Fixation of [13N]N2 and transfer of fixed nitrogen in the Anthoceros-Nostoc symbiotic association

The initial product of fixation of [¹³N]N₂ by pure cultures of the reconstituted symbiotic association between Anthoceros punctatus L. and Nostoc sp. strain ac 7801 was ammonium; it accounted for 75% of the total radioactivity recovered in methanolic extracts after 0.5 min and 14% after 10 min of incubation. Glutamine and glutamate were the primary organic products synthesized from [¹³N]N₂ after incubation times of 0.5–10 min. The kinetics of labeling of these two amino acids were characteristic of a precursor (glutamine) and product (glutamate) relationship. Results of inhibition experiments with methionine sulfoximine (MSX) and diazo-oxonorleucine were also consistent with the assimilation of N₂-derived NH ₄ ⁺ by Anthoceros-Nostoc through the sequential activities of glutamine synthetase (EC 6.3.1.2) and glutamate synthase (EC 1.4.7.1), with little or no assimilation by glutamate dehydrogenase (EC 1.3.1.3). Isolated symbiotic Nostoc assimilated exogenous ¹³NH ₄ ⁺ into glutamine and glutamate and their formation was inhibited by MSX, indicating operation of the glutamine synthetase-glutamate synthase (GS-GOGAT) pathway: However, relative to free-living cultures, isolated symbiotic Nostoc assimilated 80% less exogenous ammonium into glutamine and glutamate, implying that symbiotic Nostoc could assimilate only a fraction of N₂-derived NH ₄ ⁺ . This implication was tested by using Anthoceros associations reconstituted with wild-type or MSX-resistant strains of Nostoc incubated with [¹³N]N₂ in the presence of MSX. The results of these experiments indicated that, in situ, symbiotic Nostoc assimilated about 10% of the N₂-derived NH ₄ ⁺ and that NH ₄ ⁺ was made available to Anthoceros tissue where it was apparently assimilated by the GS-GOGAT pathway. Since less than 1% of the fixed N₂ was lost to the suspension medium, it appears that transfer of NH ₄ ⁺ from symbiont to host tissue was very efficient in this extracellular symbiotic association.Abbreviations DON 6-diazo-5-oxo-l-norleucine - GDH glutamate dehydrogenase - GOGAT glutamate synthase - GS glutamine synthetase - MSX l-methionine-dl-sulfoximine     

Assimilation of exogenous and dinitrogen-derived13NH 4 + byAnabaena azollae separated fromAzolla caroliniana Willd

Anabaena azollae was isolated fromAzolla caroliniana by the gentle roller method and differential centrifugation. Incubation of suchAnabaena preparations for 10 min with [¹³N]N₂ resulted in the formation of four radioactive compounds; ammonium, glutamine, glutamate and alanine. Ammonium accounted for 66% of the total radioactivity recovered and 58% of the ammonium was in an extracellular fraction. Since essentially no extracellular¹³N-labeled organic compounds were found, it appears that ammonium is the compound most probably made available toAzolla during dinitrogen-dependent growth of the association.The kinetics of incorporation of exogenous¹³NH ₄ ⁺ into glutamine and glutamate were characteristic of a precursor (glutamine)-product (glutamate) relationship and consistent with assimilation by the glutamine synthetase-glutamate synthase pathway. The results of experiments using the glutamine synthetase inhibitor, methionine sulfoximine, the glutamate synthase inhibitor, diazo-oxonorleucine, and increasing the ammonium concentration to greater than 1 mM, provided evidence for assimilation primarily by the glutamine synthetase-glutamate synthase pathway with little or no contribution from biosynthetic glutamate dehydrogenase.While showing that N₂ fixation and NH ₄ ⁺ assimilation were not tightly coupled metabolic processes in symbioticAnabaena, these results reflect a composite picture and do not indicate the extent to which ammonium assimilatory enzymes might be regulated in filaments associated with specific stages in theAzolla-Anabaena developmental profile.Non-standard abbreviations DON 6-Diazo-5-oxo-l-norleucine - GDH glutamate dehydrogenase - GOGAT glutamate synthase - GS glutamine synthetase - MSX l-methionine-Dl-sulfoximine     

