Irreversible Inhibitor Of Glutamine Synthetase Research Articles

Glutamine Addiction of Multiple Myeloma Cells Reprograms Mesenchymal Stromal Cell Enzymes and Transporters Towards a Pro-Tumor Phenotype

Metabolic alterations in cancer are not only functional to ensure malignant cell growth but also shape a pro-tumor behavior of normal cell populations in the tumor microenvironment. Multiple myeloma (MM) is the only human cancer that is both glutamine-addicted and glutamine-auxotroph, a feature that renders MM growth completely dependent upon extracellular glutamine availability. Indeed, plasma cells from most MM patients do not express Glutamine Synthetase (GS), the only enzyme able to synthetize glutamine from glutamate and ammonium, while express high levels of Glutaminase (GLS), which catalyze the first step of glutaminolysis by deamidating glutamine into glutamate and ammonium. As a consequence, the bone marrow (BM) plasma of MM patients has low-glutamine/high glutamate levels compared to patients with smoldering MM (SMM) and Monoclonal Gammopathy of Uncertain Significance (MGUS), as firstly demonstrated by our group. This particular metabolic microenvironment induces GS expression in mesenchymal stromal cells (MSCs) impairing osteoblast (OB) differentiation of MSCs, thus favoring MM bone lesions typical of active MM. The impact of these metabolic alterations on other BM cell populations is much less defined and it has been investigated in this study. For instance, although MM BM displays an oversized adipocyte population, which sustain MM growth by providing free fatty acids, the effects of MM metabolism on MSCs and their adipocyte differentiation remain to be characterized. First we show that 13C 5-Gln isotopomer distribution revealed that almost 50% of total glutamate in human myeloma cell lines (HMLCs) directly derives from glutamine deamidation by GLS. Furthermore, in standard culture conditions, HMLCs secreted glutamate likely through the SLC7A11 transporter, whose activity was higher in HMLCs than in MSCs. On the other hand, HMCLs were not able to take up glutamate from the extracellular space, while human primary MSCs isolated from healthy donors displayed high activity of the sodium-dependent glutamate transporters of the EAAT family. Moreover, glutamate uptake was higher in undifferentiated MSCs than in MSC incubated for 14 days in osteogenic medium (10 -8 M dexamethasone and 50 µg/ml ascorbic acid). Consistently, a public transcriptional profile of MSCs and OBs obtained from bone biopsies of healthy donors (n=7) or MM patients (n=16) revealed that the expression of the inward Glu transporter EAAT3 is higher in MSCs compared to OBs. In glutamine-free conditions, MSCs produced and secreted glutamine, a process boosted by extracellular glutamate in a dose-dependent manner. Glutamine secretion was hindered by either l-methionine sulfoximine (MSO), an irreversible inhibitor of GS, or D-aspartate (D-Asp), a high-affinity inhibitor of EAAT3 that hinders glutamate uptake. In a co-culture system, HMCLs induced the expression of SNAT5 glutamine efflux transporter in MSCs. Consistently, upon glutamine withdrawal, primary undifferentiated MSCs from healthy donors sustained HMCLs growth, while this nutritional support was markedly impaired by either inhibition or silencing of GS and EAAT3, with a substantial decrease in MM cell viability. Lastly, primary human MSCs were incubated under adipogenic conditions (0.5 mM 3-Isobutyl-1-methylxanthine, 5 µM indomethacin, 50 µM dexamethasone and 10 mg/ml human insulin) in the presence or in the absence of glutamine to mimic, respectively, normal or MM BM microenvironment. Glutamine deprivation increased lipogenesis, assessed with Oil Red O staining, as well as the expression of the adipocyte markers PPARG, LEP, and ADIPOQ. These data point to a MM-driven metabolic pro-tumor BM niche in which MSCs feed glutamine-addicted MM cells, and MSC differentiation is skewed from osteogenesis to adipogenesis. Several steps of these deranged pathways are amenable to pharmacological inhibition, pointing to possible novel therapeutic approaches to counteract MM growth and its effects on the BM niche.

