Regulation of the ammonia assimilatory enzymes in Salmonella typhimurium.

The regulation of glutamate dehydrogenase (EC 1.4.1.4), glutamine synthetase (EC 6.3.1.2), and glutamate synthase (EC 2.6.1.53) was examined for cultures of Salmonella typhimurium grown with various nitrogen and amino acid sources. In contrast to the regulatory pattern observed in Klebsiella aerogenes, the glutamate dehydrogenase levels of S. typhimurium do not decrease when glutamine synthetase is derepressed during growth with limiting ammonia. Thus, it appears that the S. typhimurium glutamine synthetase does not regulate the synthesis of glutamate dehydrogenase as reported for K. aerogenes. The glutamate dehydrogenase activity does increase, however, during growth of a glutamate auxotroph with glutamate as a limiting amino acid source. The regulation of glutamate synthase levels is complex with the enzyme activity decreasing during growth with glutamate as a nitrogen source, and during growth of auxotrophs with either glutamine or glutamate as limiting amino acids.     

Submitochondrial localization and function of enzymes of glutamine metabolism in avian liver

Glutamine synthetase (EC 6.3.1.2) was localized within the matrix compartment of avian liver mitochondria. The submitochondrial localization of this enzyme was determined by the digitonin-Lubrol method of Schnaitman and Greenawalt (35). The matrix fraction contained over 74% of the glutamine synthetase activity and the major proportion of the matirx marker enzymes, malate dehydrogenase (71%), NADP-dependent isocitrate dehydrogenase (83%), and glutamate dehydrogenase (57%). The highest specific activities of these enzymes were also found in the matrix compartment. Oxidation of glutamine by avian liver mitochondria was substantially less than that of glutamate. Bromofuroate, an inhibitor of glutamate dehydrogenase, blocked oxidation of glutamate and of glutamine whereas aminoxyacetate, a transaminase inhibitor, had little or no effect with either substrate. These results indicate that glutamine metabolism is probably initiated by the conversion of glutamine to glutamate rather than to an alpha-keto acid. The localization of a glutaminase activity within avian liver mitochondria plus the absence of an active mitochondrial glutamine transaminase is consistent with the differential effects of the transaminase and glutamate dehydrogenase inhibitors. The high glutamine synthetase activity (40:1) suggests that mitochondrial catabolism of glutamine is minimal, freeing most of the glutamine synthesized for purine (uric acid) biosynthesis.     

Activity profiles of enzymes involved in glutamine and glutamate metabolism in the apple during autumnal senescence

Seasonal changes in glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 2.6.1.53), and glutamate dehydrogenase (EC 1.4.1.3) were measured in both senescing leaf and bark tissues of ‘Golden Delicious’ apple trees (Malus domestica Borkh.). From the measured enzyme activities we attempted to estimate the in vivo catalytic potentials of the enzymes with special reference to nitrogen mobilization and conservation of senescing apple trees. The cumulative glutamine synthetase activity of leaf tissue was about three times higher than that of bark. The estimated catalytic potential of leaf glutamine synthetase was 800-fold higher than the actual protein nitrogen loss of senescing leaves. The cumulative glutamate synthase activity of bark was about six times higher than that of leaf. The estimated catalytic potential of bark glutamate synthase was 160-times higher than the actual protein nitrogen gain in that tissue. The cumulative glutamate dehydrogenase activities in leaf and bark tissue were approximately the same. However, the catalytic potential of leaf glutamate dehydrogenase was twice that of leaf glutamate synthase. It is thus concluded that the physiological role of glutamine synthetase in senescing leaf tissue is to furnish the amide(s) prior to mobilization of nitrogen to storage tissue. The higher activity of glutamate synthase in bark tissue could provide a mechanism to transform the imported amide nitrogen to amino nitrogen of glutamate for storage protein synthesis. The possible regulatory factors upon the activity of these enzymes in the tissues of senescing apple trees are discussed.     