Nitrogen Assimilation in Mycorrhizas : Ammonium Assimilation in the N-Starved Ectomycorrhizal Fungus Cenococcum graniforme

Ammonium assimilation was followed in N-starved mycelia from the ectomycorrhizal Ascomycete Cenococcum graniforme. The evaluation of free amino acid pool levels after the addition of 5 millimolar NH₄⁺ indicated that the absorbed ammonium was assimilated rapidly. Post-feeding nitrogen content of amino acids was very different from the initial values. After 8 hours of NH₄⁺ feeding, glutamine accounted for the largest percentage of free amino acid nitrogen (43%). The addition of 5 millimolar methionine sulfoximine (MSX) to NH₄⁺-fed mycelia caused an inhibition of glutamine accumulation with a corresponding increase in glutamate and alanine levels.

Using ¹⁵N as a tracer, it was found that the greatest initial labeling was into glutamine and glutamate followed by aspartate, alanine, and ornithine. On inhibiting glutamine synthetase using MSX, ¹⁵N enrichment of glutamate, alanine, aspartate, and ornithine continued although labeling of glutamine was quite low. Moreover, the incorporation of ¹⁵N label in insoluble nitrogenous compounds was lower in the presence of MSX. From the composition of free amino acid pools, the ¹⁵N labeling pattern and effects of MSX, NH₄⁺ assimilation in C. graniforme mycelia appears to proceed via glutamate dehydrogenase pathway. This study also demonstrates that glutamine synthesis is an important reaction of ammonia utilization.

     

Azolla-Anabaena Relationship : XIII. Fixation of [N]N(2)

The major radioactive products of the fixation of [¹³N]N₂ by Azolla caroliniana Willd.-Anabaena azollae Stras. were ammonium, glutamine, and glutamate, plus a small amount of alanine. Ammonium accounted for 70 and 32% of the total radioactivity recovered after fixation for 1 and 10 minutes, respectively. The presence of a substantial pool of [¹³N]N₂-derived ¹³NH₄⁺ after longer incubation periods was attributed to the spatial separation between the site of N₂-fixation (Anabaena) and a second, major site of assimilation (Azolla). Initially, glutamine was the most highly radioactive organic product formed from [¹³N]N₂, but after 10 minutes of fixation glutamate had 1.5 times more radiolabel than glutamine. These kinetics of radiolabeling, along with the effects of inhibitors of glutamine synthetase and glutamate synthase on assimilation of exogenous and [¹³N]N₂-derived ¹³NH₄⁺, indicate that ammonium assimilation occurred by the glutamate synthase cycle and that glutamate dehydrogenase played little or no role in the synthesis of glutamate by Azolla-Anabaena.     

The pathways of ammonium assimilation in Rhizobium meliloti

Two pathways of ammonium assimilation are known in bacteria, one mediated by glutamate dehydrogenase, the other by glutamine synthetase and glutamate synthase. The activities of these three enzymes were measured in crude extracts from four Rhizobium meliloti wild-type strains, 2011, M15S, 444 and 12. All the strains had active glutamine synthetase and NADP-linked glutamate synthase. Assimilatory glutamate dehydrogenase activity was present in strains 2011, M15S, 444, but not in strain 12. Three glutamate synthase deficient mutants were isolated from strain 2011. They were unable to use 1 mM ammonium as a sole nitrogen source. However, increased ammonium concentration allowed these mutants to assimilate ammonium via glutamate dehydrogenase. It was found that the sole mode of ammonium assimilation in strain 12 is the glutamine synthetase-glutamate synthase route; whereas the two pathways are functional in strain 2011.Abbreviations GS glutamine synthetase - GOGAT glutamate synthase - GDH glutamate dehydrogenase     

