15N INCORPORATION AND GLUTAMINE SYNTHETASE INHIBITION STUDIES OF NITROGEN ASSIMILATION IN LEAVES OF THE NITROPHILE, DATURA STRAMONIUM L.
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SUMMARY The N assimilation pathways in leaves of Datura stramonium L. have been investigated at low and high levels of xylem stream nitrate feeding by an 15 N infiltration technique in the presence and absence of the glutamine synthetase inhibitor, methionine sulphoximine (MSO). In the absence of MSO, glutamate showed the highest 15 N enrichment of any free amino compound at both feeding levels. At the high N feeding level, glutamine showed the highest accumulation of 15 N at all stages of the time course, with secondary accumulation in glutamate. At the low N feeding level the reverse was true. These findings indicate the operation of both the glutamine synthetase‐GOGAT and glutamate dehydrogenase pathways in Datura leaves. In the presence of MSO intracellular pools of all the amino compounds, with the exception of glutamine and threonine, became rapidly drained. There was an almost complete restriction of the supply of newly reduced 15 N to amino acid assimilation and it accumulated as ammonia. The large glutamine pool remaining contained no 15 N enrichment, and was probably an extrachloroplastic storage pool not immediately available for amino acid synthesis. These experimental results indicate the almost exclusive role of the glutamine synthetase‐GOGAT pathway in the assimilation of newly reduced nitrate into amino acid metabolism in Datura leaves.Keywords:
Glutamate synthase
Datura stramonium
Nitrogen Assimilation
Azaserine
Glufosinate
Nitrobacter agilis, a chemolithotrophic bacterium, utilizes ammonia as well as nitrite as a nitrogen source for growth. The growth yields were increased about twofold by growing the bacterium in a nitrite medium supplemented with 2 mm-ammonium chloride. Higher concentrations of ammonium chloride, however, competitively inhibited nitrite oxidation and growth of the bacterium. Washed cells readily incorporated 15NH4 +, 15NH2OH, 15NO2 − and 15NO3 − respectively (in decreasing order) into cell nitrogen. Enzyme activities in cell extracts of ammonia-supplemented cultures, compared to those without ammonia, were greater for glutamate dehydrogenase (EC 1.4.1.2) and similar for glutamine synthetase (EC 6.3.1.2), whereas glutamate synthase (EC 1.4.1.14) was barely detectable. Inhibitors of glutamine synthetase (l-methionine dl-sulphoximine) and glutamate synthase (azaserine) did not affect the incorporation of 15NH4 − and 15NO2 − into cell nitrogen of washed cells. The results indicate that glutamate dehydrogenase is a key enzyme in N. agilis for the assimilation of either nitrite or ammonia. Glutamine synthetase, which was also active in cell extracts, is probably required for the production of glutamine. Glutamate synthase, however, does not appear to be an important enzyme for ammonia assimilation.
Glutamate synthase
Nitrogen Assimilation
Azaserine
Ammonium chloride
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SUMMARY The N assimilation pathways in leaves of Datura stramonium L. have been investigated at low and high levels of xylem stream nitrate feeding by an 15 N infiltration technique in the presence and absence of the glutamine synthetase inhibitor, methionine sulphoximine (MSO). In the absence of MSO, glutamate showed the highest 15 N enrichment of any free amino compound at both feeding levels. At the high N feeding level, glutamine showed the highest accumulation of 15 N at all stages of the time course, with secondary accumulation in glutamate. At the low N feeding level the reverse was true. These findings indicate the operation of both the glutamine synthetase‐GOGAT and glutamate dehydrogenase pathways in Datura leaves. In the presence of MSO intracellular pools of all the amino compounds, with the exception of glutamine and threonine, became rapidly drained. There was an almost complete restriction of the supply of newly reduced 15 N to amino acid assimilation and it accumulated as ammonia. The large glutamine pool remaining contained no 15 N enrichment, and was probably an extrachloroplastic storage pool not immediately available for amino acid synthesis. These experimental results indicate the almost exclusive role of the glutamine synthetase‐GOGAT pathway in the assimilation of newly reduced nitrate into amino acid metabolism in Datura leaves.
Glutamate synthase
Datura stramonium
Nitrogen Assimilation
Azaserine
Glufosinate
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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.