The localisation of enzymes of nitrogen assimilation in maize leaves and their activities during greening

The activities of nitrate reductase (EC1.6.6.1), nitrite reductase (EC 1.6.6.4), glutamine synthetase (EC6.3.1.2), glutamate synthase (EC1.4.7.1) and NAD(P)H-dependent glutamate dehydrogenase (EC 1.4.1.3) were investigated in mesophyll and bundle sheath cells of maize leaves (Zea mays L.). Whereas nitrate and nitrite reductase appear to be restricted to the mesophyll and GDH to the bundle sheath, glutamine synthetase and glutamate synthase are active in both tissues.During the greening process, the activities of nitrate and nitrite reductase increased markedly, but glutamine synthetase, glutamate synthase and glutamate dehydrogenase changed little.Abbreviations BDH British Drug Houses - EDTA Ethylene diamine tetra-acetic acid - GDH Glutamate dehydrogenase - NADH Nicotinamide-adenine dinucleotide reduced form - NADPH Nicotnamide-adenine dinucleotide phosphate reduced form - PMSF Phenylmethyl sulphonyl fluoride     

Regulation of asparaginase, glutamine synthetase, and glutamate dehydrogenase in response to medium nitrogen concentrations in a euryhaline chlamydomonas species

The ammonium assimilatory enzymes glutamine synthetase (EC 6.3.1.2) and glutamate dehydrogenase (EC 1.4.1.3) were investigated for a possible role in the regulation of asparaginase (EC 3.5.1.1) in a Chlamydomonas species isolated from a marine environment. Cells grown under nitrogen limitation (0.1 millimolar NH(4) (+), NO(3) (-), or l-asparagine) possessed 6 times the asparaginase activity and approximately one-half the protein of cells grown at high nitrogen levels (1.5 to 2.5 millimolar). Biosynthetic glutamine synthetase activity was 1.5 to 1.8 times greater in nitrogen-limited cells than cells grown at high levels of the three nitrogen sources.Conversely, glutamate dehydrogenase (both NADH- and NADPH-dependent activities) was greatest in cells grown at high levels of asparagine or ammonium, while nitrate-grown cells possessed little activity at all concentrations employed. For all three nitrogen sources, glutamate dehydrogenase activity was correlated to the residual ammonium concentration of the media after growth (r = 0.88 and 0.94 for NADH- and NADPH-dependent activities, respectively).These results suggest that glutamate dehydrogenase is regulated in response to ambient ammonium levels via a mechanism distinct from asparaginase or glutamine synthetase. Glutamine synthetase and asparaginase, apparently repressed by high levels of all three nitrogen sources, are perhaps regulated by a common mechanism responding to intracellular nitrogen depletion, as evidenced by low cellular protein content.     

Ammonia assimilation by rhizobium cultures and bacteroids.

The enzymes involved in the assimilation of ammonia by free-living cultures of Rhizobium spp. are glutamine synthetase (EC. 6.o.I.2), glutamate synthase (L-glutamine:2-oxoglutarate amino transferase) and glutamate dehydrogenase (ED I.4.I.4). Under conditions of ammonia or nitrate limitation in a chemostat the assimilation of ammonia by cultures of R. leguminosarum, R. trifolii and R. japonicum proceeded via glutamine synthetase and glutamate synthase. Under glucose limitation and with an excess of inorganic nitrogen, ammonia was assimilated via glutamate dehydrogenase, neither glutamine synthetase nor glutamate synthase activities being detected in extracts. The coenzyme specificity of glutamate synthase varied according to species, being linked to NADP for the fast-growing R. leguminosarum, R. melitoti, R. phaseoli and R. trifolii but to NAD for the slow-growing R. japonicum and R. lupini. Glutamine synthetase, glutamate synthase and glutamate dehydrogenase activities were assayed in sonicated bacteroid preparations and in the nodule supernatants of Glycine max, Vicia faba, Pisum sativum, Lupinus luteus, Medicago sativa, Phaseolus coccineus and P. vulgaris nodules. All bacteroid preparations, except those from M. sativa and P. coccineus, contained glutamate synthase but substantial activities were found only in Glycine max and Lupinus luteus. The glutamine synthetase activities of bacteroids were low, although high activities were found in all the nodule supernatants. Glutamate dehydrogenase activity was present in all bacteroid samples examined. There was no evidence for the operation of the glutamine synthetase/glutamate synthase system in ammonia assimilation in root nodules, suggesting that ammonia produced by nitrogen fixation in the bacteroid is assimilated by enzymes of the plant system.     

