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.

     

Effect of Methionine Sulfoximine on the Accumulation of Ammonia in C(3) and C(4) Leaves : The Relationship between NH(3) Accumulation and Photorespiratory Activity

Additions of methionine sulfoximine (MSX), an inhibitor of glutamine synthetase (GS), result in an increase in NH₃ in seedling leaves of C₃ (wheat [Triticum aestivum cv. Kolibri] and barley [Hordeum vulgare var Perth]) and C₄ (corn [Zea mays W6A × W182E] and sorghum [Sorghum Vulgare var MK300]) plants. NH₃ accumulation is higher in C₃ (about 17.8 micromoles per gram fresh weight per hour) than in C₄ (about 4.7 micromoles) leaves. Under ideal conditions, when photosynthesis is not yet inhibited by the accumulation of NH₃, the rate of NH₃ accumulation is about 16% of the apparent rate of photosynthesis. A maximum accumulation of NH₃ was elicited by 2.5 millimolar MSX and was essentially independent of the addition of NO₃⁻ during either the growth or experimental period. When O₂ levels in the air were reduced to 2%, MSX resulted in some accumulation of NH₃ (6.0 micromoles per gram fresh weight per hour). At these levels of NH₃, there was no significant inhibition of rates of CO₂ fixation. There was also a minor, but significant, accumulation of NH₃ in corn roots treated with MSX. Inhibitors of photorespiration (isonicotinic hydrazide, 70 millimolar; 2-pyridylhydroxymethanesulfonic acid, 20 millimolar) or transaminase reactions (aminooxyacetate, 1 millimolar) inhibited the accumulation of NH₃ in both C₃ and C₄ leaves. These results support the hypothesis that GS is important in the assimilation of NH₃ in leaves and that the glycine-serine conversion is a major source of that NH₃.     

Effect of methionine-sulfoximine on nitrate uptake and photosynthesis in the cyanobacteriumAnabaena doliolum Bharadwaja

l-Methionine-dl-sulfoximine (MSX) stimulated nitrate uptake but inhibited¹⁴CO₂ fixation and O₂ evolution inAnabaena doliolum. Nitrate uptake was inhibited by ammonium (NH ₄ ⁺ ) in the absence of MSX, but not in the presence of MSX. Glutamine or a derivative of it appears to be the actual negative effector of nitrate utilization. In presence of nitrate, MSX-treated cells ofA. doliolum evolve more O₂ than do untreated cells. Our results suggest a close relation between photoassimilation of carbon and utilization of nitrogen.     

Isolation,improvement and characterization of an ammonium excreting mutant strain of the heterocytous cyanobacterium,Anabaena variabilis PCC 7937

Besides potential applications in the agriculture field as natural nitrogen fertilizer, N₂-fixing cyanobacteria have recently gained some attentions for new applications linked to the potential production of biologically active molecules or biohydrogen. Ammonium bioproduction is also gaining attention with the potential use of microalgae in biofuels production and the concerns about the increasing needs for nitrogen substrates. This study has investigated some phenotypic traits linked to biomass production and ammonium release in multicellular cyanobacteria, Anabaena variabilis PCC 7937. It confirms that this wild-type strain has no natural ability for ammonium excretion under diazotrophic conditions. A mutant strain, A. variabilis PCC 7937-C9, was obtained after double random mutagenesis treatments with ethyl methane–sulfonate and screening in batch cultures for resistance to the effect of a glutamine synthetase inhibitor, l-methionine-d,l-sulfoximine (MSX). Although significantly characterized by shorter cell filaments, the growth parameters in photobioreactors of the mutant strain cultures were in the same range of values than those of the wild type. In the presence of MSX this strain was shown to produce extracellular ammonium, with specific rates up to 4.9 μmol NH₄⁺ mg Chl a⁻¹ h⁻¹. The efficiency of this strain, estimated by its specific rate of ammonium excretion, was shown to be improved after consecutive batch cultures with increasing concentrations of MSX. Such mutant strains are of potential use for investigating ways to improve extracellular ammonium bioproduction.     

