Polarographic study of ammonia assimilation by isolated chloroplasts

Illuminated pea (Pisum sativum) chloroplasts catalyze (ammonia plus alpha-ketoglutarate [alpha-KG])-dependent O(2) evolution at rates which are commensurate with other estimates of the flux of assimilated nitrogen (mean of eight determinations, 8.3 mumole per mg chlorophyll per hour, sd 2.4). The reaction was usually initiated with 1 mm ammonia after preincubating chloroplasts in the presence of alpha-KG, ADP, pyrophosphate, and MgCl(2).Progressive increases in ammonia concentration gave V(max)/2 at 0.2 mm (approximately) and V(max) at about 1 mm. Higher concentrations were inhibitory; at 7 mm the rate was again about V(max)/2. The highest ratio of O(2) evolved per mol of ammonia supplied was 0.36.The (ammonia plus alpha-KG)-dependent reaction was inhibited by methionine sulfoximine, azaserine, and aspartate in the presence of amino-oxyacetate but not by amino-oxyacetate alone and not by l-glutamate. The rate of O(2) evolution in the presence of 1 mm ammonia and 2.5 mm alpha-KG was increased only slightly by addition of 5 mm glutamine. Similarly, the rate of O(2) evolution in the presence of 5 mm glutamine and 2.5 mm alpha-KG was increased only slightly by addition of 1 mm ammonia.The results are attributed to the incorporation of ammonia via glutamine synthetase and reductive transamination of the glutamine formed by photosynthetically coupled glutamate synthase using alpha-KG as the amino acceptor. Several lines of evidence rule out the possibility that photosynthetically coupled glutamate dehydrogenase is involved.     

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.     

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).     

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.     

Stimulation of ammonia and 2-oxoglutarate-dependent o(2) evolution in isolated chloroplasts by dicarboxylates and the role of the chloroplast in photorespiratory nitrogen recycling

Intact chloroplasts isolated from spinach (Spinacia oleracea L.) leaves showed a light-dependent O(2) evolution (5.5 +/- 0.75 micromoles per milligram chlorophyll per hour) when supplied with ammonia and 2-oxoglutarate. This (ammonia, 2-oxoglutarate)-dependent O(2) evolution was stimulated 2- to 4-fold by the dicarboxylates, malate, succinate, fumarate, glutarate, and l-tartarate. Evolution of O(2) in the presence of malate was dependent on the presence of both 2-oxoglutarate and NH(4)Cl; malate with only either 2-oxoglutarate and NH(4)Cl alone did not support O(2) evolution. Furthermore, in the presence of malate, the amount of O(2) evolved was solely dependent on the amount of NH(4)Cl or 2-oxoglutarate added and malate did not affect the ratio of O(2) evolved to NH(4)Cl or 2-oxoglutarate consumed. Studies with inhibitors (2-(3,4-dichlorophenyl)-1,1-dimethyl urea, methionine sulfoximine, and azaserine) indicated that the above activity was directly linked to glutamine synthetase and glutamate synthase activity in the chloroplast and was not caused by the metabolism of malate. The V(max)/2 of (ammonia, 2-oxoglutarate)-dependent O(2) evolution was reached at 32 micromolar NH(4)Cl and 6 millimolar (approximately) 2-oxoglutarate in the absence of malate, and at 22 micromolar NH(4)Cl and 73 micromolar 2-oxoglutarate when malate (3 millimolar) was present.Intact chloroplasts isolated from pea (Pisum sativum) leaves also showed a stimulation of (ammonia, 2-oxoglutarate)-dependent O(2) evolution by malate. However glutamine was required for this activity even though glutamine with only either NH(4)Cl or 2-oxoglutarate did not respond to malate stimulation.The measured rates of (ammonia, 2-oxoglutarate)-dependent O(2) evolution in isolated spinach chloroplasts in the presence of malate were about 19.5 +/- 4.5 micromoles O(2) evolved per milligram chlorophyll per hour. This is adequate to sustain photorespiratory NH(3) recycling and the refixation of NH(3) arising from NO(3) under ambient conditions in the light. The role of the chloroplast in photorespiratory NH(3) recycling and the nature of the associated transport of 2-oxoglutarate into the chloroplast is discussed.     

