Short-term ammonium inhibition of nitrate uptake by Azotobacter chroococcum

Addition of NH₄Cl at low concentrations to Azotobacter chroococcum cells caused an immediate cessation of nitrate uptake activity, which was restored when the added NH ₄ ⁺ was exhausted from the medium or by adding an NH ₄ ⁺ assimilation inhibitor, l-methionine-dl-sulfoximine (MSX) or l-methionine sulfone (MSF). In the presence of such inhibitors the newly-reduced nitrate was released into the medium as NH ₄ ⁺ . When the artificial electron donor system ascorbate/N-methylphenazinium methylsulfate (PMS), which is a respiratory substrate that was known to support nitrate uptake by A. chroococcum while inhibiting glutamine synthetase activity, was the energy source, externally added NH ₄ ⁺ had no effect on nitrate uptake. It is concluded that, in A. chroococcum cells, NH ₄ ⁺ must be assimilated to exert its short-term inhibitory effect on nitrate uptake. A similar proposal was previously made to explain the short-term ammonium inhibition of N₂ fixation in this bacterium.Abbreviations MOPS morpholinopropanesulfonic acid - MSX l-methionine-dl-sulfoximine - PMS N-methylphenazinium methylsulfate - MSF l-methionine sulfone     

Utilization of nitrate,nitrite and ammonium by Chlamydomonas reinhardii

In phototrophically grown Chlamydomonas cells, ammonium strongly inhibited the utilization of nitrate or nitrite. Under darkness, or in the presence of an uncoupler or inhibitor of the non-cyclic photosynthetic electron flow, the utilization of nitrate, nitrite or ammonium was suppressed. l-Methionine-d,l-sulfoximine (MSX) or azaserine, which blocks the assimilation of ammonium, inhibited the consumption of nitrate, but not nitrite, by the cells. Ammonium produced an immediate inhibition of the permease for nitrate in Chlamydomonas growing with nitrate, while ammonium-grown cells lacked this permease. The synthesis of nitrate-reductase activity was dependent on an active permease. In N-starved Chlamydomonas cells, previously treated with MSX, the permease for nitrate was insensitive to inhibition by ammonium, and a significant amount of nitrate reductase was synthetized. These cells photoproduce ammonium by reducing nitrate. Nitrogen-repleted cells, treated with MSX, actively photoproduced ammonium by reducing nitrite, but not nitrate.Abbreviations DCMU N-(3,4-dichlorophenyl)N,N-di-methyl-urea - PCCP Carbonylcyanid-p-trifluoromethoxy-phenylhydrazone - Mops 2-(N-morpholino)propanesulfonic acid - MSX l-Methionine-d,l-sulfoximine     

Short-term ammonium inhibition of nitrate utilization by Anacystis nidulans and other cyanobacteria

Ammonium at low concentrations caused a rapid and effective inhibition of nitrate utilization in the light by the cyanobacterium Anacystis nidulans without affecting the cellular level of nitrate reductase activity. The inhibition was reversible, and the ability of the cells to utilize nitrate was restored immediately after ammonium had been exhausted. The inhibitory effect was dependent on consumption by the cells of the added ammonium which was rapidly incorporated into amino acids. In the presence of L-methionine-d,l-sulfoximine (MSX) or azaserine, inhibitors of the glutamine synthetase-glutamate synthase pathway, ammonium did not exhibit any inhibitory effect on nitrate utilization. Ammonium assimilation, rather than ammonium itself, seems to regulate nitrate utilization in A. nidulans. Short-term inhibition by ammonium of nitrate utilization and its prevention by MSX were also demonstrated in the filamentous cyanobacteria Anabaena and Nostoc.Abbreviations MSX L-Methionine-d-l-sulfoximine     

Effects of methylammonium and ofL-methionine-DL-sulfoximine on the growth and nitrogen metabolism ofPhaeodactylum tricornutum

