Kinetic locking-on and auxiliary tactics for bioaffinity purification of NADP+-dependent dehydrogenases using N6-linked immobilized NADP+ derivatives: Studies with mammalian and microbial glutamate dehydrogenases

This study is concerned with the development and application of kinetic locking-on and auxiliary tactics for bioaffinity purification of NADP(+)-dependent dehydrogenases, specifically (1) the synthesis and characterization of highly substituted N(6)-linked immobilized NADP(+) derivatives using a rapid solid-phase modular approach; (2) the evaluation of the N(6)-linked immobilized NADP(+) derivatives for use with the kinetic locking-on strategy for bioaffinity purification of NADP(+)-dependent dehydrogenases: Model bioaffinity chromatographic studies with glutamate dehydrogenase from bovine liver (GDH with dual cofactor specificity, EC 1.4.1.3) and glutamate dehydrogenase from Candida utilis (GDH which is NADP(+)-specific, EC 1.4.1.4); (3) the selection of an effective "stripping ligand" for NADP(+)-dehydrogenase bioaffinity purifications using N(6)-linked immobilized NADP(+) derivatives in the locking-on mode; and (4) the application of the developed bioaffinity chromatographic system to the purification of C. utilis GDH from a crude cellular extract.Results confirm that the newly developed N(6)-linked immobilized NADP(+) derivatives are suitable for the one-step bioaffinity purification of NADP(+)-dependent GDH provided that they are used in the locking-on mode, steps are taken to inhibit alkaline phosphatase activity in crude cellular extracts, and 2',5'-ADP is used as the stripping ligand during chromatography. The general principles described here are supported by a specific sample enzyme purification; the purification of C. utilis GDH to electrophoretic homogeneity in a single bioaffinity chromatographic step (specific activity, 9.12 micromol/min/mg; purification factor, 83.7; yield 88%). The potential for development of analogous bioaffinity systems for other NADP(+)-dependent dehydrogenases is also discussed.     

NaCl stress effects on enzymes involved in nitrogen assimilation pathway in tomato "Lycopersicon esculentum" seedlings

Tomato plants (Lycopersicon esculentum Mill, cv. Chibli F1) grown for 10 days on control medium were exposed to differing concentrations of NaCl (0, 25, 50, and 100mM). Increasing salinity led to a decrease of dry weight (DW) production and protein contents in the leaves and roots. Conversely, the root to shoot (R/S) DW ratio was increased by salinity. Na(+) and Cl(-) accumulation were correlated with a decline of K(+) and NO(3)(-) in the leaves and roots. Under salinity, the activities of nitrate reductase (NR, EC 1.6.6.1) and glutamine synthetase (GS, EC 6.3.1.2) were repressed in the leaves, while they were enhanced in the roots. Nitrite reductase (NiR, EC 1.7.7.1) activity was decreased in both the leaves and roots. Deaminating activity of glutamate dehydrogenase (GDH, EC 1.4.1.2) was inhibited, whereas the aminating function was significantly stimulated by salinity in the leaves and roots. At a high salt concentration, the nicotinamide adenine dinucleotide reduced (NADH)-GDH activity was stimulated concomitantly with the increasing NH(4)(+) contents and proteolysis activity in the leaves and roots. With respect to salt stress, the distinct sensitivity of the enzymes involved in nitrogen assimilation is discussed.     