Regulation of nitrogen fixation by nitrite and glutamine in Derxia gummosa

Abstract Nitrogenase activity of cells of Derxia gummosa (30 h growth in cultures without combined nitrogen) was not inhibited on adding nitrate. However, on adding either azaserine or methionine sulfoximine (MSX) with nitrate to these cells, nitrogenase (C₂H₂ reduction) was inhibited because nitrite accumulated in the reaction mixtures. Nitrite inhibition of the in vivo C₂H₂ reduction had a K _i value of 16 μM. Both ammonia and glutamine inhibited N₂ fixation (C₂H₂ reduction) in intact cells and in those treated with toluene. This inhibition by ammonia was relieved by methionine sulfoximine but not by glutamine. Azaserine enhanced the inhibition of nitrogenase produced by either ammonia or glutamine, since these treatments resulted in an accumulation of glutamine.     

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.     

Effect of Methionine Sulfoximine and Methionine Sulfone on Glutamate Synthesis in Klebsiella aerogenes

At least two pathways exist in Klebsiella aerogenes for glutamate synthesis. A mutant blocked in one pathway due to the loss of glutamate dehydrogenase (gltD) does not require glutamate and has the same growth characteristics as the parent strain in most media; however, its growth is inhibited by the analogues methionine sulfoximine and methionine sulfone. Wild-type Klebsiella is resistant to 0.1 M methionine sulfoximine or methionine sulfone, whereas the gltD mutant is sensitive to 1 mM concentrations. Either glutamate or glutamine is effective in overcoming this inhibition. Activities of both glutamine synthetase and glutamate synthetase, two enzymes involved in the second pathway of glutamate synthesis, are inhibited by methionine sulfoximine and methionine sulfone. The primary effect of methionine sulfoximine appears to be the prevention of glutamine production necessary for subsequent glutamate synthesis via glutamate synthetase enzyme.     

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.     

Effect of aspartate on complexes between glutamate dehydrogenase and various aminotransferases.

In previous studies it was found that: (a) aspartate aminotransferase increases the aspartate dehydrogenase activity of glutamate dehydrogenase; (b) the pyridoxamine-P form of this aminotransferase can form an enzyme-enzyme complex with glutamate dehydrogenase; and (c) the pyridoxamine-P form can be dehydrogenated to the pyridoxal-P form by glutamate dehydrogenase. It was therefore concluded (Fahien, L.A., and Smith, S.E. (1974) J. Biol. Chem 249, 2696-2703) that in the aspartate dehydrogenase reaction, aspartate converts the aminotransferase into the pyridoxamine-P form which is then dehydrogenated by glutamate dehydrogenase. The present results support this mechanism and essentially exclude the possibility that aspartate actually reacts with glutamate dehydrogenase and the aminotransferase is an allosteric activator. Indeed, it was found that aspartate is actually an activator of the reaction between glutamate dehydrogenase and the pyridoxamine-P form of the aminotransferase. Aspartate also markedly activated the alanine dehydrogenase reaction catalyzed by glutamate dehydrogenase plus alanine aminotransferase and the ornithine dehydrogenase reaction catalyzed by ornithine aminotransferase plus glutamate dehydrogenase. In these latter two reactions, there is no significant conversion of aspartate to oxalecetate and other compounds tested (including oxalacetate) would not substitute for aspartate. Thus aspartate is apparently bound to glutamate dehydrogenase and this increases the reactivity of this enzyme with the pyridoxamine-P form of aminotransferases. This could be of physiological importance because aspartate enables the aspartate and ornithine dehydrogenase reactions to be catalyzed almost as rapidly by complexes between glutamate dehydrogenase and the appropriate mitochondrial aminotransferase in the absence of alpha-ketoglutarate as they are in the presence of this substrate. Furthermore, in the presence of aspartate, alpha-ketoglutarate can have little or no affect on these reactions. Consequently, in the mitochondria of some organs these reactions could be catalyzed exclusively by enzyme-enzyme complexes even in the presence of alpha-ketoglutarate. Rat liver glutamate dehydrogenase is essentially as active as thebovine liver enzyme with aminotransferases. Since the rat liver enzyme does not polymerize, this unambiguously demonstrates that monomeric forms of glutamate dehydrogenase can react with aminotransferases.     