Myeloma-Induced Alterations of Glutamine Metabolism Impair Bone Microenvironment Niche in Multiple Myeloma Patients

Multiple myeloma (MM) cells are characterized by tight dependence on the bone marrow (BM) microenvironment that exerts a permissive role on cell growth and survival. In turn, MM cells markedly modify their microenvironment leading, in particular, to the development of osteolytic bone lesions.Recently, we demonstrated that metabolic alterations is a major feature of MM cells showing that BM plasma of MM patients is characterized by lower levels of Glutamine (Gln) and higher levels of Glutamate (Glu) and ammonium when compared with patients with smoldering MM (SMM) and Monoclonal Gammopathy of Uncertain Significance (MGUS). In the majority of MM patients MM cells are Gln-addicted since they strictly depend on extracellular Gln, do not express Glutamine Synthetase (GS), the enzyme that synthetizes Gln from Glu and ammonium, and are endowed with high levels of the Gln transporter ASCT2. Based on this evidence, we have hypothesized that the peculiar Gln metabolism of MM cells may have a significant impact on the relationship with the bone microenvironment and contribute to the development of osteolytic lesions.We firstly characterized a panel of human MM cell lines (HMCLs) for their GS expression and response to decreasing levels of Gln. The majority of HMCLs, which did not express GS, consumed large amounts of extracellular Gln but secreted nearly half of the amino acid as Glu. Two HMCLs, MM1.S and U266, with a sizable GS expression, were less sensitive to Gln deprivation and secreted less Glu in the extracellular space compared with GS-negative HMCLs. Consistently, the activity of the Glu exchanger x-CT (the product of SLC7A11 gene) was lower in GS-positive than in GS-negative cells. The response to Gln starvation was then studied in mesenchymal stromal cell line (MSC), as well as in osteoblastic (HOBIT) and pre-osteocytic cells (HOB-01). HOBIT and HOB-01 were more sensitive to Gln depletion than MSC. Indeed, while MSC showed a low EC50 for Gln (0.064mM), which is 10-times lower than the physiological blood Gln concentration (around 0.6 mM), the EC50 values of HOBIT and HOB-01 cells were 0.250 mM and 0.297mM, respectively. Furthermore, L-methionine sulfoximine (MSO), an irreversible inhibitor of GS, emphasized the effects of Gln deprivation on all the cell lines tested. Indeed, Gln deprivation enhanced the expression of GS, suggesting that both stromal and osteoblastic cells exploit the enzyme to counteract Gln deprivation. On the basis of these data, we assessed the effects of Gln and Glu on osteogenic differentiation by incubating MSC, either immortalized or primary, with an osteogenic medium containing different concentrations of Gln and Glu. After 2 weeks, compared with cells differentiated in high Gln/high Glu conditions, MSC incubated in the presence of decreased Gln and increased Glu showed lower osteogenic ability, as assessed by real time PCR and ALP staining. Lastly, MSC co-cultured for 72 hours with GS-negative, but not with GS-positive HMCLs, showed reduced viability and increased GS expression.Lastly, to put in a translational perspective these in vitro observations, we analyzed the BM plasma levels of Gln and Glu in a cohort of 41 patients with newly diagnosed MM, including 9 smoldering MM (SMM) and 32 active MM patients (20 of them with osteolytic bone disease, 12 of them without bone disease). All 20 osteolytic MM patients had more than three osteolytic lesions. We found that MM patients had lower Gln levels and higher Glu levels than SMM patients. Moreover, when compared with MM patients without bone disease, MM patients with bone disease showed lower levels of Gln and higher levels of Glu. The results of these analyses are being continuously updated increasing the number of samples tested.Overall, these results indicate that MM cells are able to create a low-Gln/high-Glu bone marrow microenvironment that sustains GS expression in bone cells and impairs their differentiation and viability. Thus, the peculiar metabolic milieu in the MM bone microenvironment affects the relationship between neoplastic and bone cells and may contribute to the development of osteolytic bone disease in MM patients. DisclosuresAversa:Astellas: Honoraria; Merck: Honoraria; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Basilea: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees. Giuliani:Takeda Pharmaceutical Co: Research Funding; Celgene Italy: Other: Avisory Board, Research Funding; Janssen Pharmaceutica: Other: Avisory Board, Research Funding.