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Aspergillus nidulans
Azaserine
Nitrogen Assimilation
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Glutamine is the first major organic product of assimilation of (13)NH(4) (+) by tobacco (Nicotiana tabacum L. cv. Xanthi) cells cultured on nitrate, urea, or ammonium succinate as the sole source of nitrogen, and of (13)NO(3) (-) by tobacco cells cultured on nitrate. The percentage of organic (13)N in glutamate, and subsequently, alanine, increases with increasing periods of assimilation. (13)NO(3) (-), used for the first time in a study of assimilation of nitrogen, was purified by new preparative techniques. During pulse-chase experiments, there is a decrease in the percentage of (13)N in glutamine, and a concomitant increase in the percentage of (13)N in glutamate and alanine. Methionine sulfoximine inhibits the incorporation of (13)N from (13)NH(4) (+) into glutamine more extensively than it inhibits the incorporation of (13)N into glutamate, with cells grown on any of the three sources of nitrogen. Azaserine inhibits glutamate synthesis extensively when (13)NH(4) (+) is fed to cells cultured on nitrate. These results indicate that the major route for assimilation of (13)NH(4) (+) is the glutamine synthetase-glutamate synthase pathway, and that glutamate dehydrogenase also plays a role, but a minor one. Methionine sulfoximine inhibits the incorporation of (13)N from (13)NO(3) (-) into glutamate more strongly than it inhibits the incorporation of (13)N into glutamine, suggesting that the assimilation of (13)NH(4) (+) derived from (13)NO(3) (-) may be mediated solely by the glutamine synthetase-glutamate synthase pathway.
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Azaserine
Nitrogen Assimilation
Alanine
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Asparagine synthetase
Nitrogen Assimilation
Glutamate synthase
Assimilation (phonology)
Azaserine
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Azaserine
Nitrogen Assimilation
Glutamate synthase
Glufosinate
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Assimilation of nitrate and ammonium are vital procedures for plant development and growth. From these primary paths of inorganic nitrogen assimilation, this metabolism integrates diverse paths for biosynthesis of macromolecules, such as amino acids and nucleotides, and the central intermediate metabolism, like carbon metabolism and photorespiration. This paper reports research performed in the CitEST (Citrus Expressed Sequence Tag) database for the main genes involved in nitrogen metabolism and those previously described in other organisms. The results show that a complete cluster of genes involved in the assimilation of nitrogen and the metabolisms of glutamine, glutamate, aspartate and asparagine can be found in the CitEST data. The main enzymes found were nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), glutamate synthetase (GOGAT), glutamate dehydrogenase (GDH), aspartate aminotransferase (AspAT) and asparagine synthetase (AS). The different enzymes involved in this metabolism have been shown to be highly conserved among the Citrus and Poncirus species. This work serves as a guide for future functional analysis of these enzymes in citrus.
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Nitrogen Assimilation
Asparagine synthetase
Photorespiration
Nitrogen Cycle
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SUMMARY Ammonium‐nitrogen was assimilated rapidly by nitrogen‐replete cultures of the nitrate‐utilizing yeast, Candida nitratophila as long as a suitable source of carbon was available. These cultures contained high activities of an NADPH‐dependent glutamate dehydrogenase with a relatively high affinity for ammonium ( K m = 0.27 mM) and high glutamine synthetase activity. Both enzyme activities were apparently derepressed when glutamine‐grown cultures were starved of nitrogen or transferred to nitrate medium. Nitrogen‐deficient cultures also contained NADH‐dependent glutamate synthase activity that was inhibited by azaserine in vitro. Ammonium assimilation in vivo , was inhibited by methionine sulphoximine whilst addition of azaserine resulted in an accumulation of intracellular glutamine and an inhibition of glutamate production. Our results suggest that, in C. nitratophila , there is a potential for ammonium assimilation via both the glutamate dehydrogenase pathway and the glutamine synthetase/glutamate synthase pathway with the latter pathway predominating in nitrogen‐deficient cells.
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Nitrogen Assimilation
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Although the nitrate assimilation into amino acids in photosynthetic leaf tissues is active under the light, the studies during 1950s and 1970s in the dark nitrate assimilation provided fragmental and variable activities, and the mechanism of reductant supply to nitrate assimilation in darkness remained unclear. 15N tracing experiments unraveled the assimilatory mechanism of nitrogen from nitrate into amino acids in the light and in darkness by the reactions of nitrate and nitrite reductases, glutamine synthetase, glutamate synthase, aspartate aminotransferase, and asparagine synthetase. Nitrogen assimilation in illuminated leaves and non-photosynthetic roots occurs either in the redundant way or in the specific manner regarding the isoforms of nitrogen assimilatory enzymes in their cellular compartments. The electron supplying systems necessary to the enzymatic reactions share in part a similar electron donor system at the expense of carbohydrates in both leaves and roots, but also distinct reducing systems regarding the reactions of Fd-nitrite reductase and Fd-glutamate synthase in the photosynthetic and non-photosynthetic organs.
Glutamate synthase
Nitrogen Assimilation
Asparagine synthetase
Assimilation (phonology)
Nitrogen Cycle
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Azaserine
Glutamate synthase
Nitrogen Assimilation
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