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1.
Glutamate synthase (E.C. 1.4.1.14) (GOGAT) activity was not detectable in L3 Haemonchus contortus, but was present in L3 Teladorsagia circumcincta and adult worms of both species. GOGAT activity was inhibited by 80% by azaserine. Activity (nmol min−1 mg−1 protein) was 33–59 in adult H. contortus, 51–91 in adult T. circumcincta and 24–41 in L3 T. circumcincta, probably depending on exposure to ammonia, as incubation with 1 mM NH4Cl doubled GOGAT activity. The pH optimum was 7.5 in both species. Either NAD or NADP acted as co-factor. The mean apparent Km for 2-oxoglutarate was 0.7 (0.5–0.9) mM and for glutamine was 1.0 (0.5–1.7) mM for different homogenates. There was no detectable activity in whole parasite homogenates of glutamate decarboxylase (E.C. 4.1.1.15) or succinic semialdehyde dehydrogenase (E.C. 1.2.1.24), the first and third enzymes of the GABA shunt, respectively, suggesting that the GABA shunt is not important in general metabolism in these species. 相似文献
2.
The ornithine urea cycle, polyamine synthesis, nitric oxide synthesis and metabolism of arginine to putrescine have been investigated in L3 and adult Haemonchus contortus and Teladorsagia circumcincta. Neither parasite had a detectable arginine deiminase/dihydrolase pathway nor a functional ornithine urea cycle. Nitric oxide synthase was present in central and peripheral nerves, but was not detected in whole parasite homogenates. Both arginase (E.C. 3.5.3.1) and agmatinase (E.C. 3.5.3.11) activities were present in both species. Arginase did not require added Mn2+ and had an optimal pH of 8.5. Polyamine metabolism differed in the two species and from that in mammals. Ornithine decarboxylase (E.C. 4.1.1.17) was present in both parasites, but no arginine decarboxylase (E.C. 4.1.1.19) activity was detected in T. circumcincta. The flexibility of synthesis of putrescine in H. contortus may make this pathway less useful as a target for parasite control than in T. circumcincta, in which only the ornithine decarboxylase pathway was detected. 相似文献
3.
da Rocha IM Vitorello VA Silva JS Ferreira-Silva SL Viégas RA Silva EN Silveira JA 《Journal of plant physiology》2012,169(1):41-49
The role of the δ-ornithine amino transferase (OAT) pathway in proline synthesis is still controversial and was assessed in leaves of cashew plants subjected to salinity. The activities of enzymes and the concentrations of metabolites involved in proline synthesis were examined in parallel with the capacity of exogenous ornithine and glutamate to induce proline accumulation. Proline accumulation was best correlated with OAT activity, which increased 4-fold and was paralleled by NADH oxidation coupled to the activities of OAT and Δ1-pyrroline-5-carboxylate reductase (P5CR), demonstrating the potential of proline synthesis via OAT/P5C. Overall, the activities of GS, GOGAT and aminating GDH remained practically unchanged under salinity. The activity of P5CR did not respond to NaCl whereas Δ1-pyrroline-5-carboxylate dehydrogenase was sharply repressed by salinity. We suggest that if the export of P5C from the mitochondria to the cytosol is possible, its subsequent conversion to proline by P5CR may be important. In a time-course experiment, proline accumulation was associated with disturbances in amino acid metabolism as indicated by large increases in the concentrations of ammonia, free amino acids, glutamine, arginine and ornithine. Conversely, glutamate concentrations increased moderately and only within the first 24 h. Exogenous feeding of ornithine as a precursor was very effective in inducing proline accumulation in intact plants and leaf discs, in which proline concentrations were several times higher than glutamate-fed or salt-treated plants. Our data suggest that proline accumulation might be a consequence of salt-induced increase in N recycling, resulting in increased levels of ornithine and other metabolites involved with proline synthesis and OAT activity. Under these metabolic circumstances the OAT pathway might contribute significantly to proline accumulation in salt-stressed cashew leaves. 相似文献
4.
