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1.
Methionine sulfoximine (MSX), an irreversible inhibitor of glutamine synthetase of Rhodopseudomonas palustris restored nitrogenase activity to cells in which nitrogenase had been completely inhibited by ammonia switch-off. After addition of MSX, there was a lag period before nitrogenase activity was fully restored. During this lag, glutamine synthetase activity progressively decreased, and near the time of its complete inhibition, nitrogenase activity resumed. Nitrogenase switch-off by ammonia thus required active glutamine synthetase. Glutamine itself caused nitrogenase inhibition whose reversal by MSX depended on the relative ratio of MSX to glutamine. Unlike ammonia, glutamine inhibited nitrogenase under conditions where glutamine synthetase activity was absent. This indicates that glutamine is the effector molecule in nitrogenase switch-off, for instance by interacting with the enzymatic system for Fe protein inactivation. The effects of glutamine and MSX were also dependent on the culture age. Possible explanation for this and for the competitive effects are a common binding site within the regulatory apparatus for nitrogenase, or, in part, within a common transport system. Some observations with MSX were extended to Rhodopseudomonas capsulata and agreed with those in R. palustris. 相似文献
2.
Regulation of urease and ammonia assimilatory enzymes in Selenomonas ruminantium. 总被引:4,自引:3,他引:1
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Urease and glutamine synthetase activities in Selenomonas ruminantium strain D were highest in cells grown in ammonia-limited, linear-growth cultures or when certain compounds other than ammonia served as the nitrogen source and limited the growth rate in batch cultures. Glutamate dehydrogenase activity was highest during glucose (energy)-limited growth or when ammonia was not growth limiting. A positive correlation (R = 0.96) between glutamine synthetase and urease activities was observed for a variety of growth conditions, and both enzyme activities were simultaneously repressed when excess ammonia was added to ammonia-limited, linear-growth cultures. The glutamate analog methionine sulfoximine (MSX), inhibited glutamine synthetase activity in vitro, but glutamate dehydrogenase, glutamate synthase, and urease activities were not affected. The addition of MSX (0.1 to 100 mM) to cultures growing with 20 mM ammonia resulted in growth rate inhibition that was dependent upon the concentration of MSX and was overcome by glutamine addition. Urease activity in MSX-inhibited cultures was increased significantly, suggesting that ammonia was not the direct repressor of urease activity. In ammonia-limited, linear-growth cultures, MSX addition resulted in growth inhibition, a decrease in GS activity, and an increase in urease activity. These results are discussed with respect to the importance of glutamine synthetase and glutamate dehydrogenase for ammonia assimilation under different growth conditions and the relationship of these enzymes to urease. 相似文献
3.
Effect of methionine sulfoximine on asparaginase activity and ammonium levels in pea leaves 总被引:1,自引:0,他引:1
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In developing leaves of Pisum sativum the levels of ammonium did not change during the light-dark photoperiod even though asparaginase (EC 3.5.1.1) did; asparaginase activity in detached leaves doubled during the first 2.5 hours in the light. When these leaves were supplied with 1 millimolar methionine sulfoximine (MSX, an inhibitor of glutamine synthetase, GS, activity) at the beginning of the photoperiod, levels of ammonium increased 8-to 10-fold, GS activity was inhibited 95%, and the light-stimulated increase in asparaginase activity was completely prevented, and declined to less than initial levels. When high concentrations of ammonium were supplied to leaves, the light-stimulated increase of asparaginase was partially prevented. However, it was also possible to prevent asparaginase increase, in the absence of ammonium accumulation, by the addition of MSX together with aminooxyacetate (AOA, which inhibits transamination and some other reactions of photorespiratory nitrogen cycling). AOA alone did not prevent light-stimulated asparaginase increase; neither MSX, AOA, or elevated ammonium levels inhibited the activity of asparaginase in vitro. These results suggest that the effect of MSX on asparaginase increase is not due solely to interference with photorespiratory cycling (since AOA also prevents cycling, but has no effect alone), nor to the production of high ammonium concentration or its subsequent effect on photosynthetic mechanisms. MSX must have further inhibitory effects on metabolism. It is concluded that accumulation of ammonium in the presence of MSX may underestimate rates of ammonium turnover, since liberation of ammonium from systems such as asparaginase is reduced by the effects of MSX. 相似文献
4.
