Evidence for an involvement of glutamine synthetase in regulation of nitrogenase activity in Rhodopseudomonas capsulata

In the presnet studies with whole cells and extracts of the photosynthetic bacterium Rhodopseudomonas capsulata the rapid inhibition of nitrogenase dependent activities (i.e. N₂-fixation acetylene reduction, or photoproduction of H₂) by ammonia was investigated. The results suggest, that the regulation of the nitrogenase activity by NH ₄ ⁺ in R. capsulata is mediated by glutamine synthetase (GS). (i) The glutamate analogue methionine sulfoximine (MSX) inhibited GS in situ and in vitro, and simultaneously prevented nitrogenase activity in vivo. (ii) When added to growing cultures ammonia caused rapid adenylylation of GS whereas MSX abolished the activity of both the adenylylated and unadenylylated form of the enzyme. (iii) Recommencement of H₂ production due to an exhaustion of ammonia coincided with the deadenylylation of GS. (iv) In extracts, the nitrogenase was found to be inactive only when NH ₄ ⁺ or MSX were added to intact cells. Subsequently the cells had to be treated with cetyltrimethylammonium bromide (CTAB). (v) In extracts the nitrogenase activity declined linearily with an increase of the ration of adenylylated vs. deadenylylated GS. A mechanism for inhibition of nitrogenase activity by ammonia and MSX is discussed.Abbreviations BSA bovin serum albumine - CTAB cetyltrimethylammonium bromide - GOGAT l-glutamine: 2-oxoglutarate amino transferase - GS glutamine synthetase - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - MSX l-methionine-d,l-sulfoximine     

Photoproduction of ammonium by immobilized mutant strains of Anabaena variabilis

Summary Ethylenediamine (EDA) is toxic to the cyanobacterium Anabaena variabilis and inhibits nitrogenase activity. The inhibition of nitrogenase was prevented by pretreatment of cells with l-methionine-d,l-sulphoximine (MSX). Mutant strains of Anabaena variabilis (ED81, ED92), resistant to EDA, had low levels of glutamine synthetase (GS) biosynthetic activity compared with the wild type strain. ED92 had a low level of GS protein whereas ED81 had a similar level to that of the parent strain as estimated using antibodies against GS. Both strains fixed N₂ and liberated NH₄ ⁺ into the media. Following immobilization of the mutant strains, sustained photoproduction of NH₄ ⁺ was obtained in air-lift reactors at rates of up to 50 mol NH₄ ⁺ mg chl a^–1 h^–1, which were comparable to the rates obtained when immobilized cyanobacteria were treated with MSX.Abbreviations EDA 1,2-diaminoethane (ethylenediamine) - GS glutamine synthetase - MSX l-methionine-d,l-sulphoximine     

Biochemical characterization of glutamine synthetase from the diazotrophic cyanobacterium,Anabaena doliolum

In cyanobacteria, the glutamine synthetase-L-glutamine-2-oxoglutarate aminotransferase (GS-GOGAT) pathway is the major ammonia-assimilating route. The GS ofAnabaena doliolum was synthesized more under N₂-fixing conditions, followed by ammonium, nitrate, and nitrite as nitrogen sources. The activities of both the glutamine synthetase, Mg²⁺-dependent biosynthetic and Mn²⁺-dependent -glutamyl transferase were optimum at pH 7. The active site of the enzyme bears sulfhydryl (-SH) groups; this was confirmed with the-SH group inhibitors, para-chloromercuribenzoate (pCMB) and N-ethylmaleimide (NEM). The biosynthetic and -glutamyl transferase activities showed specificity for the divalent cations, Mg²⁺ and Mn²⁺, respectively. The other divalent cations Co²⁺, Cu²⁺, and Ni²⁺ were poor substitutes. This enzyme also required these divalent cations to stabilize its structure and function under extreme conditions such as high and low temperatures and urea denaturation. The glutamate analogl-methionine-d,l-sulfoximine, inactivated the enzyme, whereas the GOGAT inhibitor, azaserine, had no effect on the enzyme activity. The GS enzyme required de novo protein synthesis.     