Initial organic products of assimilation of [N]ammonium and [N]nitrate by tobacco cells cultured on different sources of nitrogen

Glutamine is the first major organic product of assimilation of ¹³NH₄⁺ by tobacco (Nicotiana tabacum L. cv. Xanthi) cells cultured on nitrate, urea, or ammonium succinate as the sole source of nitrogen, and of ¹³NO₃⁻ by tobacco cells cultured on nitrate. The percentage of organic ¹³N in glutamate, and subsequently, alanine, increases with increasing periods of assimilation. ¹³NO₃⁻, 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 ¹³N in glutamine, and a concomitant increase in the percentage of ¹³N in glutamate and alanine. Methionine sulfoximine inhibits the incorporation of ¹³N from ¹³NH₄⁺ into glutamine more extensively than it inhibits the incorporation of ¹³N into glutamate, with cells grown on any of the three sources of nitrogen. Azaserine inhibits glutamate synthesis extensively when ¹³NH₄⁺ is fed to cells cultured on nitrate. These results indicate that the major route for assimilation of ¹³NH₄⁺ 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 ¹³N from ¹³NO₃⁻ into glutamate more strongly than it inhibits the incorporation of ¹³N into glutamine, suggesting that the assimilation of ¹³NH₄⁺ derived from ¹³NO₃⁻ may be mediated solely by the glutamine synthetase-glutamate synthase pathway.     

The effect of Cu2+ on uptake and assimilation of ammonium by cucumber seedlings

The ammonium uptake by cucumber seedlings was estimated from ammonium ions depletion in an uptake solution. The uptake of NH ₄ ⁺ was decreased by about 60 % after one hour and by about 90 % after two hours of 100 μM Cu²⁺ treatment. On the contrary the accumulation of ammonium in roots of Cu²⁺-treated seedlings at the same time was higher than in the control. Cu²⁺ in the concentration inhibiting NH ₄ ⁺ absorption during one hour inhibited also glutamine synthetase (GS) (EC 6.3.1.2) and NADH-glutamate dehydrogenase (NADH-GDH) (EC 1.4.1.2) activities both localized in the roots of seedlings. After one hour and at least up to the 4th hour Cu²⁺ accumulated mainly in roots (95 %). It was probably the reason of the GS activity in cotyledons of seedling treated with Cu²⁺ that it was at the same level as in the control. NADH-GDH activity in cotylcdons after one hour of the Cu²⁺ treatment was lower than in the control but the influence of Cu²⁺ action on the activity of this enzyme in roots was by far stronger. 100 μM Cu²⁺ did not affect the activities of both enzymes in in vitro experiments. Copper added into the incubation medium in 1000 μM concentration decreased GS activity, but still did not change NADH-GDH activity. These results suggested the indirect Cu²⁺ action on the investigated enzymes in in vivo experiments. However, no substantial effect on enzyme activities extracted from control plants was observed after the addition of the extract from Cu²⁺-treated plants into the incubation medium. The data suggest that the influence of Cu²⁺ on uptake and assimilation of ammonium may be connected not only with changes of plasma membrane properties in the root cells of Cu²⁺ treated seedlings but also with Cu²⁺ action on two major enzymes involved in NH ₄ ⁺ assimilation: glutamate synthetase and NADH-glutamate dehydrogenase.     

