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1.
Glutamine synthetase (EC 6.3.1.2) was purified to homogeneity from a free-living nitrogen fixing bacteria, Bacillus polymyxa. The holoenzyme, relative molecular mass (Mr) of 600 000 is composed of monomeric sub-units of 60 000 (Mr). The isoelectric point of the sub-units was 5.2. The pH optimum for the biosynthetic and transferase enzyme activity was 8.2 and 7.8, respectively. The apparent K
m values (K
m
app
) in the biosynthetic reaction for glutamate, NH4Cl and ATP were 3.2, 0.22 and 1 mM, respectively. In the transferase reaction the K
m values for glutamine, hydroxylamine and ADP were 6.5, 3.5 and 8×10-4 mM respectively. L-Methionine-D-L-sulfoximine was a very potent inhibitor in both biosynthetic and transferase reactions. Similar to most Gram positive bacteria there was no evidence of in vivo adenylylation and the enzyme seemed to be mainly regulated by feed-back mechanism.Abbreviations PMSF
phenylmethylsulfonylfluoride
- TCA
trichloroacetic acid
- GS
glutamine synthetase
- MSO
L-Methionine-D-L-sulfoximine
- SDS-PAGE
sodium dodecyl sulfatepolyacrylamide gel electrophoresis
- SVPDE
snake venum phosphodiesterase 相似文献
2.
The activity of glutamine synthetase (GS) was investigated during culture development of Bacillus polymyxa CN 2219 and its asporogenous mutant deficient in protease production. At 28°C, temperature permissive for sporulation, the glutamine synthetase activity was found to decline in the wild type cells which acquire the competence for sporulation. This decline was not observed in the asporogenous mutant. Incubation at 37°C (temperature non permissive) suppressed sporulation in the wild type and maintained glutamine synthetase activity. The involvement of glutamine synthetase in the repression of sporulation was further confirmied by the action of l-methionine sulfoximine a specific inhibitor of glutamine synthetase, which overcomes the catabolite repression by ammonium and induces sporulation. Intracellular proteases were measured as early markers of the initiation of sporulation and were found to be induced during sporulation.Abbreviations GS
glutamine synthetase
- MSO
l-methionine sulfoximine
- GYS
glucose-yeast extract-salts
- GT
-glutamyltransferase
- PMSF
phenylmethylsulfonylfluoride 相似文献
3.
No active uptake of ammonium was detected in Proteus vulgaris, Bacillus pasteurii, and Sporosarcina ureae, which indicates that these bacteria depend on the passive diffusion of ammonia across the cell membrane. In P. vulgaris the glutamine synthetase-glutamate synthase (GS-GOGAT) pathway and glutamate dehydrogenase (GDH) were present, and these enzymes exhibited high affinities for ammonium. In B. pasteurii and S. ureae, however, no GS activity was detected, and GOGAT activity was only present in S. ureae. GDH enzymes were present in these two organisms, but showed only low affinity for ammonium, with apparent K
m-values of 55.2 mM in B. pasteurii and 36.7 mM in S. ureae, repectively. These observations explain why P. vulgaris is able to grow at neutral pH and low ammonium concentration (2 mM), while B. pasteurii and S. ureae require high ammonium concentration (40 mM) and alkaline pH for growth.Non-standard abbreviations GS
glutamine synthetase
- GOGAT
glutamate synthase
- GDH
glutamate dehydrogenase
- GT
glutamyl transferase
- MA
methylammonium
- NB
nutrient broth
- YE
yeast extract
- NA
nocotinic acid 相似文献
4.