Phaeodactylum tricornutum Bohlin grew well withL-methionine-DL-sulfoximine (MSX) as sole nitrogen source. Such growth helps to explain the lack of effect of MSX on ammonium assimilation by this organism. Methylammonium inhibited growth with nitrate or MSX as sole nitrogen source but not growth on ammonium. Methylammonium could not be metabolised byP. tricornutum but was accumulated in the cells, the concentration factor sometimes approaching 25,000. Ammonium addition, but not that of MSX or nitrate, displaced methylammonium from the cells and this displacement was followed by resumption of growth. Both methylammonium and ammonium inhibited the uptake of nitrate and nitrite by the cells but inhibition by methylammonium, in comparison with that by ammonium, required a higher concentration and a longer time to develop. Inhibition by methylammonium is shown to be associated with its accumulation by the cells. Methylammonium also prevented the disappearance of nitrate from the interior of the cells (presumably by nitrate assimilation) whereas ammonium did not. It is concluded that methylammonium and ammonium differ in the ways in which they inhibit nitrate metabolism inP. tricornutum.Abbreviation MSX L-methionine-DL-sulfoximine     

Regulation of assimilatory nitrate reductase in the cyanobacteriumAnabaena doliolum

The assimilatory nitrate reductase (NR) from the cyanobacteriumAnabaena doliolum was membrane bound and solubilized by sonication. The Km value of the enzyme was 870 µM for nitrate with dithionite-reduced methyl viologen (MV) as electron donor. The pH optimum was 10.5 in the MV assay. Nitrate acted as an inducer and ammonium as repressor of the enzyme synthesis. In the presence ofl-methionine-d,l-sulfoximine (MSX) or azaserine, inhibitors of the glutamine synthetase-glutamate synthase (GS-GOGAT) pathway, ammonium did not exhibit any inhibitory effect on the enzyme. The photosynthetic nature of NR was shown with PS II inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). This enzyme fromA. doliolum has been shown to be a light-driven process, requiring de novo protein synthesis. It was inhibited by chlorate, the structural analog of nitrate;p-chloromercuribenzoate, a thiol reagent; sodium tungstate; and certain cations.     

Involvement of a cadmium-induced low molecular weight protein in regulating cadmium toxicity in the diazotrophic cyanobacterium Anabaena doliolum

This study demonstrated the production of a cadmium-induced low molecular weight (3.5 kDa), buthionine sulfoximine (BSO) sensitive protein in Anabaena doliolum. Production of this protein was accompanied by a decrease in the glutathione level of the cell. Cadmium was found to be differentially toxic to carbon fixation, O₂ evolution, ATP content, nitrate reductase, nitrogenase, alkaline phosphatase and ATPase of control (untreated), BSO, cadmium and (cadmium + BSO) pre-treated A. doliolum. Toxicity was maximum in BSO-grown cells followed be control (untreated), cadmium + BSO and least in cadmium-grown A. doliolum. Cadmium and (cadmium + BSO)-grown cells registered an increased lipid production, reduced metal uptake and low K⁺, Na⁺ loss. In spite of equal cadmium uptake rates, a significant difference in toxicity between cadmium-grown and (cadmium + BSO)-grown cultures was, however, noticed. Better performance of physiological and biochemical variables of cadmium-grown A. doliolum and its tolerance to cadmium could be due to the synthesis of low molecular weight cadmium binding protein (presumably phytochelatin) as well as an increased production of lipid.     

Biosynthesis of 5-(p-aminophenyl)-1,2,3,4-tetrahydroxypentane by methanogenic bacteria

In an attempt to establish the nature of the ammonium-assimilation products which mediate the inhibition by ammonium of nitrate uptake in cyanobacteria, the effect of different amino acids on nitrate utilization by intact Anacystis nidulans cells has been assayed. To exclude an indirect inhibition of nitrate uptake through the ammonium which the amino acids might release, the cells were pretreated with l-methionine-d,l-sulfoximine (MSX), a potent inactivator of glutamine synthetase. Under these conditions, several l-amino acids, but not the corresponding d-isomers, affected nitrate utilization to a variable extent, causing inhibitions ranging between 20 and 80% when added at 20 mM concentration.For most of the inhibitory amino acids, including l-isoleucine, l-leucine and l-valine, a correlation was found between their ability to act as amino group donors to -ketoglutarate, in reactions catalyzed by A. nidulans cell-free extracts, and their inhibitory effect on nitrate utilization. l-Glutamine, l-asparagine and glycine, being effective inhibitors of nitrate utilization, were poor substrates for the transaminating activity to -ketoglutarate, however. The possible role of the latter amino acids as mediators in the ammonium-promoted inhibition of nitrate uptake is discussed.Abbreviations MSX l-methionine-d,l-sulfoximine - MTA-5 mixed alkyltrimethylammonium bromide - Mops morpholinopropane sulfonic acid     