Enzymes of ammonia assimilation in Rhizobium leguminosarum bacteroids.

The activities of the following enzymes were studied in connection with dinitrogen fixation in pea bacteroids: glutamine synthetase(L-glutamate: ammonia ligase (ADP-forming)(EC 6.3.1.2)(GS); glutamate dehydrogenase (NADP+)(L-glutamate: NADP+ oxidoreductase (deaminating)(EC 1.4.1.4)(GDH); glutamate synthase (L-glutamine: 2-exeglutarate aminotransferase (NADPH-oxidizing))(EC 2.6.1.53)(GOGAT). GS activity was high throughout the growth of the plant and GOGAT activity was always low. It is unlikely that GDH or the GS-GOGAT pathway can account for the incorporation of ammonia from dinitrogen fixation in the pea bacteroid,     

Regulation of nitrogen metabolism in Escherichia coli and Klebsiella aerogenes: studies with the continuous-culture technique.

Ammonia-nitrogen-limited continuous cultures of Escherichia coli and Klebsiella aerogenes contain induced levels of glutamine synthetase that is deadenylyated (i.e., fully active). In the presence of excess ammonia or glutamate in glucose-limited cultures of E. coli, glutamine synthetase is repressed and adenylylated (inactive). The average state of adenylylation (n) is a linear function of the specific growth rate. At low specific growth rates, glutamine synthetase is adenylylated; as the specific growth rate increases, n decreases, approaching 0 to 2 at rapid growth rates. The average state of adenylylation correlates well with the intracellular concentrations and ratios of alpha-ketoglutarate and glutamine, which are key effectors in the adenylylation-deadenylylation systems. E. coli and K. aerogenes differ markedly in their growth yields, growth rates, and enzymatic composition during nitrogen limitation. The data suggest that, unlike K. aerogenes, E. coli W uses glutamate dehydrogenase to incorporate ammonia during nitrogen limitation. In E. coli, glutamate dehydrogenase is progressively induced during nitrogen limitation when mu (growth rate) approaches mumax. In contrast, in K. aerogenes glutamate dehydrogenase is repressed during nitrogen limitation, whereas glutamate synthase, an alternative supplier of glutamate to the cell, is induced. Data are presented that support the regulatory schemes proposed for the control of glutamine synthetase activity by induction-repression phenomena and adenylylation-deadenylylation reaction. We propose that the intracellular ratio of alpha-ketoglutarate to glutamine may be the most important physiological parameter in determining the activity of glutamine synthetase.     

Assimilation of Ammonium Nitrogen by Heterotrophic and Symbiotrophic White Lupine Plants

The activities of glutamate dehydrogenase, asparagine synthetase, and total glutamine synthetase in the organs of the white lupine (Lupinus albus L.) plants were measured during plant growth and development. In addition, the dynamics of free amino acids and amides in plant organs was followed. It was shown that the change in the nutrition type was important for controlling enzyme activities in the organs examined and, consequently, for directing the pathway of ammonium nitrogen assimilation. As long as the plants remained heterotrophic, glutamine-dependent asparagine synthetase of cotyledons and glutamine synthetase of leaves apparently played a major role in the assimilation of ammonium nitrogen. In symbiotrophic plants, root nodules became an exclusive site of asparagine synthesis, and the role of leaf glutamine synthetase increased. Unlike glutamine synthetase and asparagine synthetase, glutamate dehydrogenase activity was present in all organs examined and was less dependent on the nutrition type. This was also indicated by a weak correlation of glutamate dehydrogenase activity with the dynamics of free amino acid and amide content in these organs. It is supposed that glutamine synthetase plays a leading role in both the primary assimilation of ammonium, produced during symbiotic fixation of molecular nitrogen in root nodules, and in its secondary assimilation in cotyledons and leaves. On the other hand, secondary nitrogen assimilation in the axial organs occurs via an alternative glutamate dehydrogenase pathway.     