Photorespiratory ammonium release by the cyanobacterium Anabaena cylindrica in the presence of methionine sulfoximine

A release of ammonium by non-nitrogen-fixing Anabaena cylindrica (grown on NH₄Cl) in the presence of MSX (methionine sulfoximine) and absence of any external nitrogen source was found. In the light the release was maximal at 0.2 mM MSX, a concentration which did not affect net CO₂ fixation nor the glycollate excretion, but inhibited the glutamine synthetase activity and the reassimilation of ammonium. It is suggested that the major source of the ammonium released is the photorespiratory conversion of glycine to serine as (1) the release was stimulated by increase in light intensity, (2) high CO₂ (3%) lowered the release, if not given as a longer pretreatment (as CO₂ or HCO ₃ ^- ) when a stimulation was observed, (3) glyoxylate and glutamate stimulated the release, the latter compound particularly under nitrogen-deficient conditions and (4) isonicotinic acid hydrazide caused a reduced release of ammonium. Furthermore, a substantial part of the ammonium released by N₂-fixing A. cylindrica in presence of MSX may thus originate from the glycollate pathway. The data show that in the light the glycine to serine conversion is active in cyanobacteria with a concomitant production of ammonium which is assimilated by glutamine synthetase.Abbreviations MSX L-methionine-Dl-sulfoximine - INH isonicotinic acid hydrazide - RuDP ribulose 1,5-diphosphate - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - GS glutamine synthetase - GOGAT glutamate synthase - DTT Dl-dithiothreitol     

PATHWAY OF AMMONIUM ASSIMILATION IN A MARINE DIATOM DETERMINED WITH THE RADIOTRACER 13N1

In unicellular algae, ammonium can be assimilated into glutamate through the action of glutamate dehydrogenase (GDH) or into glutamine through the sequential activities of glutamine synthetase and glutamate 2-oxoglutarate amidotransferase (GS-GOGAT pathway). We have shown that the first radio-labeled product of assimilation of ¹³NH₄⁺ (t_1/2= 10 min) was glutamine in the marine diatom Thalassiosira pseudonana (Hustedt). When GS-GOGAT was inhibited with methionine sulfoximine, the incorporation of radioactivity into both glutamine and glutamate was blocked, implying that the radio-labeled glutamate is formed from glutamine. Glutamine was also the first labeled product when the intracellular concentration of ammonium was elevated by preincubation with unlabeled ammonium. The results indicate that the GS-GOGAT pathway is the primary pathway for the assimilation of nitrogen in T. pseudonana.     

Ammonium formation and assimilation in P(SARK)∷IPT tobacco transgenic plants under low N

Wild Type (WT) and transgenic tobacco plants expressing isopentenyltransferase (IPT), a gene encoding the enzyme regulating the rate-limiting step in cytokinins (CKs) synthesis, were grown under limited nitrogen (N) conditions. We analyzed nitrogen forms, nitrogen metabolism related-enzymes, amino acids and photorespiration related-enzymes in WT and P_SARK∷IPT tobacco plants. Our results indicate that the WT plants subjected to N deficiency displayed reduced nitrate (NO₃⁻) assimilation. However, an increase in the production of ammonium (NH₄⁺), by the degradation of proteins and photorespiration led to an increase in the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle in WT plants. In these plants, the amounts of amino acids decreased with N deficiency, although the relative amounts of glutamate and glutamine increased with N deficiency. Although the transgenic plants expressing P_SARK∷IPT and growing under suboptimal N conditions displayed a significant decline in the N forms in the leaf, they maintained the GS/GOGAT cycle at control levels. Our results suggest that, under N deficiency, CKs prevented the generation and assimilation of NH₄⁺ by increasing such processes as photorespiration, protein degradation, the GS/GOGAT cycle, and the formation of glutamine.     

Pathway of ammonia assimilation in illuminated and darkened Chlamydomonas reinhardii

Evidence is presented which shows that NH₃ assimilation in Chlamydomonas occurs exclusively via the glutamate synthase cycle in illuminated and darkened cells and those in which the internal level of NH₃ is elevated. This result indicates that glutamate dehydrogenase probably plays a catabolic rather than anabolic role in the N nutrition of the alga. Glutamine synthetase and glutamate dehydrogenase were characterized and their kinetic properties shown to be consistent with these proposals. It is suggested that reversible activity modulations of glutamine synthetase regulate the operation of the glutamate synthase cycle in the light but the availability of reductant and ATP limits its activity in darkened cells. The possible involvement of the two glutamate synthase enzymes in both light and dark assimilation is discussed.     