Takeshi Omasa Mitsugu Yamanaka Naoko Tanimura Yoshio Katakura Michimasa Kishimoto Ken-ichi Suga Shin Enosawa 《Enzyme and microbial technology》2004,35(6-7):519-524
To establish the ammonia-metabolizing cell lines for a bioartificial liver support system, CHO-K1 and HepG2 were transformed with pBK-CMV-GS vector that contains glutamine synthetase (gs) gene. The recombinant cell lines were selected under the various concentrations of glutamine synthetase inhibitor, methionine sulfoximine (MSX). The host CHO-K1 and HepG2 cell lines produces ammonia, but the both MSX tolerable CHO (GS-CHO) and HepG2 (GS-HepG2) cell lines endowed with the high GS activity could metabolize the ammonium from medium. The ammonia-metabolizing activity of CHO and HepG2 cell was about one-fourth of that of primary hepatocyte. 相似文献
5.
Tabtoxin is a chlorosis-inducing toxin produced by the plant pathogenic bacterium Pseudomonas syringae pv. tabaci. Previous studies have indicated that tabtoxin inhibits glutamine synthetase (EC 6.3.1.2) in vitro. We report here that tabtoxin also inhibits glutamine synthetase in vivo. The main evidence was that assimilation of exogenous 15 NH3 into Asparagus sprengeri protein was rapidly inhibited in isolated cells exposed to tabtoxin. This was associated with an equivalent decline in glutamine synthetase activity in extracts of these cells and the accumulation of extracellular ammonia. Glutamine synthetase was also inhibited in leaves of Nicotiana tabacum L. cv. White Burley treated with tabtoxin and the affected tissue accumulated ammonia and became chlorotic. However, the development of symptoms and accumulation of ammonia was suppressed when the leaves were held in air containing 1% CO2 to reduce photorespiration. This indicates that the chlorotic symptom did not result from the inhibition of nitrogen assimilation but was a consequence of the interruption of the photorespiratory nitrogen cycle. 相似文献
6.
Methionine sulfoximine (MSX), a glutamine synthetase inhibitor,suppressed the inhibitory effect of ammonia on nitrate uptakeby Anabaena cells in both the light and dark. In the light,MSX did not inhibit nitrate uptake in the absence of ammonia,but under dark conditions, MSX above 2 µM inhibited nitrateuptake. Nitrite uptake, which is not affected by ammonia ineither the light or the dark, was inhibited by MSX in the darkbut not in the light. (Received October 3, 1984; Accepted April 22, 1985) 相似文献
7.
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 总被引:3,自引:2,他引:1
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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. 相似文献
8.
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 相似文献
9.
Francisco Romero Francisco Javier Caballero Francisco Castillo José Manuel Roldán 《Archives of microbiology》1985,143(2):111-116
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 相似文献
10.
Leaves of 15 - 30-d-old plants of sunflower and jute were harvested at 10.00 or 23.00 (local time) and measured immediately,
or those harvested at 10.00 were incubated for one hour in sunlight either in water or 5 mM methionine sulfoximine (MSX) solution
and then for three hours in dark either in water or 15 mM KNO3 solution. Nitrate feeding during dark incubation, in general,
increased nitrate reductase (NR) and nitrite reductase (NiR) activities, and NADH and soluble sugar contents. Increase in
tissue nitrate concentration in MSX fed but not in control samples suggested reduction of nitrate in dark. NADPH-dependent
NR activity increased considerably upon feeding with nitrate in dark. Concomitantly, NADPH phosphatase activity was also increased
in nitrate treated, dark incubated leaves. It is proposed that nitrate regulates dark nitrate reduction by facilitating generation
of NADH from NADPH by NADPH phosphatase. High amounts of ammonia accumulated in MSX treated, but not in control leaves, upon
dark incubation. Relative activities of NR and NADPH phosphatase, and amounts of soluble sugar and NADH were low in MSX fed
samples compared to that of control. So, high amount of ammonia might partially repress NADPH phosphatase and consequently
deprive NR of reducing equivalents.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
11.
Carmelo Rigano Vittoria Di Martino Rigano Vincenza Vona Amodio Fuggi 《Archives of microbiology》1979,121(2):117-120
Addition ofl-methionine-dl-sulphoximine to cells ofCyanidium caldarium brings about a loss of glutamine synthetase activity. Concomitantly ammonia assimilation is prevented.Under physiological conditions nitrate reductase [NAD(P)H: nitrate oxidoreductase EC 1.6.6.2] is reversibly converted into an inactive enzyme upon addition of ammonia. In the presence of methionine sulphoximine, when glutamine synthetase activity is lost, nitrate reductase is no longer inactivated by ammonia. It is suggested that ammonia itself is not the actual effector of nitrate reductase inactivation.Concomitantly with the failure of nitrate reductase to undergo ammonia-inactivation, in the presence of methionine sulphoximine nitrate reduction is an uncontrolled process, thus, in media with nitrate ammonia continues to be produced and excreted into the external medium at a constant rate.Abbreviations NR
Nitrate reductase
- GS
Glutamine synthetase
- GOGAT
Glutamate syntase
- MSX
l-methionine-dl-sulphoximine 相似文献
12.