Role of sulphur availability on cadmium-induced changes of nitrogen and sulphur metabolism in maize (Zea mays L.) leaves

The interactions between sulphur nutrition and Cd exposure were investigated in maize (Zea mays L.) plants. Plants were grown for 12 days in nutrient solution with or without sulphate. Half of the plants of each treatment were then supplied with 100 microM Cd. Leaves were collected 0, 1, 2, 3, 4 and 5 days from the beginning of Cd application and used for chemical analysis and enzyme assays. Cd exposure produced symptoms of toxicity (leaf chlorosis, growth reduction) and induced a noticeable accumulation of non-protein SH compounds. As phytochelatins are glutamate- and cysteine-rich peptides, the effect of cadmium on some enzyme activities involved in N and S metabolism of maize leaves was studied in relation to the plant sulphur supply. In vivo Cd application to S-sufficient plants resulted in a drop of all measured enzyme activities. On the other hand, S-deficient plants showed a decrease in nitrate reductase (NR; EC 1.6.6.1) and glutamine synthetase (GS; EC 6.3.1.2) activity, and an increase in NAD-dependent glutamate dehydrogenase (GDH; EC 1.4.1.2) and phosphoenolpyruvate carboxylase (PEPc; EC 4.1.1.31) activity as a result of the Cd treatment. Furthermore, in the same plants ATP sulphurylase (ATPs; EC 2.7.7.4) and O-acetylserine sulphydrylase (OASs; EC 4.2.99.8) showed a particular pattern as both enzymes exhibited a transient maximum value of activity after 4 days from the beginning of Cd exposure. Results provide evidence that the increase of ATPs, OASs, GDH and PEPc activities, observed exclusively in S-deficient Cd-treated plants, may be part of the defence mechanism based on the production of phytochelatins.     

Coordinated regulation of ammonium assimilation and carbon catabolism by glyoxylate in Saccharomyces cerevisiae

The activities of citrate synthase (EC 4.1.3.7) and NADP+-dependent glutamate dehydrogenase (GDH) (EC 1.4.1.4) of Saccharomyces cerevisiae were inhibited in vitro by glyoxylate. In the presence of glyoxylate, pyruvate and glyoxylate pools increased, suggesting that glyoxylate was efficiently transported and catabolized. Pyruvate accumulation also indicates that citrate synthase was inhibited. A decrease in the glutamate pool was also observed under these conditions. This can be attributed to an increased transamination rate and to the inhibitory effect of glyoxylate on NADP+-dependent GDH. Furthermore, the increase in the ammonium pool in the presence of glyoxylate suggests that NADP+-dependent GDH was being inhibited in vivo, since the activity of glutamine synthetase did not decrease under these conditions. We propose that the inhibition of both citrate synthase and NADP+-dependent GDH could form part of a mechanism that regulates the internal 2-oxoglutarate concentration.     

A study of the role of glutamate dehydrogenase in the nitrogen metabolism of Arabidopsis thaliana

Glutamate-dehydrogenase (GDH, EC 1.4.1.2) activity and isoenzyme patterns were investigated in Arabidopsis thaliana plantlets, and parallel studies were carried out on glutamine synthetase (GS, EC 6.3.1.2). Both NADH-GDH and NAD-GDH activities increased during plant development whereas GS activity declined. Leaves deprived of light showed a considerable enhancement of NADH-GDH activity. In roots, both GDH activities were induced by ammonia whereas in leaves nitrogen assimilation was less important. It was demonstrated that the increase in GDH activity was the result of de-novo protein synthesis. High nitrogen levels were first assimilated by NADH-GDH, while GS was actively involved in nitrogen metabolism only when the enzyme was stimulated by a supply of energy, generated by NAD-GDH or by feeding sucrose. When methionine sulfoximine, an inhibitor of GS, was added to the feeding solution, NADH-GDH activity remained unaffected in leaves whereas NAD-GDH was induced. In roots, however, there was a marked activation of GDH and no inactivation of GS. It was concluded that NADH-GDH was involved in the detoxification of high nitrogen levels while NAD-GDH was mainly responsible for the supply of energy to the cell during active assimilation. Glutamine synthetase, on the other hand was involved in the assimilation of physiological amounts of nitrogen. A study of the isoenzyme pattern of GDH indicated that a good correlation existed between the relative activity of the isoenzymes and the ratio of aminating to deaminating enzyme activities. The NADH-GDH activity corresponded to the more anodal isoenzymes while the NAD-GDH activity corresponded to the cathodal ones. The results indicate that the two genes involved in the formation of GDH control the expression of enzymes with different metabolic functions.Abbreviations GDH glutamate dehydrogenase - GS glutamine synthetase - MSO methionine sulfoximine     