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.     

Ammonia Assimilation in Alnus glutinosa and Glycine max: SHORT-TERM STUDIES USING [N]AMMONIUM

The pattern of assimilation of NH₄⁺ by Alnus glutinosa, a N₂-fixing, nonleguminous angiosperm, was examined. Detached nodules, roots, and nodulated roots of intact plants were exposed to ¹³NH₄⁺ for up to 15 minutes. Glutamine was the most highly labeled compound at all times; the only other compound labeled significantly was glutamate. Similar results were obtained after incubating soybean (L. merr) nodules and roots with ¹³NH₄⁺. These observations and the results of pulse-labeling and inhibitor studies with nodules of Alnus were distinctly different from those predicted for the assimilation of NH₄⁺ via glutamine synthetase and glutamate synthase and suggest that glutamate dehydrogenase may play a major role in the assimilation of exogenously supplied NH₄⁺.     

Ammonium assimilation by Candida albicans and other yeasts: a 13N isotope study.

Nuclear magnetic resonance spectra of cultures of Candida albicans incubated in the presence of 15N-labelled ammonium demonstrated that glutamine and glutamate were the only initial products of ammonium assimilation. The nature of the route of assimilation in the yeasts Candida albicans, Saccharomyces cerevisiae, and Candida tropicalis was further examined by the use of the short-lived isotope 13N. [13N]ammonium was generated in the reaction 16O(p,alpha)13N, induced by proton bombardment of water in tandem accelerator. High-pressure liquid chromatography was used to separate and identify the products of assimilation, and radioactivity was detected and corrected for decay, using a computer-linked NaI scintillation detector. In the three yeasts studied, the labelled ammonium was assimilated into the acid-extractable fraction of cell suspensions within 1 min, and over 75% was converted to glutamine and glutamate. Subsequent to exhaustion of the labelled ammonium, the stoichiometry of the distribution of radiolabel was consistent with a net transfer of radiolabel from glutamine to glutamate, confirming the operation of glutamate synthase (EC 1.4.1.14) in these yeasts. Initial assimilation of label was mostly into glutamine (at a maximal rate within 10 s in C. albicans), whereas accumulation in glutamate did not occur at maximal rate until more than 70% of the labelled ammonium had been assimilated (between 30 and 60 s in C. albicans). We conclude that the glutamine synthetase-glutamate synthase pathway is the major route of ammonium assimilation in C. albicans and also in nitrogen-starved cultures of S. cerevisiae and C. tropicalis.     

Short-Term Metabolite Changes during Transient Ammonium Assimilation by the N-Limited Green Alga Selenastrum minutum

In this study, we measured the total pool sizes of key cellular metabolites from nitrogen-limited cells of Selenastrum minutum before and during ammonium assimilation in the light. This was carried out to identify the sites at which N assimilation is acting to regulate carbon metabolism. Over 120 seconds following NH₄⁺ addition we found that: (a) N accumulated in glutamine while glutamate and α-ketoglutarate levels fell; (b) ATP levels declined within 5 seconds and recovered within 30 seconds of NH₄⁺ addition; (c) ratios of pyruvate/phosphoenolpyruvate, malate/phosphoenolpyruvate, Glc-1-P/Glc-6-P and Fru-1,6-bisphosphate/Fru-6-P increased; and (d) as previously seen, photosynthetic carbon fixation was inhibited. Further, we monitored starch degradation during N assimilation over a longer time course and found that starch breakdown occurred at a rate of about 110 micromoles glucose per milligram chlorophyll per hour. The results are consistent with N assimilation occurring through glutamine synthetase/glutamate synthase at the expense of carbon previously stored as starch. They also indicate that regulation of several enzymes is involved in the shift in metabolism from photosynthetic carbon assimilation to carbohydrate oxidation during N assimilation. It seems likely that pyruvate kinase, phosphoenolpyruvate carboxylase, and starch degradation are all activated, whereas key Calvin cycle enzyme(s) are inactivated within seconds of NH₄⁺ addition to N-limited S. minutum cells. The rapid changes in glutamate and triose phosphate, recently shown to be regulators of cytosolic pyruvate kinase, are consistent with them contributing to the short-term activation of this enzyme.     