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     

Assimilation of 13NH 4 + by Anthoceros grown with and without symbiotic Nostoc

The pathways of assimilation of ammonium by pure cultures of symbiont-free Anthoceros punctatus L. and the reconstituted Anthoceros-Nostoc symbiotic association were determined from time-course (5–300 s) and inhibitor experiments using ¹³NH ₄ ⁺ . The major product of assimilation after all incubation times was glutamine, whether the tissues were cultured with excess ammonium or no combined nitrogen. The ¹³N in glutamine was predominantly in the amide-nitrogen position. Formation of glutamine and glutamate by Anthoceros-Nostoc was strongly inhibited by either 1mM methionine sulfoximine (MSX) or 1 mM exogenous ammonium. These data are consistent with the assimilation of ¹³NH ₄ ⁺ and formation of glutamate by the glutamine synthetase (EC 6.3.1.2)-glutamate synthase (EC 1.4.7.1) pathway in dinitrogen-grown Anthoceros-Nostoc. However, in symbiont-free Anthoceros, grown with 2.5 mM ammonium, formation of glutamine, but not glutamate, was decreased by either MSX or exogenous ammonium. These results indicate that during short incubation times ammonium is assimilated in nitrogenreplete Anthoceros by the activities of both glutamine synthetase and glutamate dehydrogenase (EC 1.4.1.2). In-vitro activities of glutamine synthetase were similar in nitrogen-replete Anthoceros and Anthoceros-Nostoc, indicating that the differences in the routes of glutamate formation were not based upon regulation of synthesis of the initial enzyme of the glutamine synthetase-glutamate synthase pathway. When symbiont-free Anthoceros was cultured for 2 d in the absence of combined nitrogen, total ¹³NH ₄ ⁺ assimilation, and glutamine and glutamate formation in the presence of inhibitors, were similar to dinitrogen-grown Anthoceros-Nostoc. The routes of immediate (within 2 min) glutamate formation and ammonium assimilation in Anthoceros were apparently determined by the intracellular levels of ammonium; at low levels the glutamine synthetase-glutamate synthase pathway was predominant, while at high levels independent activities of both glutamine synthetase and glutamate dehydrogenase were expressed.     

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     

MSX-resistant mutants of Anabaena 7120 with derepressed heterocyst development and nitrogen fixation

To investigate the role of ammonium-assimilating enzyme in heterocyst differentiation, pattern formation and nitrogen fixation, MSX-resistant and GS-impaired mutants of Anabaena 7120 were isolated using transposon (Tn5-1063) mutagenesis. Mutant Gs1 and Gs2 (impaired in GS activity) exhibited a similar rate of nitrogenase activity compared to that of the wild type under dinitrogen aerobic conditions in the presence and absence of MSX. Filaments of Gs1 and Gs2 produced heterocysts with an evenly spaced pattern in N₂-grown conditions, while addition of MSX altered the interheterocyst spacing pattern in wild type as well as in mutant strains. The wild type showed complete repression of heterocyst development and nitrogen fixation in the presence of NO₃ ^– or NH₄ ⁺, whereas the mutants Gs1 and Gs2 formed heterocysts and fixed nitrogen in the presence of NO₃ ^– and NH₄ ⁺. Addition of MSX caused complete inhibition of glutamine synthetase activity in wild type but Gs1 and Gs2 remained unaffected. These results suggest that glutamine but not ammonium is directly involved in regulation of heterocyst differentiation, interheterocyst spacing pattern and nitrogen fixation in Anabaena.     

Nitrogen Metabolism of the Marine Microalga Chlorella autotrophica

The levels of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in Chlorella autotrophica (clone 580) are strongly regulated by the nitrogen source and salt concentration of the medium. GS is present at high levels in NO₃⁻-grown cells, and at maximum levels in nitrogen-starved cells. However, the levels of GS in these cells are somewhat decreased by increasing salinity. Cells growing on NH₄⁺ have high NADPH-GDH activity, the levels of which increase with increasing NH₄⁺ supply, while GS decreases to a very low level under these conditions. Salinity intensifies the induction of NADPH-GDH activity in NH₄⁺-grown cells. The levels of NADH-GDH are low in this alga, but present under all growth conditions. Methionine sulfoximine (MSX) has little effect on growth and nitrogen assimilation of the alga in the presence of NH₄⁺.     

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.     

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     

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     

Characterization of Novel MSX1 Mutations Identified in Japanese Patients with Nonsyndromic Tooth Agenesis

Since MSX1 and PAX9 are linked to the pathogenesis of nonsyndromic tooth agenesis, we performed detailed mutational analysis of these two genes sampled from Japanese patients. We identified two novel MSX1 variants with an amino acid substitution within the homeodomain; Thr174Ile (T174I) from a sporadic hypodontia case and Leu205Arg (L205R) from a familial oligodontia case. Both the Thr174 and Leu205 residues in the MSX1 homeodomain are highly conserved among different species. To define possible roles of mutations at these amino acids in the pathogenesis of nonsyndromic tooth agenesis, we performed several functional analyses. It has been demonstrated that MSX1 plays a pivotal role in hard tissue development as a suppressor for mesenchymal cell differentiation. To evaluate the suppression activity of the variants in mesenchymal cells, we used the myoD-promoter, which is one of convenient reporter assay system for MSX1. Although the gene products of these MSX1 variants are stable and capable of normal nuclear localization, they do not suppress myoD-promoter activity in differentiated C2C12 cells. To clarify the molecular mechanisms underlying our results, we performed further analyses including electrophoretic mobility shift assays, and co-immunoprecipitation assays to survey the molecular interactions between the mutant MSX1 proteins and the oligonucleotide DNA with MSX1 consensus binding motif or EZH2 methyltransferase. Since EZH2 is reported to interact with MSX1 and regulate MSX1 mediated gene suppression, we hypothesized that the T174I and L205R substitutions would impair this interaction. We conclude from the results of our experiments that the DNA binding ability of MSX1 is abolished by these two amino acid substitutions. This illustrates a causative role of the T174I and L205R MSX1 homeodomain mutations in tooth agenesis, and suggests that they may influence cell proliferation and differentiation resulting in lesser tooth germ formation in vivo.     