Rhodopseudomonas acidophila strain 7050 assimilated ammonia via a constitutive glutamine synthetase/glutamate synthase enzyme system.Glutamine synthetase had a K
m
for NH
4
+
of 0.38 mM whilst the nicotinamide adenine dinucleotide linked glutamate synthase had a K
m
for glutamine of 0.55 mM. R. acidophila utilized only a limited range of amino acids as sole nitrogen sources: l-alanine, glutamine and asparagine. The bacterium did not grow on glutamate as sole nitrogen source and lacked glutamate dehydrogenase. When R. acidophila was grown on l-alanine as the sole nitrogen source in the absence of N2 low levels of a nicotinamide adenine dinucleotide linked l-alanine dehydrogenase were produced. It is concluded, therefore, that this reaction was not a significant route of ammonia assimilation in this bacterium except when glutamine synthetase was inhibited by methionine sulphoximine. In l-alanine grown cells the presence of an active alanine-glyoxylate aminotransferase and, on occasions, low levels of an alanine-oxaloacetate aminotransferase were detected. Alanine-2-oxo-glutarate aminotransferase could not be demonstrated in this bacterium.Abreviations ADH
alanine dehydrogenase
- GDH
glutamate dehydrogenase
- GS
glutamine synthetase
- GOGAT
glutamate synthase
- MSO
methionine sulphoximine 相似文献
5.
The activities of nitrate reductase (EC1.6.6.1), nitrite reductase (EC 1.6.6.4), glutamine synthetase (EC6.3.1.2), glutamate synthase (EC1.4.7.1) and NAD(P)H-dependent glutamate dehydrogenase (EC 1.4.1.3) were investigated in mesophyll and bundle sheath cells of maize leaves (Zea mays L.). Whereas nitrate and nitrite reductase appear to be restricted to the mesophyll and GDH to the bundle sheath, glutamine synthetase and glutamate synthase are active in both tissues.During the greening process, the activities of nitrate and nitrite reductase increased markedly, but glutamine synthetase, glutamate synthase and glutamate dehydrogenase changed little.Abbreviations BDH
British Drug Houses
- EDTA
Ethylene diamine tetra-acetic acid
- GDH
Glutamate dehydrogenase
- NADH
Nicotinamide-adenine dinucleotide reduced form
- NADPH
Nicotnamide-adenine dinucleotide phosphate reduced form
- PMSF
Phenylmethyl sulphonyl fluoride 相似文献
6.
The Km for ammonia for glutamine synthetase and glutamate dehydrogenase was measured in enzyme extracts from Skeletonema costatum (Grev.) Cleve. At similar physiological pH and temperature the half-saturation constant for glutamine synthetase was 29 μM, whereas for GDH it was 28mM. On the basis of relative enzymic activity, as well as substrate affinity, it is suggested that glutamine synthetase is the enzyme primarily responsible for the incorporation of ammonium into the amino acid pool, when extracellular nitrogen is at ecological concentrations. 相似文献
7.
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
8.
Julie V. Cullimore 《Planta》1981,152(6):587-591
A 70% reduction in glutamine synthetase (GS) activity was observed within 5 min when 5 mM NH3 and darkness was applied to steady-state cells of Chlamydomonas utilising NO3. The enzyme was reactivated in vivo by reillumination of the culture and in vitro by treatment with thiol reagents. The activity modulations affected the synthetase and transferase activities similarly and were not influenced by protein synthesis inhibitors. Deactivation of GS was also observed when steady-state cells were treated with an uncoupler of phosphorylation, carbonylcyanide m-chlorophenylhydrazone (CCCP) or inhibitors of the electron transport chain but under these conditions the activity modulation affected over 90% of the activity and was irreversible. The mechanism of the physiological deactivation of GS is discussed in relation to both the in vivo and in vitro findings.Abbreviations GS
glutamine synthetase (EC 6.3.1.2.)
- GSs
glutamine synthetase, synthetase activity
- GSt
glutamine synthetase, transferase activity
- CAP
chloramphenicol
- CCCP
carbonylcyanide m-chlorophenyl hydrazone
- CHX
cycloheximide
- DCMU
3-(3,4-dichlorophenyl)-1,1-dimethyl urea
- DSPD
disalicylidene propanediamine
- DTT
dithiothreitol
- GSH
reduced glutathione 相似文献
9.