Regulation of nitrate assimilation in the cyanobacterium Phormidium laminosum

The intracellular ratio of 2-oxoglutarate to glutamine has been analyzed under nutritional conditions leading to different activity levels of nitrate-assimilating enzymes in Phormidium laminosum (Agardh) Gom. This non-N₂-fixing cyanobacterium adapted to the available nitrogen source by modifying its nitrate reductase (NR; EC 1.7.7.2), nitrite reductase (NiR; EC 1.7.7.1) and glutamine synthetase (GS; EC 6.3.1.2) activities. The 2-oxoglutarate/glutamine ratio was similar in cells adapted to grow with nitrate or ammonium. However, metabolic conditions that increased this ratio [i.e., nitrogen starvation or l-methionine-d,l-sulfoximine (MSX) treatment] corresponded to high activity levels of NR, NiR, GS (except in MSX-treated cells) and glutamate synthase (GOGAT; EC 1.4.7.1). By contrast, metabolic conditions that diminished this ratio (i.e., addition of ammonium to nitrate-growing cells or addition of nitrate or ammonium to nitrogen-starved cells) resulted in low activity levels. The variation in the 2-oxoglutarate/glutamine ratio preceded the changes in enzyme activities. These results suggest that changes in the 2-oxoglutarate/glutamine ratio could be the signal that triggers the adaptation of P. laminosum cells to variations in the available nitrogen source, as occurs in enterobacteria.Abbreviations Chl chlorophyll - GOGAT ferredoxin-dependent glutamate synthase (EC 1.4.7.1) - GS glutamine synthetase (EC 6.3.1.2) - MSX l-methionine-d,l-sulfoximine - NiR nitrite reductase (EC 1.7.7.1) - NR nitrate reductase (EC 1.7.7.2) - TP total protein This work has been partially supported by grants from the Spanish Ministry of Education and Science (DGICYT PB88-0300 and PB92-0464) and the University of the Basque Country (042.310-EC203/94). M.I.T. was the recipient of a fellowship from the Basque Government.     

Ammonia-related metabolism of tobacco varieties differing in their susceptibility to Alternaria alternata

Some studies report that ammonia is an important factor of disease development in tobacco plants and various post-harvest fruits. Four tobacco (Nicotiana tabacum L.) varieties resistant or susceptible to Alternaria alternata (Fries) Keissler, a tobacco pathogenic fungus, were used to investigate whether there are differences in ammonia accumulation and the related metabolism of senescing leaves. The results showed that: (a) the leaves of susceptible varieties had significantly higher apoplastic [NH ₄ ⁺ ], pH, and ammonia emission potential (??-values) than resistant varieties during the period from 40 to 60 days of leaf age; (b) leaf tissue [NH ₄ ⁺ ] and total N concentrations in the tobacco varieties were not in line with their susceptibility or resistance to disease; (c) the increases in the apoplastic pH, ??-values, and leaf [NH ₄ ⁺ ] occurred in parallel with a significant decline in glutamine synthetase activity. Compared with the resistant varieties, apoplastic pH values and ?? values were increased more rapidly in the susceptible varieties due to a steeper decline in glutamine synthetase activity and a slower increase in glutamate dehydrogenase activity. In conclusion, NH₃ accumulation or NH3-dependent alkalinization rather than [NH ₄ ⁺ ] and total N appears to be mainly attributed to the enhanced susceptibility of tobacco plants to A. alternata.     