The involvement of glutamine synthetase/glutamate synthase in ammonia assimilation by Aspergillus nidulans

Wild-type Aspergillus nidulans grew equally well on NH4Cl, KNO3 or glutamine as the only nitrogen source. NADP+-dependent glutamate dehydrogenase (EC 1.4.1.4) and glutamine synthetase (GS; EC 6.3.1.2) activities varied with the type and concentration of nitrogen source supplied. Glutamate synthase (GOGAT) activity (EC 1.4.7.1) was detected but it was almost unaffected by the type and concentration of nitrogen source supplied. Ion exchange chromatography showed that the GOGAT activity was due to a distinct enzyme. Azaserine, an inhibitor of the GOGAT reaction, reduced the glutamate pool by 60%, indicating that GOGAT is involved in ammonia assimilation by metabolizing the glutamine formed by GS.     

THE EFFECTS OF HYPERKETONEMIA ON GLUTAMATE AND GLUTAMINE METABOLISM IN DEVELOPING RAT BRAIN

Abstract— The effect of increased exposure to ketone bodies in the developing rat brain suggest that intrauterine and postnatal hyperketonemia lead to an altered metabolism of glutamine and glutamate. It is postulated that this effect is related to the delayed development of glutaminase ( l -glutamine amido-hydrolase EC 3.5.1.2) and glutamate dehydrogenase ( l -glutamate: NAD oxidoreductase EC 1.4.1.2).
The specific activities of glutamate dehydrogenase (GDH), glutaminase and glutamine synthetase ( l -glutamate: ammonia ligase EC 6.3.1.2) in the brains of newborn rats increased during early development. A positive correlation was observed between the specific activity of glutaminase and the concentration of glutamate in the brain as well as between the concentrations of blood and brain glutamine and glutamate in both control and hyperketonemic pups. This indicates a different degree of permeability and metabolism for glutamine and glutamate in the brain during the neonatal period, as compared to adulthood.
In hyperketonemic pups, glutamine and glutamate metabolism were found to differ from that in control animals. The concentrations of glutamate were higher, and glutamine lower, in both the blood and brain as compared to that in controls. The concentrations of α-ketoglutarate were also lower in their brain. In the brains of hyperketonemic and control pups, the concentration of malate was the same. During the first 3 weeks of life the increase of spec. act. of GDH and glutaminase was found to be suppressed in the brains of hyperketonemic pups. However, the spec. act. of glutamine synthetase was similar to that of the control pups.     

Effects of water stress on enzymes of ammonia assimilation in root nodules of alfalfa (Medicago sativa)

Protein content and activities of the enzymes glutamine synthetase (EC 6.3.1.2), NADH-glutamate synthase (EC 1.4.1.14), NADH-glutamate dehydrogenase (reductive amination (EC 1.4.1.2) and NAD⁺-glutamate dehydrogenase (oxidative deamination) (EC 1.4.1.2) from the plant fraction of root nodules of alfalfa ( Medicago sativa L. cv. Aragon) were determined under water stress. Only NADH-glutamate synthase activity was inhibited during drought. The results indicate that the glutamine synthetase/NADH-glutamate synthase cycle was fully operational in alfalfa nodules of control or even mildly stressed plants when N₂-fixation was not inhibited, but that the coupling between glutamine synthetase and NADH-glutamate synthase was lost as drought progressed. Patterns of glutamine synthetase and NADH-/NAD⁺-gluta-mate dehydrogenase activities reflect changes in ammonia content of nodules and/or availability of carbon substrates, and indicate that nodules maintain sufficient enzyme activity for ammonia assimilation throughout water stress.     