Zwittermicin A (ZwA) is a novel, broad-spectrum linear aminopolyol antibiotic produced by some Bacillus cereus and Bacillus thuringiensis. However, only part of its biosynthesis cluster has been identified and characterized from B. cereus UW85. To better understand the biosynthesis cluster of ZwA, a bacterial artificial chromosome (BAC) library of B. thuringiensis subsp. kurstaki strain YBT-1520, a ZwA-producing strain, was constructed. Two BAC clones, 1F8 and 5E2, were obtained by PCR, which overlap
the known ZwA biosynthesis cluster of B. cereus UW85. This ZwA biosynthesis cluster is at least 38.6 kb and is located on the chromosome, instead of the plasmid. Partial
DNA sequencing revealed both BAC clones carry three new ZwA biosynthesis-related genes, zwa6, zwa5A and zwa5B, which were found at the corresponding location of B. cereus UW85. Putative amino acid sequences of these genes shown that ZWA6 is homologous to a typical carbamoyltransferase from Streptomyces avermitilis, while ZWA5A and ZWA5B are homologs of cysteine synthetase and ornithine cyclodeaminase which jointly synthesize 2,3-diaminopropionate
in the viomycin biosynthesis pathway, respectively. The identification of these three genes further supports the hypothesized
ZwA biosynthesis pathway. 相似文献
10.
Birgitta Bergman 《Archives of microbiology》1984,137(1):21-25
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 相似文献
11.
The control of glutamine synthetase level in Lemna minor L. 总被引:1,自引:1,他引:0
Summary The specific activity of glutamine synthetase (E.C. 6.3.1.2) of Lemna minor L. is markedly reduced when either ammonium ions or glutamine are present in the growth medium. Combinations of 5 mM ammonia and 5 mM glutamic acid or 5 mM ammonia and 5 mM glutamine as nitrogen source, lead to a 4–5 fold reduction of the maximum activity measurable on 5 mM -aminobutyric acid. Analyses of the soluble pool of nitrogen indicate that the reduction in enzyme level is associated with an increase in the pool of glutamine. There is an inverse correlation between the apparent rate of synthesis of glutamine synthetase and the intracellular concentration of glutamine, and this relationship suggests that the glutamine synthetase of Lemna minor is subject to end product repression by the endogenous pool of glutamine. 相似文献
12.
The activity of glutamine synthetase fromAspergillus niger was significantly lowered under conditions of citric acid fermentation. The intracellular pH of the organism as determined
by bromophenol blue dye distribution and fluorescein diacetate uptake methods was relatively constant between 6·0–6·5, when
the pH of the external medium was varied between 2·3–7·0.Aspergillus niger glutamine synthetase was rapidly inactivated under acidic pH conditions and Mn2+ ions partially protected the enzyme against this inactivation. Mn2+-dependent glutamine synthetase activity was higher at acidic pH (6·0) compared to Mg2+-supported activity. While the concentration of Mg2+ required to optimally activate glutamine synthetase at pH 6·0 was very high (≥ 50 mM), Mn2+ was effective at 4 mM. Higher concentrations of Mn2+ were inhibitory. The inhibition of both Mn2+ and Mg2+-dependent reactions by citrate, 2-oxoglutarate and ATP were probably due to their ability to chelate divalent ions rather
than as regulatory molecules. This suggestion was supported by the observation that a metal ion chelator, EDTA also produced
similar effects. Of the end-products of the pathway, only histidine, carbamyl phosphate, AMP and ADP inhibitedAspergillus niger glutamine synthetase. The inhibitions were more pronounced when Mn2+ was the metal ion activator and greater inhibition was observed at lower pH values. These results permit us to postulate
that glutamine synthesis may be markedly inhibited when the fungus is grown under conditions suitable for citric acid production
and this block may result in delinking carbon and nitrogen metabolism leading to acidogenesis 相似文献
13.
Francisco Romero Antonio López-Ruiz Jean-Pierre Verbelen José Manuel Roldán 《Archives of microbiology》1988,149(4):330-334
Intracellular localization of glutamine synthetase has been studied by immunochemical techniques with cryosections and London Resin sections of Rhodobacter capsulatus E1F1 and Rhodopseudomonas acidophila. For immunostaining, sections were sequentially incubated with monospecific anti-glutamine synthetase antibodies (R. capsulatus) and gold labelled goat anti-rabbit antibodies. Gold label was present in the cytoplasm but not in the cell walls. The antigen is not associated with the cell membrane or with photosynthetic vesicle whether these are round and randomly distributed (R. capsulatus) or flattened and organized in well defined stacks (R. acidophila). Our results also indicate that glutamine synthetase is absent from the central, nucleoid part of the cell. The enzyme is present in dense cytoplasmic patches, which appear to be RNA-ribosome-containing areas.Abbreviations GS
glutamine synthetase
- LR
London Resin White 相似文献
14.