Ethylenediamine uptake and metabolism in the cyanobacterium Anabaena variabilis

The cyanobacterium Anabaena variabilis showed a pH dependent uptake of ethylenediamine. No uptake of ethylenediamine was detected at pH 7.0. At higher pH values (e.g. pH 8.0 and pH 9.0) accumulation did occur and was attributed to diffusion of uncharged ethylenediamine in response to a pH gradient. A biphasic pattern of uptake was observed at these higher pH values. Treatment with l-methionine-d,l-sulphoximine (MSX) to inactivate glutamine synthetase (GS) inhibited the second slower phase of uptake without any significant alteration of the initial uptake. Therefore for sustained uptake, metabolism of ethylenediamine via GS was required. NH ₄ ⁺ did not alter the uptake of ethylenediamine. Ethylenediamine was converted in the second phase of uptake to an analogue of glutamine which could not be detected in uptake experiments at pH 7.0 or in uptake experiments at pH 9.0 following pretreatment of cells with MSX. Ethylenediamine treatment inhibited nitrogenase activity and this inhibition was greatest at high pH values.Abbreviations EDA 1,2-diaminoethane (ethylenediamine) - GS glutamine synthetase - HEPES 4-(2-hydroxyethyl)-1 piperazine ethanesulphonic acid - MSX l-methionine-dl-sulphoximine - membrane potential - Tricine N-tris(hydroxymethyl) methylglycine     

Immunoelectrophoretic approach to the metabolic regulation of glutamine synthetase in Rhodopseudomonas capsulata E1F1: role of glutamine

Anti-glutamine synthetase serum was raised in rabbits by injecting purified glutamine synthetase (GS) of the phototrophic bacterium Rhodopseudomonas capsulata E1F1. The antibodies were purified to monospecificity by immunoaffinity chromatography in GS-sepharose gel. These anti-GS antibodies were used to measure the antigen levels in crude extracts from bacteria, grown phototrophically with dinitrogen, nitrate, nitrite, ammonia, glutamate, glutamine or alanine as nitrogen sources. The amount of GS detected by rocket immunoelectrophoresis was proportional to Mn²⁺-dependent transferase activity measured in the crude extracts. Addition of GS inhibitor l-methionine-d,l-sulfoximine (MSX) to the actively growing cells promoted increased antigen levels, that were not found in the presence of glutamine or chloramphenicol. The ammonia-induced decrease in GS relative levels was reverted by MSX. GS levels remained constant when phototrophically growing cells were kept in the dark.Abbreviations GS glutamine synthetase - MOPS 2-(N-morpholine) propane sulfonate - MSX l-methionine-d,l-sulfoximine     

Nitrogen assimilation in Rhodopseudomonas acidophila

Rhodopseudomonas acidophila strain 7050 assimilated ammonia via a constitutive glutamine synthetase/glutamate synthase enzyme system.Glutamine synthetase had a K _m for NH ₄ ⁺ of 0.38 mM whilst the nicotinamide adenine dinucleotide linked glutamate synthase had a K _m for glutamine of 0.55 mM. R. acidophila utilized only a limited range of amino acids as sole nitrogen sources: l-alanine, glutamine and asparagine. The bacterium did not grow on glutamate as sole nitrogen source and lacked glutamate dehydrogenase. When R. acidophila was grown on l-alanine as the sole nitrogen source in the absence of N₂ low levels of a nicotinamide adenine dinucleotide linked l-alanine dehydrogenase were produced. It is concluded, therefore, that this reaction was not a significant route of ammonia assimilation in this bacterium except when glutamine synthetase was inhibited by methionine sulphoximine. In l-alanine grown cells the presence of an active alanine-glyoxylate aminotransferase and, on occasions, low levels of an alanine-oxaloacetate aminotransferase were detected. Alanine-2-oxo-glutarate aminotransferase could not be demonstrated in this bacterium.Abreviations ADH alanine dehydrogenase - GDH glutamate dehydrogenase - GS glutamine synthetase - GOGAT glutamate synthase - MSO methionine sulphoximine     

The effects of reduced nitrogenous compounds suggests that glutamine synthetase activity is involved in the development of somatic embryos in carrot