Abstract 5245: Adipocyte production of glutamine protects leukemia cells from L-asparaginase

Abstract Obesity is associated with increased cancer incidence and mortality. Adipose tissue has been demonstrated to play a role in several types of cancer, including acute lymphoblastic leukemia (ALL). We have shown that obesity impairs the survival of mice transplanted with ALL and treated with the front-line chemotherapy, L-Asparaginase (ASNase), which acts through the deamination of asparagine (ASN) and glutamine (GLN). We have also shown that adipocytes protect ALL cells from ASNase in vitro. We hypothesized that adipocytes protect ALL cells from ASNase via production of GLN. Fat pads were excised from control and diet-induced obese mice. Glutamine synthetase, the rate-limiting enzyme for GLN synthesis, was analyzed by western blot. Amino acids secreted by fat pads were measured using HPLC. ALL cell sensitivity to ASN and/or GLN depletion, and ASNase, was determined by trypan blue exclusion in culture alone and in transwells over 3T3-L1 adipocytes. Mouse fat pads expressed high levels of glutamine synthetase protein, and this was not affected by obesity or 5 days of L-asparaginase treatment (3,000 IU/kg/day). Fat pad explants secreted GLN (251±50 nmoles/100 mg tissue), but not ASN, over 72 hours. Confirming that GLN secretion could protect ALL from ASNase, twice daily addition of 500 nmoles of GLN to murine ALL cells completely reversed ASNase cytotoxicity (64.0x10^4 vs. 7.9x10^4 viable cells after 72 hours of 1 IU/mL ASNase, p<0.01). Testing a panel of human ALL cell lines, we found that proliferation of 3 of 9 cell lines were decreased when ASN was removed from the media (RS4;11, SupB15, Molt4), while 8 of 9 cell lines were decreased when GLN was removed from the media (RS4;11, SupB15, Molt4, SEM, RCH-ACV, BV-173, SD-1, HL60). To test whether blocking adipocyte production of GLN would reverse their ability to protect ALL cells, we pretreated 3T3-L1 adipocytes with methionine sulfoximine (MSO), an irreversible inhibitor of glutamine synthetase. MSO pretreatment blocked GLN secretion by adipocytes and completely reversed adipocyte protection of ALL cells (with no adipocytes: 7.5x10^4; with untreated adipocytes: 14.8x10^4; with MSO treated adipocytes: 6.2x10^4, p=0.03 vs. untreated adipocytes). Finally, we tested whether an alternative preparation of ASNase with a higher glutaminase activity, ERWinase, could overcome adipocyte protection of three GLN dependent cell lines: SD-1, HL60, and Molt-4. Compared to E. coli ASNase, ERWinase treatment resulted in significantly decreased survival of all 3 cell lines in the presence of adipocytes (SD-1 4.2x10^4 vs. 6.7x10^4, p<0.05; HL60 4.1x10^4 vs. 8.8x10^4, p<0.05; Molt-4 16.9x10^4 vs. 26.4x10^4, p<0.05). These studies uncover a novel mechanism of adipocyte protection of leukemia cells from chemotherapy: adipocytes protect ALL cells from ASNase treatment via the production of GLN. Adipocyte production of GLN is both necessary and sufficient to protect ALL cells from ASNase treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5245. doi:1538-7445.AM2012-5245

Inhibition of glutamate/glutamine cycle in vivo results in decreased benzodiazepine binding and differentially regulated GABAergic subunit expression in the rat brain