Evaluation of the enzymes involved in primary nitrogen metabolism in Tilia platyphyllos-Tuber borchii ectomycorrhizae

No information is available about Tuber borchii Vittad. ammonium metabolism during its life cycle, which involves the succession of three distinct phases. In this direction, the levels of glutamine synthetase (GS; EC 6.3.1.2), glutamate synthase (GOGAT; EC 1.4.1.13-14) and glutamate dehydrogenase (GDH; EC 1.4.1.2-4) were evaluated in Tilia platyphyllos Scop.-Tuber borchii Vittad. ectomycorrhizae, free living mycelium and non-inoculated roots. In the plant roots, GS shows high specific activity and only NADH-GDH (EC 1.4.1.2) is detectable; on the other hand, in free living mycelium GS and NADPH-GDH (EC 1.4.1.4) can be detected. Ectomycorrhizal metabolism was found to be deeply influenced by the two symbiotic partners. In fact, GS and both forms of GDH are present and their specific activities are higher than those found in the plant root and in the mycelial cells.     

How does glutamine synthetase activity determine plant tolerance to ammonium?

The wide range of plant responses to ammonium nutrition can be used to study the way ammonium interferes with plant metabolism and to assess some characteristics related with ammonium tolerance by plants. In this work we investigated the hypothesis of plant tolerance to ammonium being related with the plants’ capacity to maintain high levels of inorganic nitrogen assimilation in the roots. Plants of several species (Spinacia oleracea L., Lycopersicon esculentum L., Lactuca sativa L., Pisum sativum L. and Lupinus albus L.) were grown in the presence of distinct concentrations (0.5, 1.5, 3 and 6 mM) of nitrate and ammonium. The relative contributions of the activity of the key enzymes glutamine synthetase (GS; under light and dark conditions) and glutamate dehydrogenase (GDH) were determined. The main plant organs of nitrogen assimilation (root or shoot) to plant tolerance to ammonium were assessed. The results show that only plants that are able to maintain high levels of GS activity in the dark (either in leaves or in roots) and high root GDH activities accumulate equal amounts of biomass independently of the nitrogen source available to the root medium and thus are ammonium tolerant. Plant species with high GS activities in the dark coincide with those displaying a high capacity for nitrogen metabolism in the roots. Therefore, the main location of nitrogen metabolism (shoots or roots) and the levels of GS activity in the dark are an important strategy for plant ammonium tolerance. The relative contribution of each of these parameters to species tolerance to ammonium is assessed. The efficient sequestration of ammonium in roots, presumably in the vacuoles, is considered as an additional mechanism contributing to plant tolerance to ammonium nutrition.     

Ammonium and amino acid metabolism in excised leaves of wheat (Triticum aestivum) senescing in the dark