The addition of l-glutamine, -alanine or l-glutamic acid strongly stimulates somatic embryo formation in carrot, not only in the number of somatic embryos formed but also with respect to their development. The effects of the amino acids on somatic embryogenesis were stronger than that of ammonium ion. In particular, l-glutamine strongly stimulated the development of somatic embryos. To clarify the different effects of amino acids and ammonium ion, the activity of glutamine synthetase (GS; EC 6.3.1.2), a key enzyme involved in nitrogen assimilation, was measured. Its activity decreased during the later stages of embryo development.Abbreviations -Ala -alanine - Glu l-glutamic acid - Gln l-glutamine - 2,4-D 2, 4-dichlorophenoxyacetic acid - -GHA l-glutamic acid -monohydroxamate - GS glutamine synthetase - MS medium Murashige & Skoog (1962) medium - MS-NH₄ medium MS medium without NH₄NO₃ - MS+NH₄ medium MS-NH₄ medium with 10 mM NH₄Cl - MS+ala medium MS-NH₄ medium with 10 mM -alanine - MS+GLU medium MS-NH₄ medium with 10 mM l-glutamic acid - MS+GLN medium MS-NH₄ medium with 10 mM l-glutamine - NIR nitrite reductase - NR nitrate reductase     

Isolation and characterisation of nitrate reductase mutants and regulation of nitrate reductase and nitrogenase in the cyanobacterium Nostoc muscorum

Summary Chlorate resistant mutants of the cyanobacterium Nostoc muscorum isolated after N-methyl-N-nitro-N-nitrosoguanidine (MNNG) mutagenesis were found to be defective/blocked in nitrate reductase (NR).The parent strain possessed active NR in the presence of nitrogen as nitrate and only basal levels of activity in ammonia and N-free grown cultures. Addition of ammonia suppressed the NR activity in the parent strain whereas addition of L-methionine DL-sulphoximine (MSX) restored NR activity. A similar repression by ammonia, glutamine and derepression with MSX were also observed for nitrogenase synthesis.One class of mutants lacked NR activity (nar ^-) whereas the specific activity of NR was low in another class of mutants (nar ^def). Unlike the parent, the mutants synthesized nitrogenase and differentiated heterocysts in the presence of nitrate nitrogen. Uptake studies of nitrite and ammonia in mutants revealed that they possessed both nitrite reductase and glutamine synthetases (GS) at low levels, and the same level respectively in comparison with the parent.     

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     

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.     

Role of glutamine synthetase in the initiation of sporulation in Bacillus polymyxa

The activity of glutamine synthetase (GS) was investigated during culture development of Bacillus polymyxa CN 2219 and its asporogenous mutant deficient in protease production. At 28°C, temperature permissive for sporulation, the glutamine synthetase activity was found to decline in the wild type cells which acquire the competence for sporulation. This decline was not observed in the asporogenous mutant. Incubation at 37°C (temperature non permissive) suppressed sporulation in the wild type and maintained glutamine synthetase activity. The involvement of glutamine synthetase in the repression of sporulation was further confirmied by the action of l-methionine sulfoximine a specific inhibitor of glutamine synthetase, which overcomes the catabolite repression by ammonium and induces sporulation. Intracellular proteases were measured as early markers of the initiation of sporulation and were found to be induced during sporulation.Abbreviations GS glutamine synthetase - MSO l-methionine sulfoximine - GYS glucose-yeast extract-salts - GT -glutamyltransferase - PMSF phenylmethylsulfonylfluoride     

Glutamine synthetase/glutamate synthase ammonium-assimilating pathway in Schizosaccharomyces pombe

Kinetic parameters of glutamine synthetase (GS) and glutamate synthase (glutamineoxoglutarate aminotransferase) (GOGAT) activities, including initial velocity, pH, and temperature optima, as well as K _m values, were estimated in Schizosaccharomyces pombe crude cell-free extracts. Five glutamine auxotrophic mutants of S. pombe were isolated following MNNG treatment. These were designated gln1-1,2,3,4,5, and their growth could be repaired only by glutamine. Mutants gln1-1,2,3,4,5 were found to lack GS activity, but retained wild-type levels of NADP-glutamate dehydrogenase (GDH), NAD-GDH, and GOGAT. One further glutamine auxotrophic mutant, gln1-6, was isolated and found to lack both GS and GOGAT but retained wild-type levels of NADP-GDH and NAD-GDH activities. Fortuitously, this isolate was found to harbor an unlinked second mutation (designated gog1-1), which resulted in complete loss of GOGAT activity but retained wild-type GS activity. The growth phenotype of mutant gog1-1 (in the absence of the gln1-6 mutation) was found to be indistinguishable from the wild type on various nitrogen sources, including ammonium as a sole nitrogen source. Double-mutant strains containing gog1-1 and gdh1-1 or gdh2-1 (mutations that result specifically in the abolition of NADP-GDH activity) result in a complete lack of growth on ammonium as sole nitrogen source in contrast to gdh or gog mutants alone.     