Suppression of the enzymes of glutamate metabolism in cortical synaptosomes in methionine sulfoximine toxicity

Enzymes of glutamate metabolism were studied in synaptosomes prepared from normal rats and those treated with acute (300 mg/kg) and subacute (150 mg/kg) doses of the convulsant methionine sulfoximine (MSO). The activities of glutamine synthetase, glutamate dehydrogenase and aspartate aminotransferase were inhibited in the synaptosomes of drug treated animals. It is suggested that MSO would suppress the formation of glutamine and glutamate and consequently the releasable pool of glutamate, aspartate and GABA. These neurotransmitters would be depleted irom the nerve endings. It is also indicated that the ammonia accumulated would affect the cerebral functioning by interfering with the maintenance of ionic gradients.     

Amino acid concentrations in Rhodospirillum rubrum during expression and switch-off of nitrogenase activity.

The amino acid concentrations in the phototrophic bacterium Rhodospirillum rubrum were measured during growth under nif-repressing and nif-derepressing conditions. The effects of ammonium, glutamine, darkness, phenazine methosulfate, and the inhibitors methionine sulfoximine and azaserine on amino acid levels of cells were tested. The changes were compared to changes in whole-cell nitrogenase activity and ADP-ribosylation of dinitrogenase reductase. Glutamate was the dominant amino acid under every growth condition. Glutamine levels were equivalent when cells were grown on high-ammonia (nif-repressing) medium or glutamate (nif-derepressing) medium. Thus, glutamine is not the solitary agent that controls nif expression. No other amino acid correlated with nif expression. Glutamine concentrations rose sharply when either glutamate-grown or N-starved cells were treated with ammonia, glutamine, or azaserine. Glutamine levels showed little change upon treatment of the cells with darkness or ammonium plus methionine sulfoximine. Treatment with phenazine methosulfate resulted in a decrease in glutamine concentration. The glutamine concentration varied independently of dinitrogenase reductase ADP-ribosylation, and it is concluded that an increase in glutamine concentration is neither necessary nor sufficient to initiate the modification of dinitrogenase reductase. No other amino acid exhibited changes in concentration that correlated consistently with modification. Glutamine synthetase activity and nitrogenase activity were not coregulated under all conditions, and thus the two regulatory cascades perceive different signal(s) under at least some conditions.     

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.     

Regulation of Neurotransmitter Aspartate Metabolism by Glial Glutamine Synthetase

Aspartate levels and release from rat striatal slices following the inhibition of glutamine synthetase (GS) by methionine sulfoximine (MSO) were studied. Striatal levels of aspartate and glutamine were decreased over time in a manner that correlated with GS inhibition. Ca2+-dependent, K+-stimulated aspartate release was diminished in striatal tissue slices from animals pretreated with MSO. The decreased release of aspartate correlated over time with the inhibition of GS. The addition of glutamine to the perfusion medium completely reversed the effects of MSO on calcium-dependent aspartate release. It is suggested that glutamine is a major precursor for transmitter aspartate.     

The pathway of glutamine and glutamate oxidation in isolated mitochondria from mammalian cells

1. Pyruvate strongly inhibited aspartate production by mitochondria isolated from Ehrlich ascites-tumour cells, and rat kidney and liver respiring in the presence of glutamine or glutamate; the production of (14)CO(2) from l-[U-(14)C]glutamine was not inhibited though that from l-[U-(14)C]glutamate was inhibited by more than 50%. 2. Inhibition of aspartate production during glutamine oxidation by intact Ehrlich ascites-tumour cells in the presence of glucose was not accompanied by inhibition of CO(2) production. 3. The addition of amino-oxyacetate, which almost completely suppressed aspartate production, did not inhibit the respiration of the mitochondria in the presence of glutamine, though the respiration in the presence of glutamate was inhibited. 4. Glutamate stimulated the respiration of kidney mitochondria in the presence of glutamine, but the production of aspartate was the same as that in the presence of glutamate alone. 5. The results suggest that the oxidation of glutamate produced by the activity of mitochondrial glutaminase can proceed almost completely through the glutamate dehydrogenase pathway if the transamination pathway is inhibited. This indicates that the oxidation of glutamate is not limited by a high [NADPH]/[NADP(+)] ratio. 6. It is suggested that under physiological conditions the transamination pathway is a less favourable route for the oxidation of glutamate (produced by hydrolysis of glutamine) in Ehrlich ascites-tumour cells, and perhaps also kidney, than the glutamate dehydrogenase pathway, as the production of acetyl-CoA strongly inhibits the first mechanism. The predominance of the transamination pathway in the oxidation of glutamate by isolated mitochondria can be explained by a restricted permeability of the inner mitochondrial membrane to glutamate and by a more favourable location of glutamate-oxaloacetate transaminase compared with that of glutamate dehydrogenase.     