Mohamed SA Mohamed TM Fahmy AS El-Badry MO Abdel-Gany SS 《Experimental parasitology》2008,118(1):47-53
Ornithine aminotransferase (OAT), proline oxidase (PO), Delta 1-pyrroline-5-carboxylate reductase (P5CR), and Delta 1-pyrroline-5-carboxylate dehydrogenase (P5CD) were assessed in Fasciola gigantica. All enzymes are involved in the conversion of ornithine into glutamate and proline. High levels of P5CD suggest that the direction of the metabolic flow from ornithine is more toward glutamate than proline. F. gigantica P5CD1 and P5CD2 were separated from the majority of contaminating proteins in crude homogenate using a CM-cellulose column. A Sephacryl S-200 column was employed for P5CD2 to obtain pure enzyme with increased specific activity. The molecular mass of P5CD2 was estimated to be 50kDa using a Sephacryl S-200 column and SDS-PAGE. It migrated as a single band on SDS-PAGE, indicating a monomeric enzyme. P5CD2 had Km values of 1.44mM and 0.37mM for NAD and P5C, respectively. P5CD2 oxidized a number of aliphatic and aromatic aldehydes, where the aromatic compounds had higher affinity toward the enzyme. All amino acids examined had partial inhibitory effects on the enzyme. While 3mM AMP caused 31% activation of enzyme, 3mM ADP and ATP inhibited activity by 18% and 23%, respectively. Apart from Cu2+, the divalent cations that were studied caused partial inhibitory effects on the enzyme. 相似文献
5.
Umair S Knight JS Patchett ML Bland RJ Simpson HV 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2012,161(3):255-260
The expression of glutamate dehydrogenase (GDH; EC 1.4.1.3) in L3 of the nematode Haemonchus contortus was confirmed by detecting GDH mRNA, contrary to earlier reports. The enzyme was active in both L3 and adult H. contortus homogenates either with NAD+/H or NADP+/H as co-factor. Although it was a dual co-factor GDH, activity was greater with NAD+/H than with NADP+/H. The rate of the aminating reaction (glutamate formation) was approximately three times higher than for the deaminating reaction (glutamate utilisation). GDH provides a pathway for ammonia assimilation, although the affinity for ammonia was low. Allosteric regulation by GTP, ATP and ADP of L3 and adult H. contortus and Teladorsagia circumcincta (Nematoda) GDH depended on the concentration of the regulators and the direction of the reaction. The effects of each nucleotide were qualitatively similar on the mammalian and parasite GDH, although the nematode enzymes were more responsive to activation by ADP and ATP and less inhibited by GTP under optimum assay condition. GTP inhibited deamination and low concentrations of ADP and ATP stimulated weakly. In the reverse direction, GTP was strongly inhibitory and ADP and ATP activated the enzyme. 相似文献
6.
Asish Kumar Parida Vipin S. Dagaonkar Manoj S. Phalak Laxman P. Aurangabadkar 《Acta Physiologiae Plantarum》2008,30(5):619-627
The relative water content (RWC), free proline levels and the activities of enzymes involved in proline metabolism were studied
in drought tolerant (Ca/H 680) and drought sensitive (Ca/H 148) genotypes of cotton (Gossypium hirsutum L.) during induction of water stress and posterior recovery. Water stress caused a significant increase in proline levels
and P5CS activity in leaves of both tolerant and sensitive genotypes, whereas the activity of P5CR increased minimally and
the activity of OAT remains unchanged. The activity of PDH decreased under drought stress in both the genotypes. The leaf
of tolerant genotype maintained higher RWC, photosynthetic activity and proline levels, as well as higher P5CS and P5CR activities
under water stress than that of drought sensitive genotype. The drought induced proline levels and activities of P5CS and
P5CR declined and tend to be equal to their respective controls, during recovery, whereas the PDH activity tends to increase.
These results indicate that induction of proline levels by up regulation of P5CS and down regulation of PDH may be involved
in the development of drought tolerance in cotton. 相似文献
7.