Several parameters of amino acid metabolism were studied in detached primary leaves of wheat (Triticum aestivum L. cv. Castell) during a 14 day incubation period in the dark. Protein loss was accompanied by a 5-fold increase in the total amount of free amino acids during the first 4 days of the incubation period with asparagine being the most important. Beyond this stage a pronounced intracellular accumulation of ammonium occured. A gradual decrease in the levels of free amino acids and ammonium at the later stages of senescence could in part be accounted for by leakage from the leaves. Additionally, some nitrogen was lost due to ammonia volatilization. The rapid decay of the glutamine synthetase (GS; EC 6.3.1.2)-glutamate synthase (Fd-GOGAT; EC 1.4.7.1) system and the fast decline of glutamate-pyruvate transaminase (GPT; EC 2.6.1.2) activity appear to be predominant features of senescence in the dark. Decreasing Fd-GOGAT activity was slightly compensated by a small and temporary increase in the activity of NADH-GOGAT (EC 1.4.1.14). Glutamateoxalocetate transaminase (GOT: EC 2.6.1.1) activity, although declining continuously, proved to be much more persistent. Changes in glutamate dehydrogenase (GDH; EC 1.4.1.3) activity closely resembled the profile of ammonium evolution in the leaves and NADP-isocitrate dehydrogenase (IDH; EC 1.1.1.42) activity revealed a temporary maximum during the period of rapid increase in GDH activity. Increased activity of GDH could also be induced by exogenous ammonium. Ammonium accumulation could, at least partly, be caused by increased asparaginase (EC 3.5.1.1) activity which accompanied the rapid conversion of asparagine to aspartic acid. Asparagine aminotransferase (EC 2.6.1.14) activity declined sharply from the beginning of the senescence period. Although the activity profile of glutaminase (EC 3.5.1.2) was similar to that of asparaginase, glutamine was of little importance quantitatively and an analogous relationship between glutamine and glutamic acid could not be detected.     

Salt stress affects glutamine synthetase activity and mRNA accumulation on potato plants in an organ-dependent manner

Ammonium assimilation into glutamine and glutamate is vital for plant growth as these are precursors for almost all nitrogenous compounds. Ammonium can be assimilated onto nitrogenous organic compounds by the concerted action of two enzymes that compose the glutamine synthetase (GS, EC 6.3.1.2) – glutamate synthase (Fd-GOGAT, EC 1.4.7.1; NADH–GOGAT, EC 1.4.1.14) cycle. Ammonium may also be directly incorporated into glutamate by the glutamate dehydrogenase (GDH, EC 1.4.1.2) aminating reaction. However, as GDH reversibly deaminates glutamate, its physiological role in vivo remains controversial. Potato has been classified as moderately tolerant to salinity. Potato GS is encoded by a small multigene family which is differentially regulated in an organ and age-dependent way. In this study, the effect of increasing concentrations of salinity in the soil in GS activity and gene-specific mRNA accumulation levels were studied on potato leaves and roots, as well as the biochemical parameters protein, chlorophyll, lipid peroxidation and proline levels, in order to evaluate the severity of the imposed stress. The data obtained suggests that when potato plants are subjected to salt stress, increased ammonium assimilation occurs in roots, due to an increased GS accumulation, along with a decreased assimilation in leaves. Regarding GS gene-specific mRNA accumulation, an organ-dependent response was also observed that contributes for the detected alteration in the ammonium assimilatory metabolism. This response may be a key feature for future genetic manipulations in order to increase crop productivity in salty soils. The possible contribution of GDH for ammonia assimilation was also investigated.     

Cold acclimation of wheat (Triticum aestivum): Properties of enzymes involved in proline metabolism

Two cultivars of wheat ( Triticum aestivum L.), a winter wheat, Kharkov, and a spring wheat, Glenlea, were acclimated under controlled conditions at 2 temperatures, 5°C and 25°C with a 12-h photoperiod. Water content, protein and proline concentrations were determined. Enzymatic properties (activity and apparent energy of activation) were investigated for enzymatic systems involved in 2 pathways of proline metabolism, the glutamic acid and ornithine pathways. Four enzymes were studied, proline dehydrogenase (PDH, EC 1.5.1.2), glutamate dehydrogenase (GDH, EC 1.4.1.2-4), glutamine synthetase (GS, EC 6.3.1.2) and ornithine transaminase (OT, EC 2.6.1.13). Cold acclimation led to an accumulation of proline, a decrease in water content and an increase in soluble protein, especially in winter wheat. For both cultivars, cold acclimation modulated enzyme properties of PDH and GDH. Increased activities of GS and OT were observed as a result of cold acclimation in both cultivars, with the greatest increase in Kharkov. The apparent energy of activation of these 2 enzymes decreased, particularly for Kharkov, which accumulated proline in cold conditions.     