Nitrate Uptake by the Halotolerant Cyanobacterium <Emphasis Type="Italic">Aphanothece halophytica</Emphasis> Grown Under Non-Stress and Salt-Stress Conditions

We have compared the characteristics of nitrate uptake by Aphanothece halophytica grown under non-stress and salt-stress conditions. Both cell types showed essentially similar patterns of nitrate uptake toward ammonium, nitrite, and DL-glyceraldehyde. Although the affinities of nitrate to non-stress cells and salt-stress cells were not significantly different, i.e., K_s = 416 and 450 µM, respectively, the V_max value for non-stress cells was about twofold of that for salt-stress cells (9.1 vs 5.3 µmol min^–1 mg^–1 Chl). Nitrate uptake by A. halophytica was found to be dependent on Na⁺. Ammonium inhibited nitrate uptake, and the presence of methionine sulfoximine could not release the inhibition by ammonium. Nitrite appeared to competitively inhibit nitrate uptake with a K_i value of 84 µM. Both chloride and phosphate anions did not affect nitrate uptake. DL-Glyceraldehyde, an inhibitor of CO₂ fixation, caused a reduction in the uptake of nitrate.Received: 22 October 2002 / Accepted: 6 December 2002     

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     

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     

Evidence for the nitrate assimilation-dependent nitrite excretion in cyanobacterium Nostoc MAC

Nitrate uptake and nitrite efflux patterns in Nostoc MAC showed a rapid phase followed by their saturation. Nitrite efflux was maximum in nitrate medium whereas the cells incubated in N₂ and NH ₄ ⁺ media exhibited a decreased nitrite efflux activity. The simultaneous presence of NH ₄ ⁺ and nitrate significantly decreased nitrite efflux. L-Methionine-Dl-sulphoximine (MSX) prevented inhibition of nitrite efflux by NH ₄ ⁺ . In the dark there was negligible nitrite efflux, whereas illumination increased the rate of nitrite efflux significantly. The nitrite efflux system was maximally operative at pH 8.0, 30°C and a photon fluence rate of 50 mol m^-2. s^-1. These results confirm that (i) the nitrite efflux system in Nostoc MAC is dependent upon nitrate uptake and assimilation and is repressible by NH ₄ ⁺ ; (ii) NH ₄ ⁺ itself is not the actual repressor of nitrite efflux; a product of NH ₄ ⁺ assimilation via glutamine synthetase (GS) is required for repression to occur; (iii) the catalytic function of GS does not appear to be involved in nitrate assimilation-dependent nitrite efflux, and (iv) the optimum pH, temperature and illumination for maximum nitrite efflux were found to be 8.0, 30°C and 50mol m^-2. s respectively.B.B. Singh, P.K. Pandey and P.S. Bisen are with the Department of Microbiology, Barkatullah University. Bhopal 462026, India. S.Singh is with the Department of Microbiology, School of Life Sciences, North Maharashtra University, Jalgaon, India     

CONTRASTING EFFECTS OF METHIONINE SULFOXIMINE ON UPTAKE AND ASSIMILATION OF AMMONIUM IN ULVA INTESTINALIS (CHLOROPHYCEAE)1

Ammonium is assimilated in algae by the glutamine synthetase (GS)–glutamine:2‐oxoglutarate aminotransferase pathway. In addition to the assimilation of external ammonium taken up across the cell membrane, an alga may have to reassimilate ammonium derived from endogenous sources (i.e. nitrate reduction, photorespiration, and amino acid degradation). Methionine sulfoximine (MSX), an irreversible inhibitor of GS, completely inhibited GS activity in Ulva intestinalis L. after 12 h. However, assimilation of externally derived ammonium was completely inhibited after only 1–2 h in the presence of MSX and was followed by production of endogenous ammonium. However, endogenous ammonium production in U. intestinalis represented only a mean of 4% of total assimilation attributable to GS. The internally controlled rate of ammonium uptake (V_i) was almost completely inhibited in the presence of MSX, suggesting that V_i is a measure of the maximum rate of ammonium assimilation. After complete inhibition of ammonium assimilation in the presence of MSX, the initial or surge (V_s) rate of ammonium uptake in the presence of 400 μM ammonium chloride decreased by only 17%. However, the amount that the rate of ammonium uptake decreased by was very similar to the uninhibited rate of ammonium assimilation. In addition, the decrease in the rate of ammonium uptake in darkness (in the absence of MSX) in the presence of 400 μM ammonium chloride matched the decrease in the rate of ammonium assimilation. However, in the presence of 10 μM ammonium chloride, MSX completely inhibited ammonium assimilation but had no effect on the rate of uptake.     