Glutamate Metabolism in Rat Cortical Astrocyte Cultures

Glutamate metabolism in rat cortical astrocyte cultures was studied to evaluate the relative rates of flux of glutamate carbon through oxidative pathways and through glutamine synthetase (GS). Rates of 14CO2 production from [1-14C]glutamate were determined, as was the metabolic fate of [14C(U)]glutamate in the presence and absence of the transaminase inhibitor aminooxyacetic acid and of methionine sulfoximine, an irreversible inhibitor of GS. The effects of subculturing and dibutyryl cyclic AMP treatment of astrocytes on these parameters were also examined. The vast majority of exogenously added glutamate was converted to glutamine and exported into the extracellular medium. Inhibition of GS led to a sustained and greatly elevated intracellular glutamate level, thereby demonstrating the predominance of this pathway in the astrocytic metabolism of glutamate. Nevertheless, there was some glutamate oxidation in the astrocyte culture, as evidenced by aspartate production and labeling of intracellular aspartate pools. Inhibition of aspartate aminotransferase caused a greater than 70% decrease in 14CO2 production from [1-14C]glutamate. Inhibition of GS caused an increase in aspartate production. It is concluded that transamination of glutamate rather than oxidative deamination catalyzed by glutamate dehydrogenase is the first step in the entry of glutamate carbon into the citric acid cycle in cultured astrocytes. This scheme of glutamate metabolism was not qualitatively altered by subculturing or by treatment of the cultures with dibutyryl cyclic AMP.     

Amino Acid Metabolism of Lemna minor L. : III. Responses to Aminooxyacetate

Aminooxyacetate, a known inhibitor of transaminase reactions and glycine decarboxylase, promotes rapid depletion of the free pools of serine and aspartate in nitrate grown Lemna minor L. This compound markedly inhibits the methionine sulfoximine-induced accumulation of free ammonium ions and greatly restricts the methionine sulfoximine-induced depletion of amino acids such as glutamate, alanine, and asparagine. These results suggest that glutamate, alanine, and asparagine are normally catabolized to ammonia by transaminase-dependent pathways rather than via dehydrogenase or amidohydrolase reactions. Aminooxyacetate does not inhibit the methionine sulfoximine-induced irreversible deactivation of glutamine synthetase in vivo, indicating that these effects cannot be simply ascribed to inhibition of methionine sulfoximine uptake by amino-oxyacetate. This transaminase inhibitor promotes extensive accumulation of several amino acids including valine, leucine, isoleucine, alanine, glycine, threonine, proline, phenylalanine, lysine, and tyrosine. Since the aminooxyacetate induced accumulations of valine, leucine, and isoleucine are not inhibited by the branched-chain amino acid biosynthesis inhibitor, chlorsulfuron, these amino acid accumulations most probably involve protein turnover. Depletions of soluble protein bound amino acids are shown to be approximately stoichiometric with the free amino acid pool accumulations induced by aminooxyacetate. Aminooxyacetate is demonstrated to inhibit the chlorsulfuron-induced accumulation of α-amino-n-butyrate in L. minor, supporting the notion that this amino acid is derived from transamination of 2-oxobutyrate.     