Changes induced by Fe deficiency and Fe resupply in the organic acid metabolism of sugar beet (Beta vulgaris) leaves

The effects of iron deficiency and iron resupply on the metabolism of leaf organic acids have been investigated in hydroponically grown sugar beet. Organic acid concentrations and activities in leaf extracts of several enzymes related to organic acid metabolism were measured. Enzymes assayed included phosphoenol pyruvate carboxylase (PEPC; EC 4.1.1.31), different Krebs cycle enzymes: malate dehydrogenase (MDH; EC 1.1.1.37), aconitase (EC 4.2.1.3), fumarase (EC 4.2.1.2), citrate synthase (CS; EC 4.1.3.7) and isocitrate dehydrogenase (ICDH; EC 1.1.1.42), glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) and two enzymes related to anaerobic metabolism (lactate dehydrogenase [LDH]; EC 1.1.1.27, and pyruvate decarboxylase [PDC]; EC 4.1.1.1). Iron concentration in leaves was severely decreased by iron deficiency. Iron resupply caused an increase in iron concentrations, reaching levels similar to the controls in 96 h. Iron deficiency induced a 2.3-fold (from 16 to 37 mmol m⁻²) increase in leaf total organic acid concentration. Organic anion concentrations were still 4-fold higher than the controls 24 h after resupply and decreased to values similar to those found in the controls after 96 h. All measured enzymes had increased activities in extracts of iron-deficient leaves when compared to the controls and generally decreased to control values 24 h after iron addition. These data provide evidence that organic acid accumulation in iron-deficient leaves is likely not due to an enhancement in leaf carbon fixation. Instead, this accumulation could be associated with organic acid export from the roots to the leaves via xylem.     

NaCl对水稻谷氨酸合酶和谷氨酸脱氢酶的胁迫作用

在ＮａＣｌ的胁迫下,水稻幼苗根和叶的谷氨酸合酶和谷氨酸脱氢酶的活性随着营养液中的ＮａＣｌ浓度的升高而降低;游离ＮＨ４＾＋在叶中积累,在根中未见明显变化。与根相比,叶对ＮａＣｌ的胁迫作用更为敏感。叶的ＮＡＤＨ－ＧＯＧＡＴ和ＮＡＤＨ－ＧＤＨ活性在ＮａＣｌ胁迫降低的程度明显大于根。无论是否有ＮａＣｌ存在,根的ＮＡＤＨ－ＧＤＨ活性明显高于叶。ＧＳ／ＧＤＨ比值分析提示,对对照下,根中的ＮＨ４＾存在,根的ＮＡ     

Regulation of carbon and nitrogen metabolisms in rice roots by 2-oxoglutarate at the level of hexokinase

Feeding experiments were designed, to investigate the role of 2-oxoglutarate (2-OG) in regulation of carbon and nitrogen metabolisms in non-photosynthetic tissues of rice ( Oryza sativa L.), and enzyme activities involved in the metabolisms as well as contents of several relating metabolites were determined in the roots. The enhancement of 2-OG level by feeding 2-OG or metabolizable sugars [sucrose (Suc) or glucose (Glc)], rather than by feeding non-metabolizable carbon sources (mannose or mannitol), led to increase in enzyme activities, including hexokinase (HXK, EC 2.7.1.1), nicotinamide adenine dinucleotide phosphate (NADP)⁺-dependent isocitrate dehydrogenase (NADP⁺-ICDH, EC 1.1.1.42), phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), glutamine synthetase (GS, EC 6.3.1.2) and the reduced form of nicotinamide adenine dinucleotide (NADH)-dependent glutamate synthase (NADH-GOGAT, EC 1.4.1.14). In addition, the increase in ammonium uptake, glutamine and glutamate (Glu) as well as the decrease in soluble carbohydrates were observed. The effects of feeding 2-OG or metabolizable sugars were reversed by feeding of N- acetyl-glucosamine (NAG; a HXK inhibitor). The decreased 2-OG level by the feeding of NAG alone led to increase in soluble carbohydrates and decrease in the enzyme activities, ammonium uptake as well as Glu content. The effects of NAG were reversed by supply of 2-OG, Suc and Glc. These results suggest that nitrogen uptake and assimilation as well as their related carbohydrate metabolism in rice roots were regulated in coordination by 2-OG level, and HXK activity was involved in the regulation of 2-OG.     

