [2-¹⁵N]glutamine. Higher concentrations of KIC in the medium had correspondingly larger effects. The presence of KIC in the medium did not affect flux through glutaminase, which was measured using [2-¹⁵N]glutamine as a tracer. Nor did KIC inhibit the activity of glutamine synthetase that was purified from sheep brain. Addition of KIC to the medium caused no increased release of lactate dehydrogenase from the astrocytes, suggesting that the ketoacid was not toxic to the cells. KIC treatment was associated with an approximately twofold increase in the formation of ¹⁴CO₂ from [U-¹⁴C]glutamate, indicating that transamination of glutamate with KIC increases intraastrocytic α-ketoglutarate, which is oxidized in the tricarboxylic acid cycle. KIC inhibited glutamine synthesis more than any other ketoacid tested, with the exception of hydroxypyruvate. The data indicate that KIC diminishes flux through glutamine synthetase by lowering the intraastrocytic glutamate concentration below the K_m of glutamine synthetase for glutamate, which we determined to be ～7 mM.     

Ammonia assimilation and oxygen evolution by a reconstituted chloroplast system in the presence of 2-oxoglutarate and glutamate

(Ammonia plus 2-oxoglutarate)-dependent O₂ evolution by intact chloroplasts was enhanced three- to five fold by 2 mM L- and D-malate, attaining rates of 9–15 μmol mg^-1 Chl h^-1. Succinate and fumarate also promoted activity but D-aspartate and, in the presence of aminooxyacetate, L-aspartate inhibited the malate-promoted rate. A reconstituted chloroplast system supported (ammonia plus 2-oxoglutarate)-dependent O₂ evolution at rates of 6-11 μmol mg^-1 Chl h^-1 in the presence of MgCl₂, NADP(H), ADP plus Pi (or ATP), ferredoxin and L-glutamate. The concentrations of L-glutamate and ATP required to support 0.5 V _max were 5 mM and 0.25 mM, respectively. When the reaction was initiated with NH₄Cl, O₂ evolution was preceded by a lag phase before attaining a constant rate. The lag phase was shortened by addition of low concentrations of L-glutamine or by preincubating in the dark in the presence of glutamate, ATP and NH₄Cl. Oxygen evolution was inhibited by 2 mM azaserine and, provided it was added initially, 2 mM methionine sulphoximine. The (ammonia plus 2-oxoglutarate)-dependent O₂ evolution was attributed to the synthesis of glutamine from NH₄Cl and glutamate which reacted with 2-oxoglutarate in a reaction catalysed by ferredoxin-specific glutamate synthase using H₂O as the ultimate electron donor. The lag phase was attributed to the establishment of a steady-state pool of glutamine. L-Malate did not affect the activity of the reconstituted system.     

Oxygen evolution by a reconstituted spinach chloroplast system in the presence ofl-glutamine and 2-oxoglutarate

Intact chloroplasts prepared from summer-grown spinach plants supported (aspartate plus 2-oxoglutarate)-dependent O₂ evolution but not (glutamine plus 2-oxoglutarate)-dependent O₂ evolution. The former activity, which was sensitive to amino oxyacetate, was attributed to transaminase activity and reduction of the resulting oxalo-acetate to malate using H₂O as eventual electron donor. A reconstituted chloroplast system which included chloroplast stroma, thylakoid membranes, ferredoxin and NADP(H) supported O₂ evolution in the presence ofl-glutamine and 2-oxoglutarate at rates of 15–22 μmol mg^-1 chlorophyll h^-1 although lower rates were obtained with material from winter-grown plants. Activity was not observed in the absence of ferredoxin and omission of NADP(H) decreased activity by 40%. The reaction was associated with the production of 0.49 mol O₂ mol^-1 2-oxoglutarate consumed and up to 0.46 mol O₂ mol^-1 glutamine supplied. The reaction, which was inhibited by azaserine but not by methionine sulphoximine or amino oxyacetate, was attributed to light-coupled glutamate synthase (EC 1.4.1.13) with H₂O serving as eventual electron donor. Activity was not affected significantly byl-malate. The reconstituted system also supported O₂ evolution in the presence of nitrite, oxaloacetate, (aspartate plus 2-oxoglutarate) and oxidised glutathione.     

Reductant for glutamate synthase in generated by the oxidative pentose phosphate pathway in non-photosynthetic root plastids

Purified pea root plastids were supplied with glutamine, 2-oxoglutarate and phosphorylated sugars. Formation of glutamate was linear for 75 min and dependent upon the intactness of the organelle. Glucose-6-phosphate and ribose-5-phosphate were the most effective substrates in supporting glutamate synthesis. Flux through the oxidative pentose phosphate pathway during glutamate synthesis in purified plastids was followed by monitoring the release of ¹⁴CO₂ from [1-¹⁴C]glucose-6-phosphate. ¹⁴CO₂ evolution from C-1 was dependent upon the presence of both glutamine and 2-oxoglutarate and could be inhibited by the application of azaserine. The data are discussed in view of the role of the oxidative pentose phosphate pathway in non-photosynthetic plastids.     