The astrocytic enzyme glutamine synthetase (GS) is a key regulator of glutamate and γ-aminobutyric acid (GABA) metabolism in the glutamate/glutamine cycle (GGC). Inhibition of GS results in changes of neurotransmitter release and recycling. However, little is known about the influence of GGC on neurotransmitter receptor expression. In the pentylenetetrazole model of epilepsy, GS becomes nitrated and partially inhibited, and we demonstrated alterations of neurotransmitter receptor expression in the same model. Therefore, we hypothesized similar changes of neurotransmitter receptor expression when GS is inhibited in vivo. Rats were treated with a single dose (100 mg/kg bodyweight) of l-methionine sulfoximine (MSO), an irreversible inhibitor of GS. We used ³H-receptor autoradiography to measure glutamatergic [α-amino-3-hydroxy-5-methyl-4-isoxazol-propionic acid (AMPA), kainate, N-methyl-D-aspartate (NMDA)], GABAergic (GABA(A) , GABA(B) and GABA(A) -associated benzodiazepine (BZ) binding sites], dopamine D₁, and adenosine A₁ receptor subtypes. In addition, we performed saturation analysis of BZ binding sites on cerebral membrane homogenates and investigated the expression of GABA(A) α₁ and γ₂ subunits (which primarily mediate BZ binding) by western blot analysis. We demonstrated a significant reduction of BZ binding in the somatosensory, piriform, and entorhinal cortices and in the amygdala, 24 and 72 h after MSO treatment. Saturation analysis revealed decreased BZ binding (B(max)) on cerebral membrane homogenates 72 h after MSO treatment, without changes in binding site affinity (K(D)). Furthermore, we found differential changes of α₁ , γ₂ , and phosphorylated γ₂ subunits following MSO treatment. On the basis of our findings, we conclude that the glutamate/glutamine cycle directly influences GABAergic neurotransmission by regulating GABA(A) subunit composition, thereby affecting its modulation by endogenous benzodiazepines.

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 (Vi) was almost completely inhibited in the presence of MSX, suggesting that Vi 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 (Vs) 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.

Inhibition of glutamine synthetase in a549 cells during hyperoxia.

High oxygen concentrations are used in the treatment of acute respiratory distress syndrome and hyaline membrane disease. Hyperoxia, however, can damage alveolar epithelial cells through the release of free oxygen radicals. Supplemental glutamine (Gln) has recently been shown to increase survival of A549 cells, a distal epithelial cell line, during hyperoxia (). We found that supplemental Gln (Gln+) is essential for cell growth in A549 cells. In room air, cells without supplemental Gln (Gln-) survived with BCL-2 levels similar to those of Gln+ cells, but cell growth was minimal. We also evaluated the role of glutamine synthetase (GS) in A549 cells during hyperoxia. L-methionine sulfoximine (MSO), an irreversible inhibitor of GS, was added to Gln+ and Gln- cells. In hyperoxia, Gln- cells had greater survival then Gln- cells treated with MSO. Supplemental Gln could rescue cells in hyperoxia from the effect of MSO, suggesting that GS, through the endogenous synthesis of Gln, could attenuate hyperoxic cell injury. In hyperoxia, cells treated with 10-mM concentrations of Gln had increased survival compared with cells receiving 2-mM concentrations. The higher concentration of Gln, however, did not decrease the percentage of cells undergoing necrosis.

Chronic inhibition of glutamine synthetase is not associated with impairment of learning and memory in mice

The convulsant methionine sulfoximine (MSO) is a byproduct of the agenized flour commonly used for feeding domestic animals decades ago. MSO is a powerful glycogenic and epileptogenic agent, and it is an irreversible inhibitor of glutamine synthetase. This latter effect was hypothesized to be responsible for the increase in the incidence of some neuropathologies in humans, such as Alzheimer’s disease or Parkinson’s disease. In order to test this hypothesis, we chronically administered MSO to two inbred strains of mice, C57BL/6J and BALB/cJ, and analyzed possible alterations in learning and memory features of these mice. Mice were given 20 mg/kg of MSO three times a week for 10 weeks. Spatial learning capabilities assessed with a radial maze were not affected by the long-term MSO treatment, although activity was significantly decreased in BALB/cJ mice. Thus, our data suggest that long-term administration of nonconvulsive and nonglycogenic doses of MSO do not alter the spatial memory of mice. Our results do not support the hypothesis that chronic treatment with MSO influences hippocampus-dependent learning abilities in mice.