Activity of enzymes of carbon metabolism during the induction of Crassulacean acid metabolism in Mesembryanthemum crystallinum L.

The maximum extractable activities of twenty-one photosynthetic and glycolytic enzymes were measured in mature leaves of Mesembryanthemum crystallinum plants, grown under a 12 h light 12 h dark photoperiod, exhibiting photosynthetic characteristics of either a C₃ or a Crassulacean acid metabolism (CAM) plant. Following the change from C₃ photosynthesis to CAM in response to an increase in the salinity of in the rooting medium from 100 mM to 400 mM NaCl, the activity of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) increased about 45-fold and the activities of NADP malic enzyme (EC 1.1.1.40) and NAD malic enzyme (EC 1.1.1.38) increased about 4- to 10-fold. Pyruvate, Pi dikinase (EC 2.7.9.1) was not detected in the non-CAM tissue but was present in the CAM tissue; PEP carboxykinase (EC 4.1.1.32) was detected in neither tissue. The induction of CAM was also accompanied by large increases in the activities of the glycolytic enzymes enolase (EC 4.2.1.11), phosphoglyceromutase (EC 2.7.5.3), phosphoglycerate kinase (EC 2.7.2.3), NAD glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), and glucosephosphate isomerase (EC 2.6.1.2). There were 1.5- to 2-fold increases in the activities of NAD malate dehydrogenase (EC 1.1.1.37), alanine and aspartate aminotransferases (EC 2.6.1.2 and 2.6.1.1 respectively) and NADP glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13). The activities of ribulose-1,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39), fructose-1,6-bisphosphatase (EC 3.1.3.11), phosphofructokinase (EC 2.7.1.11), hexokinase (EC 2.7.1.2) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) remained relatively constant. NADP malate dehydrogenase (EC 1.1.1.82) activity exhibited two pH optima in the non-CAM tissue, one at pH 6.0 and a second at pH 8.0. The activity at pH 8.0 increased as CAM was induced. With the exceptions of hexokinase and glucose-6-phosphate dehydrogenase, the activities of all enzymes examined in extracts from M. crystallinum exhibiting CAM were equal to, or greater than, those required to sustain the maximum rates of carbon flow during acidification and deacidification observed in vivo. There was no day-night variation in the maximum extractable activities of phosphoenolpyruvate carboxylase, NADP malic enzyme, NAD malic enzyme, fructose-1,6-bisphosphatase and NADP malate dehydrogenase in leaves of M. crystallinum undergoing CAM.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - RuBP ribulose-1,5-bisphosphate     

Induction of a specific isoenzyme of glutamate dehydrogenase during isolation and the first 48 h of culture of Brassica napus leaf protoplasts

The specific activity of NADH‐glutamate dehydrogenase (GDH, EC 1.4.1.2) in leaf protoplasts ( Brassica napus L. cv. Bronowski) was initially low and progressively increased during culture in Murashige and Skoog (MS) medium and MS (−NH₄) (ammonium nitrate‐free MS) medium in the dark. Native polyacrylamide gel electrophoresis (PAGE) and tetrazolium staining revealed that the high specific activity of NAD‐GDH (deamination) in leaves correlated with the cathodal isoenzymes, and the high specific activity of NADH‐GDH (amination) in leaf protoplasts to the anodal ones. Changes in isoenzyme pattern were correlated with an increase in the specific activity of NADH‐GDH but not with the NADH‐GDH/NAD‐GDH ratio. The increase in NADH‐GDH (amination) activity of leaf protoplasts was correlated with the occurrence of the isoenzyme GDH7, which was not detected in leaves.     