The Role of Ammonium Assimilating Enzymes in Lentil Roots and Nodules

Activities of ammonium assimilating enzymes glutamate dehydrogenase (GDH), glutamine synthetase (GS), glutamate synthase (GOGAT), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) as well as the amino acid content were higher in nodules compared to roots. Their activities increased at 40 and 60 d after sowing, with a peak at 90 d, a time of maximum nitrogenase activity. The GS/GOGAT ratio had a positive correlation with the amino acid content in nodules. Higher activities of AST than ALT may be due to lower glutamine and higher asparagine content in xylem. The data indicated that glutamine synthetase and glutamate synthase function as the main route for the assimilation of fixed N, while NADH-dependent glutamate dehydrogenase may function at higher NH₄ ⁺ concentration in young and senescing nodules. Enzyme activities in lentil roots reflected a capacity to assimilate N for making the amino acids they may need for both growth and export to upper parts of the plant. This revised version was published online in July 2006 with corrections to the Cover Date.     

Glutamine synthetase and glutamate synthase from Sclerotinia sclerotiorum

Glutamine synthetase (GS, EC 6.3.1.2) and glutamate synthase (GOGAT, EC 1.4.1.13) were purified from Sclerotinia sclerotiorum and some of their properties studied. The GS transferase and biosynthetic activities, as well as GOGAT activity, were sensitive to feedback inhibition by amino acids and other metabolites. GS showed a marked dependence on ADP in the transferase reaction and on ATP in the Mg²⁺-dependent biosynthetic reaction. Regulation of GS activity by adenylylation/deadenylylation was demonstrated by snake venom phosphodiesterase treatment of the purified enzyme. GOGAT required NADPH as an electron donor; NADH was inactive. GOGAT was strongly inhibited by p-chloromercuribenzoate and the inhibition was reversed by cysteine. The enzyme was also markedly inhibited by o-phenanthroline, 2,2′-bipyridyl and azaserine. l-Methionine-dl-sulphoximine (MSX) and azaserine inhibited the incorporation of ¹⁵N-labelled ammonium sulphate into washed cells of S. sclerotiorum. MSX and azaserine respectively also inhibited purified GS and GOGAT activities. GDH activity was not detected in cell-extracts. Thus the GS/GOGAT pathway is the main route for the assimilation of ammonium compounds in this fungus.     

N-ammonia assimilation, 2-oxoglutarate transport, and glutamate export in spinach chloroplasts in the presence of dicarboxylates in the light

The direct incorporation of ¹⁵NH₄Cl into amino acids in illuminated spinach (Spinacia oleracea L.) chloroplasts in the presence of 2-oxoglutarate plus malate was determined. The amido-N of glutamine was the most highly labeled N-atom during ¹⁵NH₄ assimilation in the presence of malate. In 4 minutes the ¹⁵N-label of the amido-N of glutamine was 37% enriched. In contrast, values obtained for both the N-atom of glutamate and the amino-N of glutamine were only about 20% while that of the N-atom of aspartate was only 3%. The addition of malate during the assimilation of ¹⁵NH₄Cl and Na¹⁵NO₂ greatly increased the ¹⁵N-label into glutamine but did not qualitatively change the order of the incorporation of ¹⁵N-label into all the amino acids examined. This evidence indicates the direct involvement of the glutamine synthetase/glutamate synthase pathway for ammonia and nitrite assimilation in isolated chloroplasts. The addition of malate or succinate during ammonia assimilation also led to more than 3-fold increase in [¹⁴C]2-oxoglutarate transport into the chloroplast as well as an increase in the export of [¹⁴C]glutamate out of the chloroplast. Little [¹⁴C]glutamine was detected in the medium of the chloroplast preparations. The stimulation of ¹⁵N-incorporation and [¹⁴C]glutamate export by malate could be directly attributed to the increase in 2-oxoglutarate transport activity (via the 2-oxoglutarate translocator) observed in the presence of exogenous malate.