Resistance to l -methionine-S-sulfoximine in Chlamydomonas reinhardtii is due to an alteration in a general amino acid transport system

It was suggested that the mutant ARF1 of Chlamydomonas reinhardtii is resistant to l-methionine-S-sulfoximine (MSX, an irreversible inhibitor of glutamine synthetase, EC 6.3.1.2) because this strain degraded and utilized this compound as a nitrogen source for growth (A.R. Franco et al., 1996, Plant Physiol 110: 1215–1222). Resistance to MSX has now been characterized in a double mutant of this alga, called MPA1, which is resistant to MSX and lacks l-amino acid oxidase (LAO activity, EC 1.4.3.2). Biochemical and genetic evidence indicate that the mutant MPA1 is altered in the same MSX-resistance locus as mutant ARF1. However, mutant MPA1 neither degraded nor utilized MSX as a nitrogen source. This led us to conclude that (i) resistance to MSX is not linked to its utilization, and (ii) that LAO activity accounts for the degradation of MSX in mutant ARF1. Data indicate that C. reinhardtii possesses a broad-specificity carrier system responsible for the transport of arginine and other amino acids, including MSX. We propose that the alteration of this carrier confers resistance to MSX in mutants ARF1 and MPA1.

Differential response in damage and repair of wild-type Anacystis nidulans and its UV-B plus heat shock tolerant (UV-HS t) strain under UV-B and heat shock stress

Both ultraviolet-B (UV-B) and heat shock (HS) tolerant (UV-HS t) strain of the unicellular cyanobacterium Anacystis nidulans has been developed which showed remarkable growth under UV-B and heat shock (high temperature) compared to negligible growth of wild type cells under the conditions. In terms of oxygen production and Hill activity UV-HS t strain and wild type cells exhibited opposite responses at higher exposures to UV-B, heat shock or UV-HS beyond the normal condition. Photo-heat-shock-inactivation of the electron transport process occurred in both the strains beyond their respective photosynthesis-saturated normal light intensity. In wild type cells, both the oxidizing and reducing sides of the photosynthetic electron transport chain were found to be damaged by UV-HS exposure, unlike the UV-HS t strain in which only the oxidizing side of photosystem II (PS II) was inactivated. With the help of exogenous electron donors hydroxylamine (NH 2OH) and diphenyl carbazide (DPC), photo-heat-shock-inactivated sites were delineated. The wild type cells showed quick and fast recovery within 24 h after imposition of the normal light condition compared to the UV-HS t strain. The significant recovery in NH 2OH added wild type cells compared to DPC incubated cells suggested that only the reducing side of PS II could be repaired. The addition of L-methionine-DL-sufoximine (MSX), a glutamate analogue and irreversible inhibitor of glutamine synthetase (GS) inhibitor and protein synthesis inhibitors such as chloramphenicol (CAP) and rifampicin (RFM), suggested the vital role of nitrogen source and de novo protein synthesis in the regulation of the recovery process.

NO 2 ? Efflux and its regulation in cyanobacterium Nostoc MAC

NO2−efflux and its regulation have been studied in the cyanobacterium Nostoc MAC. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU), carbonyl cyanide-m-chlorophenyl hydrazone (CCCP), sodium azide, p-chloromercuribenzoate (PCMB), and dicyclohexylcarbodiimide (DCCD), a specific inhibitor of bacterial ATPase, inhibited the NO2−efflux activity singificantly. No NO2−efflux activity was observed under dark-aerobic as well as under dark-anaerobic conditions; however, the addition of ATP resulted in NO2−efflux even under dark-aerobic condition. Maximum NO2−efflux activity was observed when NO3−served as the sole nitrogen source. However, NH4+ions inhibited the NO2−efflux activity when both NO3−and NH4+wer simulatneously available to the cells. The NO2−efflux was freed from NH4+repression by l-methionine-dl-sulfoximine (MSX), an irreversible inhibitor of glutamine synthetase (GS). Chloramphenicol, a protein synthesis inhibitor, inhibited the derepression of NO2−efflux system when NH4+-incubated cells were transferred to NO3−medium. Tungstate-treated cells lacking functional NO3−reductase but having NO3−uptake activity also lacked NO2−efflux activity. These results suggest that (i) NO2−efflux in Nostoc MAC is NO3−dependent and an energy-dependent process that can be regulated at the levels of NO3−uptake and NO3−reductase; (ii) NO2−efflux system is NH4+repressible; however, the product of NH4+assimilation via GS is being required for repression to occur; (iii) de novo protein synthesis is required for derepression of the NO2−efflux system; and (iv) the catalytic activity of NO2−reductase also seems to play an important role in the regulation of NO2−efflux system.