Implication of glutamate, isocitrate and malate deshydrogenases in nitrogen assimilation in the cadmium-stressed tomato

Tomato seedlings grown on nitric medium and treated with various cadmium concentrations (0 to 50 microM) were used. Results obtained show that cadmium remains predominantly located in the roots, which then seem to play the role of trap-organs. Increasing cadmium concentration in the medium leads particularly to a decrease in NO3- accumulation, together with a decrease in the activity of glutamine synthetase and in the quantity of plastidic isoform ARNm (GS2), and, on the contrary, to an increase of the cytosolic isoform ARNm (GS1). On the other hand, stimulations were observed for NADH-dependent glutamate synthase, NADH-dependent glutamate dehydrogenase, ARNm quantity of this enzyme, ammonium accumulation, and protease activity. In parallel, stimulations were observed for NAD+ and NADP+-dependent malate dehydrogenase and NADP+-dependent isocitrate dehydrogenase. These results were discussed in relation to the hypothesis attributing to the dehydrogenase enzymes (GDH, MDH, ICDH) an important role in the plant defence processes against cadmium-induced stresses.     

The changes in glutamic dehydrogenase activity following illumination of etiolated barley seedlings

Summary Changes in NADH- and NADPH-linked glutamic dehydrogenase (GDH) activity have been measured following illumination of etiolated barley leaves. Both activities initially increase, then decrease to below the etiolated value. The latter change is dependent upon prior illumination but independent of the presence of roots or added ammonium sulphate. Subsequently a sustained increase of both activities occurs. Possible reasons for these changes and alterations in the ratio of the two GDH activities are discussed.     

Differential role of glutamate dehydrogenase in nitrogen metabolism of maize tissues

Both calli and plantlets of maize (Zea mays L. var Tuxpeño 1) were exposed to specific nitrogen sources, and the aminative (NADH) and deaminative (NAD⁺) glutamate dehydrogenase activities were measured at various periods of time in homogenates of calli, roots, and leaves. A differential effect of the nitrogen sources on the tissues tested was observed. In callus tissue, glutamate, ammonium, and urea inhibited glutamate dehydrogenase (GDH) activity. The amination and deamination reactions also showed different ratios of activity under different nitrogen sources. In roots, ammonium and glutamine produced an increase in GDH-NADH activity whereas the same metabolites were inhibitory of this activity in leaves. These data suggest the presence of isoenzymes or conformers of GDH, specific for each tissue, whose activities vary depending on the nutritional requirements of the tissue and the state of differentiation.     

Disturbed ammonium assimilation is associated with growth inhibition of roots in rice seedlings caused by NaCl

The effects of NaCl on changes in ammonium level and enzyme activities of ammonium assimilation in roots growth of rice (Oryza sativa L.) seedlings were investigated. NaCl was effective in inhibiting root growth and stimulated the accumulation of ammonium in roots. Accumulation of ammonium in roots preceded inhibition of root growth caused by NaCl. Both effects caused by NaCl are reversible. Exogenous ammonium chloride and methionine sulfoximine (MSO), which caused ammonium accumulation in roots, inhibited root growth of rice seedlings. NaCl decreased glutamine synthetase and glutamate synthase activities in roots, but increased glutamate dehydrogenase activity. The growth inhibition of roots by NaCl or MSO could be reversed by the addition of L-glutamic acid or L-glutamine. The current results suggest that disturbance of ammonium assimilation in roots may be involved in regulating root growth reduction caused by NaCl.Abbreviations GDH glutamate dehydrogenase - GOGAT glutamate synthase - GS glutamine synthetase - MSO methionine sulfoximine