The uptake and metabolism of methylamine by N2-fixing cyanobacteria

The cyanobacteria Anabaena variabilis and Nostoc CAN showed a biphasic pattern of ¹⁴CH₃NH ₃ ⁺ uptake at external pH values of 7.0 and 9.0. The initial phase of uptake, which was independent of metabolism of ¹⁴CH₃NH ₃ ⁺ , was attributed to uptake via a CH₃NH ₃ ⁺ (NH ₄ ⁺ ) transport system at pH 7.0 and probably to passive diffusion of uncharged CH₃NH₂ and trapping by protonation at pH 9.0. The second slower phase of uptake was attributed to metabolism of CH₃NH ₃ ⁺ via glutamine synthetase to form -methylglutamine which accumulates. Anabaena cylindrica showed an initial rapid uptake at pH 7.0 and pH 9.0 but metabolism of ¹⁴CH₃NH ₃ ⁺ was undetectable at pH 7.0 and was barely detectable at pH 9.0. Pretreatment of A. variabilis with l-methionine-d,l-sulphoximine to inactivate glutamine synthetase, inhibited the second phase of ¹⁴CH₃NH ₃ ⁺ uptake at both pH 7.0 and pH 9.0 and the accumulation of -methylglutamine but had no effect on the first phase of uptake. Following transfer of A. variabilis to darkness the initial phase of ¹⁴CH₃NH ₃ ⁺ uptake at pH 7.0 and 9.0 was unaffected but the subsequent metabolism via glutamine synthetase was inhibited.Abbreviations MSX l-methionine-d,l-sulphoximine - GS glutamine synthetase     

The role fo glutamine synthetase and glutamine metabolism in nitrogen metabolite repression, a regulatory phenomenon in the lower eukaryote Neurospora crassa

Summary Growth of Neurospora crassa on media containing NH ₄ ⁺ leads to the repression of a variety of permeases and alternative pathways which would generate NH ₄ ⁺ , so called ammonium repression. The mutant am ₂ which lacks NADP-GDH is not subject to ammonium repression of nitrate reductase or urea permease, but like the wild type has repressed levels of these systems when grown in the presence of proline, glutamate or glutamine. The glutamine synthetase (GS) mutant gln-la has derepressed levels of the aforementioned systems unless grown with glutamine.The oligomeric state of GS depends upon the nitrogen sufficiency of the cell, a tetrameric form predominates under conditions of nitrogen limitation and an octameric form under conditions of nitrogen sufficiency. We have found that the tetrameric form GS predominates in the mutants am ₂ and gln-la when they are ammonium derepressed.The mechanism of NH ₄ ⁺ repression in N. crassa is thought to entail a cessation of positive gene action by the product of the nit-2 regulatory gene. We propose that under conditions of NH ₄ ⁺ sufficiency, and hence glutamine sufficiency, the octameric form of GS represses nit-2 gene expression and thereby achieves ammonium repression.     

In vivo inhibition of glutamine synthetase induces sporulation in a protease deficient asporogenous mutant of Bacillus polymyxa

In vivo inhibition of glutamine synthetase (GS) by l-methionine sulfoximine induces sporulation in a protease deficient mutant of Bacillus polymyxa. This induction of sporulation is accompanied by derepression of EDTA insensitive proteases(s) which seems to be specific for differentiation. Some amino acid analogues derepress proteolytic activity without inducing sporulation, but these proteases are sensitive to metal chelators like those in the vegetative cells. When the proteolytic activity is restored, the mutant cells, which are smaller than the parental strain, regain their normal size.Abbreviations GS glutamine synthetase - GYS glucose-yeast extract-salts - MSO l-methionine sulfoximine - Pr protease deficient mutant - DON 6-diazo-5-oxo-l-norleucine - EDTA ethylene-diaminetetraacetic acid - EGTA ethylene glycol-bis (-aminoethyl ether) N,N,N,N-tetraacetic acid - Tris tris-(hydroxymethyl)-aminomethane     

Transient change in the ATP Pool ofAnabaena cylindrica associated with ammonia assimilation