Excised Tassel-Seed Tunicate (Ts-5 Tu) Kernels as a Model In vitro System for Studying Amino Acid Metabolism in Developing Maize Seeds

Summary Previous studies suggest that nitrogenous transport compounds may be metabolized in the pedicel (basal maternal tissue) of the developing maize ( Zea mays L.) kernel. In this report, excised tassel-seed tunicate ( Ts-5 Tu ) kernels were tested as a model in vitro kernel system to study the metabolic fate of [ l4 C]aspartate within the pedicel and the endosperm. Glume-covered kernels of Ts-5 Tu maize are born on individual elongated stalks and can be removed from the parent plant without damage to the basal kernel tissues. Radiolabeled aspartate supplied to excised Ts-5 Tu kernels in a 30-min pulse was rapidly metabolized within the pedicel, with 60 % of the 14 C recovered in the acidic (organic-acid-containing) fraction at the beginning of the chase period. By 1 h into the chase period, both glutamine and glutamate were more heavily labeled than aspartate. In the endosperm, 60 % of the ethanol-soluble, aspartate-derived 14 C was recovered in the basic (amino acid-containing) fraction, 35% in the acidic fraction, and the remainder in the neutral (sugar-containing) fraction. Radioactivity in glutamate plus glutamine accounted for 70% of the 14 C contained in the basic fraction of the endosperm. Inclusion of 2 mM methionine sulfoximine, an irreversible inhibitor of glutamine synthetase (GS), had little effect on pedicel aspartate catabolism but did inhibit incorporation of aspartate-derived 14 C into glutamine in the pedicel, resulting in more 14 C being taken up by the endosperm as organic acids and less being incorporated into the prolamin fraction. It was concluded that the results obtained using Ts-5 Tu kernels incubated in vitro reflect more closely what occurs in the intact plant than those obtained with cob-borne kernels. The role of pedicel-amino-acid metabolism in maize-kernel nitrogen assimilation is also discussed.

Regulation of Nitrite Reductase Cellular Levels in the Cyanobiont Nostoc ANTH

Summary The cellular nitrite reductase activity in response to different nitrogen sources has been studied in the cyanobiont Nostoc ANTH isolated from the liverwort Anthoceros. De novo protein synthesis of nitrite reductase occurred even in the absence of added nitrogen source, although enzyme activity was higher when nitrite or nitrate served as the sole nitrogen source. In tungstate-treated cells, nitrate did not show any positive effect on nitrite reductase activity, suggesting that the stimulatory effect was not due to nitrate itself but it requires its reduction to nitrite. Thus, nitrite may be the actual inducer in this case. Ammonium-grown cells showed reduced level of nitrite reductase activity. The nitrite reductase activity was freed from ammonium repression by L-methionine-DL-sulphoximine (MSX), an irreversible inhibitor of glutamine synthetase (GS). Thus ammonium metabolism through GS is required for the ammonium repression of nitrite reductase to occur.

Glutamate metabolism in rat cortical astrocyte cultures.

Glutamate metabolism in rat cortical astrocyte cultures was studied to evaluate the relative rates of flux of glutamate carbon through oxidative pathways and through glutamine synthetase (GS). Rates of 14CO2 production from [1-14C]glutamate were determined, as was the metabolic fate of [14C(U)]glutamate in the presence and absence of the transaminase inhibitor aminooxyacetic acid and of methionine sulfoximine, an irreversible inhibitor of GS. The effects of subculturing and dibutyryl cyclic AMP treatment of astrocytes on these parameters were also examined. The vast majority of exogenously added glutamate was converted to glutamine and exported into the extracellular medium. Inhibition of GS led to a sustained and greatly elevated intracellular glutamate level, thereby demonstrating the predominance of this pathway in the astrocytic metabolism of glutamate. Nevertheless, there was some glutamate oxidation in the astrocyte culture, as evidenced by aspartate production and labeling of intracellular aspartate pools. Inhibition of aspartate aminotransferase caused a greater than 70% decrease in 14CO2 production from [1-14C]glutamate. Inhibition of GS caused an increase in aspartate production. It is concluded that transamination of glutamate rather than oxidative deamination catalyzed by glutamate dehydrogenase is the first step in the entry of glutamate carbon into the citric acid cycle in cultured astrocytes. This scheme of glutamate metabolism was not qualitatively altered by subculturing or by treatment of the cultures with dibutyryl cyclic AMP.