d-Glycerate Transport by the Pea Chloroplast Glycolate Carrier : Studies on [1-C]d-Glycerate Uptake and d-Glycerate Dependent O(2) Evolution

Rapid direct conversion of exogenously supplied [¹⁴C]aspartate to [¹⁴C] asparagine and to tricarboxylic cycle acids was observed in alfalfa (Medicago sativa L.) nodules. Aspartate aminotransferase activity readily converted carbon from exogenously applied [¹⁴C]aspartate into the tricarboxylic acid cycle with subsequent conversion to the organic acids malate, succinate, and fumarate. Aminooxyacetate, an inhibitor of aminotransferase activity, reduced the flow of carbon from [¹⁴C]aspartate into tricarboxylic cycle acids and decreased ¹⁴CO₂ evolution by 99%. Concurrently, maximum conversion of aspartate to asparagine was observed in aminooxyacetate treated nodules (30 nanomoles asparagine per gram fresh weight per hour. Metabolism of [¹⁴C]aspartate and distribution of nodulefixed ¹⁴CO₂ suggest that two pools of aspartate occur in alfalfa nodules: (a) one involved in asparagine biosynthesis, and (b) another supplying a malate/aspartate shuttle. Conversion of [¹⁴C]aspartate to [¹⁴C]asparagine was not inhibited by methionine sulfoximine, a glutamine synthetase inhibitor, or azaserine, a glutmate synthetase, inhibitor. The data did not indicate that asparagine biosynthesis in alfalfa nodules has an absolute requirement for glutamine. Radioactivity in the xylem sap, derived from nodule ¹⁴CO₂ fixation, was markedly decreased by treating nodulated roots with aminooxyacetate, methionine sulfoximine, and azaserine. Inhibitors decreased the [¹⁴C]aspartate and [¹⁴]asparagine content of xylem sap by greater than 80% and reduced the total amino nitrogen content of xylem sap (including nonradiolabeled amino acids) by 50 to 80%. Asparagine biosynthesis in alfalfa nodules and transport in xylem sap are dependent upon continued aminotransferase activity and an uninterrupted assimilation of ammonia via the glutamine synthetase/glutamate synthase pathway. Continued assimilation of ammonia apparently appears crucial to continued root nodule CO₂ fixation in alfalfa.     

Oxidation of glutamine in HeLa cells: Role and control of truncated TCA cycles in tumour mitochondria

The oxidative metabolism of glutamine in HeLa cells was investigated using intact cells and isolated mitochondria. The concentrations of the cytoplasmic amino acids were found to be aspartate, 8.0 mM; glutamate, 22.2 mM; glutamine, 11.3 mM; glycine, 9.8 mM; taurine, 2.3 mM; and alanine, <1 mM. Incubation of the cells with [¹⁴C]glutamine gave steady-state recoveries of ¹⁴C-label (estimated as exogenous glutamine) in the glutamine, glutamate, and aspartate pools, of 103%, 80%, and 25%, respectively, indicating that glutamine synthetase activity was absent and that a significant proportion of glutamate oxidation proceeded through aspartate aminotransferase. No label was detected in the alanine pool, suggesting that alanine aminotransferase activity was low in these cells. The clearance rate of [¹⁴C]glutamine through the cellular compartment was 65 nmol/min per mg protein. There was a 28 s delay after [¹⁴C]glutamine was added to the cell before ¹⁴C-label was incorporated into the cytoplasm, while the formation of glutamate commenced 10 s later. Aspartate was the major metabolite formed when the mitochondria were incubated in a medium containing either glutamine, glutamate, or glutamate plus malate. The transaminase inhibitor AOA inhibited both aspartate efflux from the mitochondria and respiration. The addition of 2-oxoglutarate failed to relieve glutamate plus malate respiration, indicating that 2-oxoglutarate is part of a well-coupled truncated cycle, of which aspartate aminotransferase has been shown to be a component [Parlo and Coleman (1984): J Biol Chem 259:9997–10003]. This was confirmed by the observation that, although it inhibited respiration, AOA did not affect the efflux of citrate from the mitochondria. Thus citrate does not appear to be a cycle component and is directly transported to the medium. Therefore, it was concluded that the truncated TCA cycle in HeLa cells is the result of both a low rate of citrate synthesis and an active citrate transporter. DNP (10 μM) induced a state III-like respiration only in the presence of succinate, which supports the evidence that NAD-linked dehydrogenases were not coupled to respiration, and suggests that these mitochondria may have a defect in complex I of the electron transport chain. Arising from the present results with HeLa cells and results extant in the literature, it has been proposed that a major regulating mechanism for the flux of glutamate carbon in tumour cells is the competitive inhibition exerted by 2-oxoglutarate on aspartate and alanine aminotransferases. This has been discussed and applied to the data. J. Cell. Biochem. 68:213–225, 1998. © 1998 Wiley-Liss, Inc.     

Detection of Glutamate Synthase in Heterocysts of Anabaena sp. Strain 7120

The formation of [¹⁴C]glutamate from [¹⁴C]glutamine in the presence of α-oxoglutarate was observed in extracts of heterocysts of Anabaena sp. strain 7120 under conditions that indicate the operation of glutamate synthase.