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     

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     

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.     

Ammonia uptake and retention in some cyanobacteria

The internal pool of ammonia in strains of unicellular and filamentous cyanobacteria was found to be 6–12 nmol·mg^-1 protein. In nitrate grown Anacystis nidulans R-2 the pool size averaged 12 nmol·mg^-1 protein, which corresponds to 2.3 mM, and was little affected by N-source or medium pH during growth. Cells from NH ₄ ⁺ -limited continuous culture contained comparable pools, and cell yield was independent of medium pH (7.2–8.5). The internal pool was not bound to macromolecules. The pool fell transiently to about one-third within 2 h on shifting cells to N-free medium, but was slowly regenerated over 24 h.Added ammonia was removed from solution by illuminated cell suspensions at a linear rate, adequate to supply biosynthetic needs, to residual concentrations less than 5 M. An apparent K _m of less than 1 M can be inferred. Uptake rates were independent of N-source during growth, and of assay pH over the range 6.2–8.7. Bicarbonate was needed for uptake, but the rate of uptake was not influenced by the simultaneous presence of NaNO₃ (10 mM) or CH₃NH₃Cl (0.15 mM). Uptake was energydependent, and was eliminated in dark, anaerobic conditions or by the addition of protonophores. Uptake was also strongly inhibited by dicyclohexylearbodiimide, an ATPase inhibitor, by — SH reagents and methionine sulfoximine, suggesting that interference with energy supply or with ammonia metabolism prevented further entry into the cells.Non-standard abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DCCD dicyclohexylcarbodiimide - DCMU dichlorophenyl dimethylurea - NEM N-ethylmaleimide - pCMB p-chloromercuribenzoate - MSX L-methionine Dl-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     

Photoproduction of ammonium ion from N2 inRhodospirillum rubrum

NH ₄ ⁺ excretion was undetectable in N₂-fixing cultures ofRhodospirillum rubrum (S-1) and nitrogenase activity in these cultures was repressed by the addition of 10 mM NH ₄ ⁺ to the medium. The glutamate analog,l-methionine-dl-sulfoximine (MSX), derepressed N₂ fixation even in the presence of 10 mM extracellular NH ₄ ⁺ . When 10 mg MSX/ml was added to cultures just prior to nitrogenase induction they developed nitrogenase activity (20% of the control activities) and excreted most of their fixed N₂ as NH ₄ ⁺ . Nitrogenase activities and NH ₄ ⁺ production from fixed N₂ were increased considerably when a combined nitrogen source, NH ₄ ⁺ (>40 moles NH ₄ ⁺ /mg cell protein in 6 days) orl-glutamate (>60 moles NH ₄ ⁺ /mg cell protein in 6 days) was added to the cultures together with MSX.Biochemical analysis revealed thatR. rubrum produced glutamine synthetase and glutamate synthase (NADP-dependent) but no detectable NADP-dependent glutamate dehydrogenase. The specific activity of glutamine synthetase was observed to be maximal when nitrogenase activity was also maximal. Nitrogenase and glutamine synthetase activities were repressed by NH ₄ ⁺ as well as by glutamate.The results demonstrate that utilization of solar energy to photoproduce large quantities of NH ₄ ⁺ from N₂ is possible with photosynthetic bacteria by interfering with their regulatory control of N₂ fixation.     

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.     

Effects of the glutamine synthetase inhibitor methionine sulfoximine on CO2 fixation inLemna gibba

Summary Net CO₂ fixation inLemna gibba L. was inhibited by 0.5 mM L-methionine D,L-sulfoximine (MSX) both under photorespiratory conditions (21% O₂) and in 2% O₂. The inhibition was noticeably delayed by addition of 5 mM glutamine. Glutamine also delayed MSX-induced inactivation of glutamine synthetase. An increase in intracellular NH ₄ ⁺ concentration was noted in the presence of MSX only, and in the presence of 10 mM NH ₄ ⁺ only. However, presence of 10 mM NH ₄ ⁺ did not cause any inhibition of CO₂ fixation.