Illumination of a colorless mutant of Chlorella vulgaris 1lh(M125) with blue light enhanced both the uptake of nitrate andthe release of ammonia. These effects were not observed underillumination with red light. The release of ammonia was alsoenhanced by the addition of methionine sulphoximine (MSX), aninhibitor of glutamine synthetase (GS). Addition of MSX to culturesin the dark increased the rate of breakdown of starch. Algal cells grown in nitrate-containing medium did not showthe aminating activity of glutamate dehydrogenase (GDH). Additionof large (millimolar) amounts of ammonia in the dark resultedin the induction of NADPH-GDH activity and, in addition, a decreasein GS activity. From these results it appears that GS catalyzesthe primary step in the assimilation of ammonia in algal cellsgrown in nitrate-containing medium. Two isoforms (GS1 and GS2)of GS have been separated by ion exchange chromatography. Theactivities of both isoforms were decreased upon the additionof ammonia. Illumination of the alga with blue light at intensities up to10,000 mW m2 enhanced the measurable activity of GS invitro, while higher intensities were ineffective. In red lightno such effect was observed. The effects of blue light and ammonia on nitrogen metabolismin algal cells are discussed. (Received November 25, 1988; Accepted March 6, 1989) 相似文献
13.
Activity of key nitrogen assimilating enzymes was studied in developing grains of high-lysine opaque sorghum P-721 and normal
sorghum CSV-5. The higher percentage of protein in opaque sorghum was mainly due to lower starch content since protein per
grain was less than in CSV-5. During grain development, albufn and globulin decreased while prolafne and glutelin increased.
Prolafne content in CSV-5 was higher than in opaque sorghum. Average nitrate reductase activity in flag and long leaf were
similar in both the varieties. The nitrate reductase activity decreased during grain development. Glutamate dehydrogenase
activity was higher during early development and lower at later stages in opaque sorghum than in CSV-5. Glutamate oxaloacetate
transaminase activity was higher and glutamine synthetase lower in opaque sorghum than in CSV-5 grains during development.
Glutamate synthase activity was higher in opaque sorghum up to day 20 and lower thereafter than in CSV-5. It is suggested
that reduced activities of glutamine synthetase as well as glutamate synthase in opaque sorghum as compared to CSV-5 during
later stages of development may restrict protein accumulation in the former. 相似文献
14.
A spontaneous double mutant of Chlamydomonas reinhardtii, designated ARF3, was resistant to L-methionine-S-sulfoximine (MSX), lacked chloroplastic glutamine synthetase (GS2) activity,
and grew very poorly in all media tested. In segregants obtained after genetic crosses, the poor-growth phenotype was always
linked to the lack of GS2 and to a diminished rate of consumption of ammonium, even under conditions where photorespiration
was minimized. The ammonium permeases in mutant ARF3, however, were not altered. This indicates that, unlike in higher plants,
GS2 contributes substantially to the primary assimilation of ammonia in this alga, and that its function cannot be replaced
by the cytosolic glutamine synthetase. In genetic crosses, the MSX resistance and the lack of GS2 segregated independently,
indicating that resistance was not due to an altered form of GS2.
Received: 5 June 1998 / Accepted: 10 September 1998 相似文献
15.
A strain of Chlamydomonas reinhardtii, named ARF-1, which grows with the glutamine synthetase (GS) inhibitor L-methionine-S-sulfoximine (MSX), has been isolated and characterized. Mutant ARF-1 is affected at a single and dominant gene, tentatively assigned to the allele msr-1-2. Neither the uptake of ammonia nor the two GS isoenzyme activities of the mutant were affected by MSX in vivo. GS activities, however, were fully abolished in vitro, thus suggesting that neither GS isoform was an altered enzyme resistant to the inhibitor. Resistance to MSX does not seem to be due to either a defect in a permease responsible for the transport of MSX or over-expression of GS activity, nor did we find an alternative enzymatic pathway for the assimilation of ammonium. Resistance was independent of the nitrogen source used and was strongly enhanced by the addition of acetate. Unlike the parental strain, mutant ARF-1 can degrade and utilize MSX as the sole nitrogen source for growth, which could account for the observed resistance. Thus, this mutant can be classified as a novel type of MSX-resistant mutant. This mutant can also use phosphinothricin, methionine sulfone, or methionine sulfoxide as the sole sources of nitrogen. This capability cosegregated in the genetic crosses and was also observed in all the diploids isolated. An MSX/[alpha]-ketoglutarate aminotransferase activity, not present in the parental strain 305, was detected in mutant ARF-1 cells. Therefore, we propose that the locus msr-1-2 either codes for this transaminase activity or its product gene is necessary to express this transaminase activity. 相似文献
16.