An Aberrant Splice Acceptor Site Due to a Novel Intronic Nucleotide Substitution in MSX1 Gene Is the Cause of Congenital Tooth Agenesis in a Japanese Family

Congenital tooth agenesis is caused by mutations in the MSX1, PAX9, WNT10A, or AXIN2 genes. Here, we report a Japanese family with nonsyndromic tooth agenesis caused by a novel nucleotide substitution in the intronic region between exons 1 and 2 of the MSX1 gene. Because the mutation is located 9 bp before exon 2 (c.452-9G>A), we speculated that the nucleotide substitution would generate an abnormal splice site. Using cDNA analysis of an immortalized patient blood cell, we confirmed that an additional 7-nucleotide sequence was inserted at the splice junction between exons 1 and 2 (c.451_452insCCCTCAG). The consequent frameshift generated a homeodomain-truncated MSX1 (p.R151fsX20). We then studied the subcellular localization of truncated MSX1 protein in COS cells, and observed that it had a whole cell distribution more than a nuclear localization, compared to that of wild-type protein. This result suggests a deletion of the nuclear localization signal, which is mapped to the MSX1 homeodomain. These results indicate that this novel intronic nucleotide substitution is the cause of tooth agenesis in this family. To date, most MSX1 variants isolated from patients with tooth agenesis involve single amino acid substitutions in the highly conserved homeodomain or deletion mutants caused by frameshift or nonsense mutations. We here report a rare case of an intronic mutation of the MSX1 gene responsible for human tooth agenesis. In addition, the missing tooth patterns were slightly but significantly different between an affected monozygotic twin pair of this family, showing that epigenetic or environmental factors also affect the phenotypic variations of missing teeth among patients with nonsyndromic tooth agenesis caused by an MSX1 haploinsufficiency.     

Role of RANKL (TNFSF11)-Dependent Osteopetrosis in the Dental Phenotype of Msx2 Null Mutant Mice

The MSX2 homeoprotein is implicated in all aspects of craniofacial skeletal development. During postnatal growth, MSX2 is expressed in all cells involved in mineralized tissue formation and plays a role in their differentiation and function. Msx2 null (Msx2 ^−/−) mice display complex craniofacial skeleton abnormalities with bone and tooth defects. A moderate form osteopetrotic phenotype is observed, along with decreased expression of RANKL (TNFSF11), the main osteoclast-differentiating factor. In order to elucidate the role of such an osteopetrosis in the Msx2 ^−/− mouse dental phenotype, a bone resorption rescue was performed by mating Msx2 ^−/− mice with a transgenic mouse line overexpressing Rank (Tnfrsf11a). Msx2 ^−/− Rank^Tg mice had significant improvement in the molar phenotype, while incisor epithelium defects were exacerbated in the enamel area, with formation of massive osteolytic tumors. Although compensation for RANKL loss of function could have potential as a therapy for osteopetrosis, but in Msx2 ^−/− mice, this approach via RANK overexpression in monocyte-derived lineages, amplified latent epithelial tumor development in the peculiar continuously growing incisor.     

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 proliferation in developing human tooth germs by MSX homeodomain proteins and cyclin-dependent kinase inhibitor p19INK4d

Before the secretion of hard dental tissues, tooth germs undergo several distinctive stages of development (dental lamina, bud, cap and bell). Every stage is characterized by specific proliferation patterns, which is regulated by various morphogens, growth factors and homeodomain proteins. The role of MSX homeodomain proteins in odontogenesis is rather complex. Expression domains of genes encoding for murine Msx1/2 during development are observed in tissues containing highly proliferative progenitor cells. Arrest of tooth development in Msx knockout mice can be attributed to impaired proliferation of progenitor cells. In Msx1 knockout mice, these progenitor cells start to differentiate prematurely as they strongly express cyclin-dependent kinase inhibitor p19^INK4d. p19^INK4d induces terminal differentiation of cells by blocking the cell cycle in mitogen-responsive G1 phase. Direct suppression of p19^INK4d by Msx1 protein is, therefore, important for maintaining proliferation of progenitor cells at levels required for the normal progression of tooth development. In this study, we examined the expression patterns of MSX1, MSX2 and p19^INK4d in human incisor tooth germs during the bud, cap and early bell stages of development. The distribution of expression domains of p19^INK4d throughout the investigated period indicates that p19^INK4d plays active role during human tooth development. Furthermore, comparison of expression domains of p19^INK4d with those of MSX1, MSX2 and proliferation markers Ki67, Cyclin A2 and pRb, indicates that MSX-mediated regulation of proliferation in human tooth germs might not be executed by the mechanism similar to one described in developing tooth germs of wild-type mouse.     

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.