C. van der Drift R. A. M. M. Smits G. A. M. Michiels H. J. M. Op den Camp 《Archives of microbiology》1986,146(3):292-294
Bacillus fastidiosus was able to grow on glycerol as a carbon source when allantoin or urate was used as nitrogen source. The primary assimilatory enzyme for glycerol was glycerol kinase; glycerol dehydrogenase could not be detected. The glycerol kinase activity was increased 30-fold in allantoin/glycerol-grown cells as compared to alantoin-grown cells. Under both growth conditions high levels of glutamate dehydrogenase were found. Glutamine synthetase and glutamate synthase activities could not be demonstrated, while low levels of alanine dehydrogenase were present. It is concluded that B. fastidiosus assimilates ammonia by the NADP-dependent glutamate dehydrogenase.Abbreviations GS
glutamine synthetase
- GOGAT
glutamate synthase
- GDH
glutamate dehydrogenase
- ADH
alanine dehydrogenase 相似文献
15.
M. J. Hasmann P. H. Seidl G. Engelhardt K. H. Schleifer 《Archives of microbiology》1986,146(3):275-279
The raw extracts of a series of microorganisms were screened for the presence of acetyl-coenzyme A: arylamine N-acetyltransferase (AAAT) using a radioactive assay with 3H-acetyl-coenzyme A and aniline as substrates. Enzyme activities were primarily detected in the soluble fractions of Bacillus and Nocardia species, and in some further soil organisms. Only strains of Bacillus cereus were able to acetylate 4-nitroaniline and 3,5-dimethyl-4-nitroaniline. The fermentation conditions for the production of the enzyme were optimized. The AAAT from one strain of Bacillus cereus was purified 24-fold and characterized.Abbreviations AAAT
acetyl-coenzyme A: arylamine N-acetyltransferase
- AcP
acetylphosphate
- CoA
coenzyme A
- EDTA
ethylenediaminetetra-acetic acid
- PTA
phosphotransacetylase 相似文献
16.
L. Turbanti M. Bazzicalupo E. Casalone R. Fani E. Gallori M. Polsinelli 《Archives of microbiology》1988,150(5):421-425
One hundred and twenty-nine mutants of Azospirillum brasilense strain Sp6, resistant to methylammonium, were isolated. Three of the mutants were found to be able to reduce acetylene in the presence of 4 mM ammonium or 120mM methylammonium, concentrations which strongly reduced the nitrogenase activity of the parental strain. Under N2-fixing conditions, two mutants failed to switch off nitrogenase when NH4Cl was added. Moreover, the three mutants showed a reduced capacity to incorporate [14C]methylammonium. The level of glutamine synthetase activity found in the mutants was not reduced as compared to that of the parental strain. All of the data indicate an impairement in the mechanism of ammonium uptake by the bacterial cell.Abbreviations MEA
Methylammonium
- MSP
minimal medium (ammonium free)
- PY
complete medium
- GS
glutamine synthetase 相似文献
17.
Investigating the effect of patulin,penicillic acid and EDTA on biofilm formation of isolates from dental unit water lines 总被引:1,自引:0,他引:1
Liaqat I Bachmann RT Sabri AN Edyvean RG Biggs CA 《Applied microbiology and biotechnology》2008,81(2):349-358
This study investigated the effect of patulin and penicillic acid, two known quorum-sensing inhibitors, and the common biocide
ethylenediaminetetraacetic acid (EDTA) on the biofilm formation and auto-inducer (AI)-2 production of three isolates from
dental unit water lines, Klebsiella sp., Bacillus subtilis and Bacillus cereus. Penicillic acid on its own had no effect on the biofilm formation of all isolates, whereas in combination with EDTA, it
enhanced biofilm formation significantly in Klebsiella sp. and B. cereus. EDTA at concentrations greater than 10 μM promoted biofilm formation in B. cereus and B. subtilis. Patulin was found to promote biofilm formation in B. cereus up to 25 μM. A significant increase in biofilm formation was observed in B. cereus and B. subtilis at concentrations greater than 10 μM of patulin when combined with EDTA. The Vibrio harveyi BB170 AI-2 bioassay showed a positive response for Klebsiella sp. AI-2 production with a maximum fold induction at the late exponential growth phase. Addition of glucose prolonged the
AI-2 production phase considerably. No significant effect of patulin, penicillic acid alone as well as in combination with
EDTA was observed on AI-2 production by Klebsiella sp. The findings have important implications for the design of biofilm prevention and eradication strategies.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
18.