When N₂-grown cells ofAnabaena cylindrica were exposed to ammonia (50 M to 5 mM) in the dark, the size of the ATP pool was reduced by 40% within 1 min, but restored after 5 or 6 min. The decrease in ATP was accompanied by increases in ADP and AMP, while the total adenylate content remained unaltered. The ammonia-induced change in the ATP pool was completely eliminated when algal cells were treated withl-methionine-dl-sulfoximine, an inhibitor of glutamine synthesis. These results suggest that ammonia is rapidly assimilated through the pathway mediated by glutamine synthetase accompanied by reduction of the ATP pool.Abbreviations GS Glutamine synthetase - MSX l-methionine-dl-sulfoximine - CCCP carbonyl cyanidem-chlorophenyl-hydrazone     

Transport of glutamine inXenopus laevis oocytes: Relationship with transport of other amino acids

Summary We have investigated transport of the amino acid glutamine across the surface membranes of prophase-arrestedXenopus laevis oocytes. Glutamine accumulation was linear with time for 30 min; it was stereospecific with aK _m of 0.12±0.02mm andV _max of 0.92±0.17 pmol/oocyte · min forl-glutamine. Transport ofl-glutamine was Na⁺-dependent, the cation not being replaceable with Li⁺, K⁺, choline, tris(hydroxymethyl)-aminomethane (Tris), tetramethylammonium (TMA) or N-methyld-glucamine NMDG); external Cl^– appeared to be necessary for full activation of Na⁺-dependent glutamine transport. Two external Na⁺ may be required for the transport of one glutamine molecule.l-glutamine transport (at 50 m glutamine) was inhibited by the presence of other amino acids:l-alanine,d-alanine,l-leucine,l-asparagine andl-arginine (about 60% inhibition at 1mm);l-histidine,l-valine and glycine (25 to 40% inhibition at 1mm);l-serine,l-lysine,l-phenylalanine andl-glutamate (45 to 55% inhibition at 10mm). N-methylaminoisobutyric acid (meAIB) had no effect at 10mm, but 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) inhibited Na⁺/glutamine transport by about 50% at 10mm.l-glutamine was a competitive inhibitor of the Na⁺-dependent transport ofl-alanine,d-alanine andl-arginine; this evidence is consistent with the existence of a single system transporting all four amino acids. Glutamine uptake in oocytes appears to be catalyzed by a transport system distinct from the cotransport Systems A, ASC, N and Gly, although it resembles System B^0,+.     

Utilization of l-alanine by Rhodopseudomonas acidophila

Rhodopseudomonas acidophila strain 7050 can satisfy all its nitrogen and carbon requirements from l-alanine. Addition of 100 M methionine sulfoximine to alanine grown cultures had no effect on growth rate indicating that deamination of alanine via alanine dehydrogenase and re-assimilation of the released NH ₄ ⁺ by glutamine synthetase/glutamate synthase was an insignificant route of nitrogen transfer in this bacterium. Determination of aminotransferase activities in cell-free extracts failed to demonstrate the presence of direct routes from alanine to either aspartate or glutamate. The only active aminotransferase involving l-alanine was the alanine-glyoxylate enzyme (114–167 nmol·min^–1·mg^–1 protein) which produced glycine as end-product. The amino group of glycine was further transaminated to yield aspartate via a glycineoxaloacetate aminotransferase (117–136 nmol·min^–1 ·mg^–1 protein). No activity was observed when 2-oxoglutarate was substituted for oxaloacetate. The formation of glutamate from aspartate was catalysed by aspartate-2-oxoglutarate aminotransferase (85–107 nmol·min^–1·mg^–1 protein). Determinations of free intracellular amino acid pools in alanine and alanine+100 M methionine sulfoximine grown cells showed the predominance of glutamate, glycine and aspartate, providing further evidence that in alanine grown cultures R. acidophila satisfies its nitrogen requirements for balanced growth by transamination.Abbreviations ADH alanine dehydrogenase - GDH glutamate dehydrogenase - GS glutamine synthetase - GOGAT glutamate synthase - MSO methionine sulfoximine - GOT glutamate-oxaloacetate aminotransferase - GPT glutamate-pyruvate amino-transferase - AGAT alanine-glyoxylate aminotransferase - GOAT glycine-oxaloacetate aminotransferase - GOTAT glycine-2-oxoglutarate aminotransferase - AOAT alanine-oxaloacetate aminotransferase     

Different ammonium-ion uptake,metabolism and detoxification efficiencies in two Lemnaceae