Y. J. Wu T. Z. Yang Y. Y. Song X. Q. Zhang S. X. Xu G. Xue X. X. Xing 《Biologia Plantarum》2016,60(1):190-194
In order to reveal the character of ammonia emission in senescent tobacco (Nicotiana tabacum), the content of NH4+, total nitrogen, and soluble protein, and the activities of nitrogen metabolism-related enzymes were measured in leaves of a quick-leaf-senescence phenotype ZY90 and a slow-leaf-senescence phenotype NC89. Compared with NC89, ZY90 had a higher NH4+ accumulation, a lower glutamine synthetase activity, and a significantly higher stomatal ammonia compensation point, and ammonia emission during 40 to 60 d after leaf emergence. During senescence, the quick-leafsenescence phenotype was characterized by nitrogen re-transfer by ammonia emmission, whereas the slow-leafsenescence phenotype by nitrogen re-assimilation. The ammonia emission was primarily regulated by glutamine synthetase activity, apoplastic pH, and NH4+ content. 相似文献
17.
Summary. Accumulation of amino acids was studied in rice roots of 3-day-old seedlings subjected for 48 h to anaerobic conditions.
Alanine and Gaba were the main amino acids accumulated under anoxia. Their synthesis was strongly inhibited by MSX and AZA,
inhibitors of glutamine synthetase and glutamate synthase. These activities increased after 8 h of anaerobic treatment and,
by immunoprecipitation of 35S-labeled proteins, it was shown that glutamine synthetase and ferredoxin-dependent glutamate synthase were synthesized during
the treatment. These findings indicate that the glutamine synthetase/glutamate synthase cycle play an important role in anaerobic
amino acid accumulation.
Received April 5, 1999 相似文献
18.
本文测定了浑球红假单胞菌(Rhodobacter sphaeroides)菌株601谷氨酰胺合成酶(GS)、谷氨酸合酶(GOGAT)、谷氨酸脱氢酶(GDH)和丙氨酸脱氢酶(ADH)的活性。低氨时,GS/GOGAT活力高,GDH活力低,高氨时,GS/GOGAT活力低,GDH活力高。在以分子氮或低浓度氨为氮源的培养条件下,加入GS抑制刑MSX(L—methionine—DL—sulphoximine),细菌生长受到抑制。但是,生长在以谷氨酸为氮源的细菌则不受影响。上述结果表明,浑球红假单胞菌菌株601氨同化是通过GS/GOGAT途径和GDH途径。 相似文献
19.
Surendra Singh 《FEMS microbiology letters》1992,98(1-3):245-248
Abstract Glutamine uptake in the cyanobiont Nostoc ANTH was energy-dependent and repressed in ammonia-grown cells. l -Methionine- dl -sulphoximine (MSX), a glutamate analogue and an inhibitor of glutamine synthetase (GS), did not affect glutamine uptake whereas azaserine, an inhibitor of glutamate synthase (GOGAT) did, suggesting that GS activity is not necessarily involved in the glutamine uptake system and that increased intracellular glutamine level regulates its own uptake. Repression of glutamine uptake by ammonia did not require de novo protein synthesis but required GS activity, suggesting that ammonia itself was not the repressor signal. The derepression of the glutamine uptake system did not require GS activity but required de novo protein synthesis. 相似文献
20.
Inhibition of Photosynthesis in Barley with Decreased Levels of Chloroplastic Glutamine Synthetase Activity 总被引:9,自引:4,他引:5
Mutant barley plants containing only 8%, 16% or 38% of the wildtype level of glutamine synthetase activity have been isolated.The level of glutamine synthetase activity in the roots of themutant containing only 8% leaf activity was not affected bythis mutation. The plants accumulated high levels of ammoniain leaves exposed to air and although they were able to carryout photosynthetic CO2fixation normally at low levels of atmosphericO2, they were unable to maintain wild type rates of CO2fixationin air. The extent of this inhibition and the extent to whichammonia accumulated in the leaves was dependent on the photonfluence rate intercepted by the plant. When leaves from themutant plant were fed glutamine under non-photorespiratory conditionsfor 40 min before they were transferred to air, the plants exhibitedwild type rates of CO2 fixation in air but the ammonia contentof the leaves increased to an even higher level. At least inthe short term, therefore, ammonia accumulation was not responsiblefor the dramatic decline in the fixation rate of these mutantsin air. The most probable explanation is that as the supplyof potential amino donors diminished on transfer to air, therewas a restriction on the return of glycerate to the Calvin cyclewithin the chloroplast. Key words: Ammonia toxicity, photorespiration, photosynthesis, GS-deficient barley 相似文献