Regulation of Urease Cellular Levels in the Cyanobacteria Anacystis nidulans and Nostoc muscorum

Summary The effect of nitrogen source on the cellular activity level of urease has been examined in the cyanobacteria Anacystis nidulans and Nostoc muscorum. In both, the N2-fixing N. muscorum as well as non N2-fixing A. nidulans, urea did not behave as an nutritional inducer of urease. NH4+ promoted repression of urease was evident in both the cyanobacteria which was freed by Lmethionine, dl-sulphoximine (MSX), an irreversible inhibitor of glutamine synthetase. NH4, must therefore, be metabolised through glutamine synthetase before repressing the urease activity.

Effect of phosphinothricin (glufosinate) on photosynthesis and photorespiration of C3 and C4 plants

Phosphinothricin (glufosinate), an irreversible inhibitor of glutamine synthetase, causes an inhibition of photosynthesis in C3 (Sinapis alba) and C4 (Zea mays) plants under atmospheric conditions (400 ppm CO2, 21% O2). This photosynthesis inhibition is proceeding slower in C4 leaves. Under non-photorespiratory conditions (1000 ppm CO2, 2% O2) there is no inhibition of photosynthesis. The inhibition of glutamine synthetase by phosphinothricin results in an accumulation of NH4 (+). The NH4 (+)-accumulation is lower in C4 plants than in C3 plants. The inhibition of glutamine synthetase through phosphinothricin in mustard leaves results in a decrease in glutamine, glutamate, aspartate, asparagine, serine, and glycine. In contrast to this, a considerable increase in leucine and valine following phosphinothricin treatment is measured. With the addition of either glutamine, glutamate, aspartate, glycine or serine, photosynthesis inhibition by phosphinothricin can be reduced, although the NH4 (+)-accumulation is greatly increased. This indicates that NH4 (+)-accumulation cannot be the primary cause for photosynthesis inhibition by phosphinothricin. The investigations demonstrate the inhibition of transmination of glyoxylate to glycine in photorespiration through the total lack of amino donors. This could result in a glyoxylate accumulation inhibiting ribulose-1,5-bisphosphate-carboxylase and consequently CO2-fixation.

THE SYNTHESIS AND BIOLOGICAL ACTIVITY OF S-ALKYL PHOSPHOHOMOCYSTEINE SULFOXIMIDES

Abstract The phosphonate analogues of methionine and buthionine sulfoximide were synthesized by the action of NaN3 on the corresponding sulfoxides. Analogues 3 and 4 were prepared to determine the effect of altering the carboxyl portion of known inhibitors in the glutamate synthase cycle. Results of biological testing revealed that 3 and 4 did not posses the potency found with buthionine and methionine sulfoximide in cellular systems. However 3 was found to be an effective and irreversible inhibitor of glutamine synthetase.

The biosynthesis of tabtoxinine-beta-lactam. Use of specifically 13C-labeled glucose and 13C NMR spectroscopy to identify its biosynthetic precursors.

Tabtoxinine-beta-lactam, an irreversible inhibitor of glutamine synthetase is produced by several pathovars of Pseudomonas syringae. We have examined tabtoxinine-beta-lactam biosynthesis, an important and poorly characterized step in pathogenesis caused by this organism. We have identified the biosynthetic precursors of tabtoxinine-beta-lactam by incorporating 13C from specifically 13C-labeled D-glucose precursors and determining the labeling pattern using 13C NMR spectroscopy. Tabtoxinine-beta-lactam is generated by combining a 4-carbon fragment, a 2-carbon fragment, and a single carbon. The 4-carbon fragment arises from aspartic acid, and the 2-carbon unit is donated from carbons 2 and 3 of pyruvate. The 6-carbon backbone of tabtoxinine-beta-lactam arises from the condensation of fragments from aspartate and pyruvate, probably using reactions analogous to the initial steps in the pathway of lysine biosynthesis.