The pathway of glutamate metabolism in rat brain mitochondria

1. The pathway of glutamate metabolism in non-synaptic rat brain mitochondria was investigated by measuring glutamate, aspartate and ammonia concentrations and oxygen uptakes in mitochondria metabolizing glutamate or glutamine under various conditions. 2. Brain mitochondria metabolizing 10mm-glutamate in the absence of malate produce aspartate at 15nmol/min per mg of protein, but no detectable ammonia. If amino-oxyacetate is added, the aspartate production is decreased by 80% and ammonia production is now observed at a rate of 6.3nmol/min per mg of protein. 3. Brain mitochondria metabolizing glutamate at various concentrations (0-10mm) in the presence of 2.5mm-malate produce aspartate at rates that are almost stoicheiometric with glutamate disappearance, with no detectable ammonia production. In the presence of amino-oxyacetate, although the rate of aspartate production is decreased by 75%, ammonia production is only just detectable (0.3nmol/min per mg of protein). 4. Brain mitochondria metabolizing 10mm-glutamine and 2.5mm-malate in States 3 and 4 were studied by using glutamine as a source of intramitochondrial glutamate without the involvement of mitochondrial translocases. The ammonia production due to the oxidative deamination of glutamate produced from the glutamine was estimated as 1nmol/min per mg of protein in State 3 and 3nmol/min per mg of protein in State 4. 5. Brain mitochondria metabolizing 10mm-glutamine in the presence of 1mm-amino-oxyacetate under State-3 conditions in the presence or absence of 2.5mm-malate showed no detectable aspartate production. In both cases, however, over the first 5min, ammonia production from the oxidative deamination of glutamate was 21-27nmol/min per mg of protein, but then decreased to approx. 1-1.5nmol/min per mg. 6. It is concluded that the oxidative deamination of glutamate by glutamate dehydrogenase is not a major route of metabolism of glutamate from either exogenous or endogenous (glutamine) sources in rat brain mitochondria.     

Ammonia Fixation via Glutamine Synthetase and Glutamate Synthase in the CAM Plant Cissus quadrangularis L

Succulent stems of Cissus quadrangularis L. (Vitaceae) contain glutamine synthetase, glutamate synthase, and glutamate dehydrogenase. The CO₂ and water gas exchanges of detached internodes were typical for Crassulacean acid metabolism plants. During three physiological phases, e.g. in the dark, in the early illumination period after stomata closure, and during the late light phase with the stomata wide open, ¹⁵NH₄Cl was injected into the central pith of stem sections. The kinetics of ¹⁵N labeling in glutamate and glutamine suggested that glutamine synthetase was involved in the initial ammonia fixation. In the presence of methionine sulfoximine, an inhibitor of glutamine synthetase, the incorporation of ¹⁵N derived from ¹⁵NH₄Cl was almost completely inhibited. Injections of amido-¹⁵N glutamine demonstrated a potential for ¹⁵N transfer from the amido group of glutamine into glutamate which was suppressed by the glutamate synthase inhibitor, azaserine. The evidence indicates that glutamine synthetase and glutamate synthase could assimilate ammonia and cycle nitrogen during all phases of Crassulacean acid metabolism.     

Action of Inhibitors of Ammonia Assimilation on Amino Acid Metabolism in Hordeum vulgare L. (cv Golden Promise)

Barley (Hordeum vulgare L. cv Golden Promise) plants were grown in a continuous culture system in which the root and shoot ammonia and amino acid levels were constant over a 6-hour experimental period. Methionine sulfoximine (MSO), 1 millimolarity when added to the culture medium, caused a total inactivation of root glutamine synthetase with little effect on the shoot enzyme. Root ammonia levels increased and glutamine levels decreased, irrespective of whether the plants were grown in 1 millimolar nitrate or 1 millimolar ammonia. Levels of glutamate, aspartate, serine, threonine, and asparagine all increased. There was little alteration in the amino acid and ammonia levels in the shoot, suggesting that MSO is not rapidly transported.

The addition of azaserine (25 micrograms per milliliter) to nitrate-grown plants caused a rapid increase in root ammonia, glutamine, and serine levels with a corresponding decrease in glutamate, aspartate, and alanine. Glutamine levels also increased in the shoot.

The in vivo effect of MSO and azaserine was as would be predicted by their known in vitro inhibitory action if the glutamine synthetase/glutamate synthase pathway of ammonia assimilation was in operation.