Various enzymes involved in the initial metabolic pathway for ammonia assimilation by Methanobacterium ivanovii were examined. M. ivanovii showed significant activity of glutamine synthetase (GS). Glutamate synthase (GOGAT) and alanine dehydrogenase (ADH) were present, wheras, glutamate dehydrogenase (GDH) was not detected. When M. ivanovii was grown with different levels of NH
+
4
(i.e. 2, 20 or 200 mM), GS, GOGAT and ADH activities varied in response to NH
+
4
concentration. ADH was not detected at 2 mM level, but its activity increased with increased levels of NH
+
4
in the medium. Both GS and GOGAT activities increased with decreasing concentrations of NH
+
4
and were maximum when ammonia was limiting, suggesting that at low NH
+
4
levels, GS and GOGAT are responsible for ammonia assimilation and at higher NH
+
4
levels, ADH might play a role. Metabolic mutants of M. ivanovii that were auxotrophic for glutamine were obtained and analyzed for GS activity. Results indicate two categories of mutants: i) GS-deficient auxotrophic mutants and ii) GS-impaired auxotrophic mutants.Abbreviations GS
Glutamine synthetase
- GOGAT
glutamate synthase
- GDH
glutamate dehydrogenase
- ADH
alanine dehydrogenase 相似文献
19.
Durban MA Silbersack J Schweder T Schauer F Bornscheuer UT 《Applied microbiology and biotechnology》2007,74(3):634-639
Twenty-two Bacillus cereus strains were screened for phospholipase C (PLC, EC 3.1.4.3) activity using p-nitrophenyl phosphorylcholine as a substrate. Two strains (B. cereus SBUG 318 and SBUG 516) showed high activity at elevated temperatures (>70°C) at acidic pH (pH 3.5–6) and were selected for
cloning and functional expression using Bacillus subtilis. The genes were amplified from B. cereus DNA using primers based on a known PLC sequence and cloned into the expression vector pMSE3 followed by transformation into
B. subtilis WB800. On the amino acid level, one protein (PLC318) was identical to a PLC described from B. cereus, whereas PLC516 contained an amino acid substitution (E173D). PLC production using the recombinant strains was performed
by an acetoin-controlled expression system. For PLC516, 13.7 U g−1 wet cell weight was determined in the culture supernatant after 30 h cultivation time. Three purification steps resulted
in pure PLC516 with a specific activity of 13,190 U mg−1 protein. 相似文献
20.
Alexander Ya. Strongin Lara S. Izotova Zakhar T. Abramov Lidia M. Ermakova Dmitrii I. Gorodetsky Valentin M. Stepanov 《Archives of microbiology》1978,119(3):287-293
While about 80% of the cell-bound intracellular serine protease of Bacillus subtilis A-50 have been recovered in the soluble fraction upon disruption of cells, the rest of the enzyme was found to be associated with the membrane fraction. Soluble cytoplasmic intracellular serine protease, as well as membrane-bound serine protease liberated by nonionic detergent treatment, have been isolated in a pure state and shown to be identical. The same protease might also be found extracellularly, due presumably to cell lysis or altered membrane permeability. Intracellular serine protease of Bacillus subtilis A-50 was clearly related to Bacillus subtilis serine proteases W1 and bacillopeptidase F described as extracellular enzymes.Abbreviations ISP
intracellular serine protease
- ISP-A-Bsu A-50 and ISP-B-Bsu A-50
molecular forms A and B of B. subtilis A-50 intracellular serine protease, respectively
- SDS
sodium dodecyl sulfate
- PMSF
phenylmethyl sulfonylfluoride
- pNA
p-nitroanilide
- Buffer A
50 mM Tris-(hydroxymethyl)aminomethane-1 mM CaCl2 adjusted to pH 8.5 with HCl 相似文献