The Effect of Phosphinothricin (Glufosinate) on Photosynthesis I. Inhibition of Photosynthesis and Accumulation of Ammonia

Phosphinothricin (glufosinate) is an irreversible inhibitor of glutamine synthetase in plants. En­zyme inhibition results in substantial light-dependent accumulation of ammonia, inhibition of photosynthesis and ultimately the death of the plants. First of all the metabolic processes in which NH3 is liberated were established. Comparative investigations under photorespiratory and non- photorespiratory conditions with Sinapis alba plants revealed that about 60% of the accumulated NH3 derives from photorespiration. Trials with PPT and KNO3 showed that nitrate assimilation is insignificant as a source of NH3, and so the remaining 40% is accumulated in catabolic or anabolic processes. Under photorespiratory conditions CO2 fixation after treatment with 1 mᴍ PPT finally fell below the compensation point, and this was independent of the addition of nitrate. In non- photorespiratory conditions, by contrast, photosynthesis continued at a level of over 80% for several hours in entire plants and in severed leaves, although at the same time substantial amounts of ammonia had accumulated.

Inactivation of pea seed glutamine synthetase by the toxin, tabtoxinine-beta-lactam.

Glutamine synthetase of plants is the physiological target of tabtoxinine-beta-lactam, a toxin produced by several disease-causing pathovars of Pseudomonas syringae. This toxin, a unique amino acid, is an active site-directed, irreversible inhibitor of glutamine synthetase from pea. ATP is required for inactivation. Neither ADP, AMP, nor adenosine 5'-(beta,gamma-methylene)triphosphate (AMP-PCP) supports inactivation. Adenyl-5'-yl imidophosphate (AMP-PNP) is slowly hydrolyzed by glutamine synthetase to produce adenyl-5'-yl phosphoramidate (AMP-PN) and inorganic phosphate as identified by 31P NMR spectroscopic analysis. AMP-PNP also supports a slow inactivation of glutamine synthetase by tabtoxinine-beta-lactam. These data are consistent with gamma-phosphate transfer being involved in the inactivation. Completely inactivated glutamine synthetase has 0.9 mumol of toxin bound/mumol of subunit. One mumol of ATP is bound per mumol of subunit of glutamine synthetase in the absence of either the toxin or another active site-directed inhibitor, methionine sulfoximine; whereas, a 2nd mumol of either [alpha- or gamma-32P]ATP is bound per mumol of subunit when glutamine synthetase is incubated in the presence of either toxin or methionine sulfoximine until all enzyme activity is lost. These data suggest that the gamma-phosphate hydrolyzed from ATP during inactivation remains with the enzyme-inhibitor complex, as well as the ADP. The open chain form, tabtoxinine, was neither a reversible nor an irreversible inhibitor of glutamine synthetase, suggesting that the beta-lactam ring is necessary for inhibition. The inactivation of glutamine synthetase with tabtoxinine-beta-lactam is pseudo-first-order when done in buffer containing 15% (v/v) ethylene glycol. The rate constant for this reaction is 3 X 10(-2) S-1, and the Ki for the toxin is 1 mM. Removal of the ethylene glycol from the buffer allows the reaction to proceed in a non-first-order manner with the apparent rate constant decreasing with time. As the enzyme is inactivated in these conditions, the binding affinity for the toxin appears to decrease, while the Km observed for glutamate does not change.

Regulation of Nitrite Reductase in the Cyanobacterium Anacystis nidulans

Summary: The effect of the nitrogen source on the activity of ferredoxin-dependent nitrite reductase has been studied in the cyanobacterium Anacystis nidulans. De novo synthesis of nitrite reductase occurred in the absence of an added nitrogen source, although enzyme activity was higher when the medium contained either NO3 − or NO2 −. The positive effect of NO3 − on nitrite reductase was also evident in tungstate-treated A. nidulans, which lacked an active nitrate reductase, indicating that the stimulatory effect was due to NO3 − itself and not to the NO2 − resulting from its intracellular reduction. NH4 + acted as a repressor, overriding any positive effect of NO3 − or NO2 −. Nitrite reductase synthesis was freed from NH4 + repression by l-methionine-DL-sulphoximine, an irreversible inhibitor of glutamine synthetase. NH4 + must therefore be metabolized through glutamine synthetase before repressing nitrite reductase. The kinetics of nitrate reductase and nitrite reductase development were similar in cells transferred from NH4 +- to NO3 −-containing media, suggesting a coordinate regulation of synthesis.