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.     

Amino acid metabolism associated with N-mobilization from the flag leaf of wheat (Triticum aestivum L.) during grain development

Field-grown winter wheat (Triticum aestivum L. cv. Castell) was used to study changes in the free amino acid pools of different plant parts and related enzyme activities in the flag leaf throughout the grain-filling period in three consecutive growing seasons. Amino acid analysis data indicated that, during senescence, the nitrogen flow in the flag leaf was directed towards the synthesis of glutamine as a specific nitrogen transport form. Of the enzymes involved, total glutamine synthetase (GS; EC 6.3.1.2) and especially ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) activities declined continuously as senescence progressed. Unlike (chloroplastic) GS₂, (cytosolic) GS₁ was shown to be very persistent suggesting a special role for this isoenzyme in the N-reallocation process. Glutamate-oxaloacetate transaminase (GOT; EC 2.6.1.1), glutamate-pyruvate transaminase (GPT; EC 2.6.1.2) and isocitrate dehydrogenase (IDH; EC 1.1.1.42) showed a characteristic biphasic activity profile after anthesis. It is proposed that these enzymes, for each of which at least two isoenzymes were demonstrated, are involved in glutamate synthesis at the later stages of leaf senescence. Ammonium levels were fairly constant throughout the flag leafs life span, an ultimate rise often following peak values of glutamate dehydrogenase (GDH; EC 1.4.1.4) activity. The enzymology of flag leaf amino acid metabolism during grain development is further discussed in relation to observations of NH₃-volatilization from naturally senescing wheat plants.     

Enzymes of Nitrogen Assimilation Undergo Seasonal Fluctuations in the Roots of the Persistent Weedy Perennial Cichorium intybus

Chicory (Cichorium intybus), a deep rooted weed, grows in regions with temperate climates. Seasonal partitioning of compounds between the root and shoot results in fluctuations in the soluble carbohydrate, nitrate, amino acid, and protein pools within the roots. The activities of nitrate reductase (NR) (EC 1.6.6.1), glutamine synthetase (EC 6.3.1.2), NADH (EC 1.4.1.14), ferrodoxin glutamate synthase (EC 1.4.7.1), and glutamate dehydrogenase (GDH) (EC 1.4.1.2-4) vary throughout the year and coincide with seasonal alterations in nitrate, fructose, and sucrose. During the winter, NR, glutamine synthetase and ferrodoxin glutamate synthase activities increase in the root, while GDH displays the opposite trend with elevated activity in the summer months. All of these enzymes exhibit seasonal alterations in abundance as detected by Western blot analysis, increasing during the winter and, therefore, contributing to the seasonally dynamic protein pool. Extensive fluctuations in abundance and activity of these enzymes in the root occur during the spring and fall and coincide with shoot growth and senescence, respectively. Several observations indicate that posttranslational modifications of NR and GDH are taking place throughout the year; for example, NR is particularly unstable during the spring and fall, and seasonal GDH activity does not correlate with protein abundance.     

Enzymes of glutamine metabolism in testa-pericarp and endosperm of developing wheat grain

Glutamate dehydrogenase, glutamine synthetase, glutamate synthase, glutamate puruvate transaminase and glutamate oxaloacetate transaminase have been assayed in developing testa-pericarp and endosperm of two wheat varieties, namely Shera (11.6% protein) and C-306 (9.8% protein). On per organ basis, activities of all the enzymes studied, except glutamine synthetase, increased during development. Glutamine synthetase activity decreased during development in the testa-pericarp, whereas, no glutamine synthetase activity could be detected in endosperm of either variety at any stage of development. Compared to testa-pericarp, endosperm had higher activities of glutamate synthase and glutamate pyruvate transaminase. On the whole, enzyme activities in Shera were higher, as compared to C-306. Developmental patterns and relative levels of enzyme activities in the two varieties were more or less the same, when expressed on dry weight basis or as specific activities. The results suggest that ammonia assimilation in developing wheat grain takes place by the glutamate dehydrogenase pathway in the endosperm; and both by the glutamate dehydrogenase and glutamine synthetase—glutamate synthase pathways in the testa-pericarp.     