¹⁵N-Nuclear magnetic resonance spectroscopy was used to follow nitrogen assimilation and amino-acid production in Wolffia arrhiza (L.) Hork. ex. Wimmer, clone Golan exposed to 4.0 mM ¹⁵NH₄Cl solutions for 24 h. The main ¹⁵N-labelled metabolites were asparagine and glutamine, as well as substantial amounts of unreacted, intracellular NH ₄ ⁺ . These results were compared with those of a previous study on Lemna gibba L. clone Hurfeish (Monselise et al., 1987, New Phytol. 10, 341–345) with regard to NH ₄ ⁺ uptake, assimilation and detoxification efficiencies. Both species, grown under continuous white light, were capable of preferential uptake of NH ₄ ⁺ in the presence of nitrate. Relative growth rates indicate that both species tolerate increased levels of NH ₄ ⁺ , up to 10^–2 mol · 1^–1, with L. gibba showing a slightly greater tolerance. No ¹⁵N-labelled free NH ₄ ⁺ was detectable in L. gibba, while in W. arrhiza excess NH ₄ ⁺ was found within the cells. This fact indicates that L. gibba is more efficient in detoxification than W. arrhiza, presumably because of inability of W. arrhiza to regenerate the NH ₄ ⁺ traps, glutamate and aspartate, rapidly enough. This is also evident from the observation that addition of -ketoglutarate to the medium caused nearly complete assimilation of intracellular NH ₄ ⁺ in W. arrhiza. In both plants, addition of -ketoglutarate increased both NH ₄ ⁺ uptake and assimilation. Addition of l-methionine dl-sulfoximine, an inhibitor of glutamine synthetase inhibited NH ₄ ⁺ assimilation, while addition of azaserine, an inhibitor of glutamate synthase, resulted in ¹⁵N incorporation into the glutamine-amide position only. These results are consistent with the glutamine synthetase-glutamate synthase pathway being the major route of NH ₄ ⁺ assimilation in the two plants under the conditions used.Abbreviations AZA azaserine (O-diazoacetyl-l-serine) - GOGAT glutamine oxoglutarate amine transferase=]glutamate synthase E.C. 1.4.7. and E.C. 1.4.1.13. - GS glutamine synthetase E.C. 6.3.1.2. - -KG -ketoglutarate=2-oxoglutarate - MSO l-methionine dl-sulphoximine - NMR nuclear magnetic resonance - RGR relative growth rate This article is dedicated to Professor Bernhard Schrader on the occasion of his 60th birthdayWe wish to thank Professor Robert Glaser for helpful discussions, and Mrs. Aliza Levkoviz and Mr. Gideon Raziel for skillful assistance.     

Alanine dehydrogenase of the N2-fixing blue-green alga,Anabaena cylindrica

The l-alanine dehydrogenase (ADH) of Anabaena cylindrica has been purified 700-fold. It has a molecular weight of approximately 270000, has 6 sub-units, each of molecular weight approximately 43000, and shows activity both in the aminating and deaminating directions. The enzyme is NADH/NAD⁺ specific and oxaloacetate can partially substitute for pyruvate. The K _m ^app for NAD⁺ is 14 M and 60 M at low and high NAD⁺ concentrations, respectively. The K _m ^app for l-alanine is 0.4 mM, that for pyruvate is 0.11 mM, and that for oxaloacetate is 3.0 mM. The K _m ^app for NH ₄ ⁺ varies from 8–133 mM depending on the pH, being lowest at high pH levels (pH 8.7 or above). Alanine, serine and glycine inhibit ADH activity in the aminating direction. The enzyme is active both in heterocysts and vegetative cells and activity is higher in nitrogen-starved cultures than in N₂-fixing cultures. The data suggest that although alanine is formed by the aminating activity of ADH, entry of newly fixed ammonia into organic combination does not occur primarily via ADH in N₂-fixing cultures of A. cylindrica. Ammonia assimilation via ADH may be important in cultures with an excess of available nitrogen. The deaminating activity of the enzyme may be important under conditions of nitrogen-deficiency.Abbreviations ADH alanine dehydrogenase - DEAE diethylamino ethyl cellulose - EDTA ethylenediamine tetraacetic acid - GDH glutamic dehydrogenase - GS glutamine synthetase - GOT aspartate-glutamate aminotransferase - NAD⁺ nicotinamide adenine dinucleotide - NADH reduced nicotinamide adenine dinucleotide - NADP⁺ nicotinamide adenine dinucleotide phosphate - NADPH reduced nicotinamide adenine dinucleotide phosphate - SDS sodium dodecyl sulphate - Tris tris(hydroxymethyl) aminomethane