Methionine sulfoximine and phosphinothricin--glutamine synthetase inhibitors and activators and their herbicidal activity (A review)

Derivatives of methionine sulfoximine (MSO) and phosphinothrycin (PPT), which are analogues of glutamate, exhibit selective herbicidal activity. This effect is accounted for by impairments of nitrogen metabolism, resulting from inhibition of its key enzyme in plants, glutamine synthetase (EC 6.3.1.2). Inhibition of the enzyme causes ammoniac nitrogen to accumulate and terminates the synthesis of glutamine. Changes in the content of these two metabolites (excess ammonium and glutamine deficiency) act in a concert to cause plant death. However, low concentrations of MSO, PPT, and their metabolites produce an opposite effect: glutamine synthetase is activated, with concomitant stimulation of plant growth and productivity. The mechanisms whereby MSO and PPT affect glutamine synthetase activity are discussed in the context of nitrogen metabolism in plants.     

The intracellular location of the enzymes of nitrate assimilation in the apices of seedling pea roots

The intracellular distribution of the enzymes of nitrate and ammonia assimilation in apical cells of pea (Pisum sativum L.) roots is described. Nitrate reductase (EC 1.6.6.2) was found to have no organelle association, and is considered to be located in the cytosol or possibly loosely bound to the outside of an organelle. Nitrite reductase and glutamate synthase (EC 2.6.1.53) are plastid located, as is glutamine synthetase (EC 6.3.1.2) although this enzyme also has activity in the cytosol. Glutamate dehydrogenase (EC 1.4.1.3) was found only in the mitochondrion.     

Activity and distribution of nitrogen-metabolism enzymes in the developing maize kernel

The activities of glutamine synthetase (EC 6.3.1.2), glutamate dehydrogenase (EC 1.4.1.2), aspartate aminotransferase (EC 2.6.1.1), alanine aminotransferase (EC 2.6.1.2) and soluble protein content in the developing endosperm and embryo of normal (Oh-43) and mutant (Oh-430₂) maize were investigated. Maize inbred lines were grown under field conditions and all plants were self-pollinated. Ears for experiments were harvested over the period of 15 lo 45 days after pollination. After pollination kernel capacity for soluble protein synthesis is located mainly in the endosperm. This progressively decreases and about 40 days after pollination soluble protein synthesis is taken over by the embryo. Comparative data on the activity of the investigated enzymes in the embryo and endosperm indicate that the capacity for synthesis of glutamine and glutamate predominates in the embryo tissue, whereas transamination processes at the initial stages of the embryo development are less intensive than their counterparts in the endosperm. The roles of embryo and endosperm subsequently interchange. Biosynthetic processes of soluble precursors for protein synthesis in the embryo and endosperm of the developing kernel are mutually coordinated.     

Methionine Sulfoximine and Phosphinothrycin: A Review of Their Herbicidal Activity and Effects on Glutamine Synthetase

Derivatives of methionine sulfoximine (MSO) and phosphinothrycin (PPT), which are analogues of glutamate, exhibit selective herbicidal activity. This effect is accounted for by impairment of nitrogen metabolism, resulting from inhibition of its key enzyme in plants, glutamine synthetase (EC 6.3.1.2). Inhibition of the enzyme causes ammoniac nitrogen to accumulate and terminates the synthesis of glutamine. Changes in the content of these two metabolites (excess ammonium and glutamine deficiency) act in concert to cause plant death. However, low concentrations of MSO, PPT, and their metabolites produce an opposite effect: glutamine synthetase is activated, with concomitant stimulation of plant growth and productivity. The mechanisms whereby MSO and PPT affect glutamine synthetase activity are discussed in the context of nitrogen metabolism in plants.