We have developed two new continuous coupled assays for ornithine-δ-aminotransferase (OAT) that are more sensitive than previous methods, measure activity in real time, and can be carried out in multiwell plates for convenience and high throughput. The first assay is based on the reduction of Δ1-pyrroline-5-carboxylate (P5C), generated from ornithine by OAT, using human pyrroline 5-carboxylate reductase 1 (PYCR1), which results in the concomitant oxidation of NADH (nicotinamide adenine dinucleotide, reduced form) to NAD+ (nicotinamide adenine dinucleotide, oxidized form). This procedure was found to be three times more sensitive than previous methods and is suitable for the study of small molecules as inhibitors or inactivators of OAT or as a method to determine OAT activity in unknown samples. The second method involves the detection of l-glutamate, produced during the regeneration of the cofactor pyridoxal 5’-phosphate (PLP) of OAT by an unamplified modification of the commercially available Amplex Red l-glutamate detection kit (Life Technologies). This assay is recommended for the determination of the substrate activity of small molecules against OAT; measuring the transformation of l-ornithine at high concentrations by this assay is complicated by the fact that it also acts as a substrate for the l-glutamate oxidase (GluOx) reporter enzyme. 相似文献
8.
The effects of salt stress were studied on the accumulation and metabolism of proline and its correlation with Na+ and K+ content in shoots and callus tissue of four potato cultivars, viz., Agria, Kennebec (relatively salt tolerant), Diamant and Ajax (relatively salt sensitive). Na+ and proline contents increased in all cultivars under salt stress. However, K+ and protein contents decreased in response to NaCl treatments. The activities of enzymes involved in proline metabolism,
Δ1-pyrroline-5-carboxylate synthetase (P5CS) and proline dehydrogenase (ProDH) increased and decreased, respectively, in response
to elevated NaCl concentrations. The changes of P5CS and ProDH activities in more salt sensitive cultivars (Diamant, Ajax)
were more than those in the tolerant ones. Then the stimulation of synthesis in combination with a partially increase of protein
proteolysis, a decrease in proline utilization and inhibition of oxidation resulted in high proline contents in seedlings
and calli under salt stress. In callus tissue, reduced growth and cell size may be partially responsible for high proline
accumulation in response to high NaCl levels. However, although the basic proline contents in the seedlings of more salt tolerant
cultivars were higher than the sensitive ones, a clear relationship was not generally observed between accumulation of proline
and salt tolerance in potato. 相似文献
9.
A full length cDNA encoding glutamate dehydrogenase was cloned from Teladorsagia circumcincta (TcGDH). The TcGDH cDNA (1614 bp) encoded a 538 amino acid protein. The predicted amino acid sequence showed 96% and 93% similarity with Haemonchus contortus and Caenorhabditis elegans GDH, respectively. A soluble N-terminal 6xHis-tagged GDH protein was expressed in the recombinant Escherichia coli strain BL21 (DE3) pGroESL, purified and characterised. The recombinant TcGDH had similar kinetic properties to those of the enzyme in homogenates of T. circumcincta, including greater activity in the aminating than deaminating reaction. Addition of 1 mM ADP and ATP increased activity about 3-fold in the deaminating reaction, but had no effect in the reverse direction. TcGDH was a dual co-factor enzyme that operated both with NAD+ and NADP+, GDH activity was greater in the deaminating reaction with NADP+ as co-factor and more with NADH in the aminating reaction. 相似文献
10.
δ1-Pyrroline-5-carboxylate (P5C) dehydrogenase (EC 1.5.1.12) activity was measured in extracts from cultured tobacco (Nicotiana plumbaginifolia Viviani) cells. Two putative isozymes were resolved by anion-exchange fast protein liquid chromatography. These enzyme forms
showed different patterns of expression during the culture growth cycle: activity-I increased in exponentially growing cells
and declined rapidly in late logarithmic phase, while activity-II was found at substantial level only in cells which were
entering the stationary phase. Both P5C dehydrogenases were partially purified and characterized with respect to kinetic and
biochemical properties. They showed similar molecular masses as judged from retention patterns upon gel-filtration chromatography.
The in vitro activity of both enzymes had a broad maximum around pH 7.4, and was progressively inhibited by Cl− at concentrations ranging from 0.1 to 1 M. A pronounced difference was found between their apparent K
m values for the two substrates, P5C and NAD+, the higher affinities being shown by activity-I. Regulation of P5C dehydrogenase during salt-stress-induced proline accumulation
was investigated. Following the addition of 175 mM NaCl to the culture medium the level of activity-I was substantially unaffected,
while the specific activity of the other isozyme failed to increase even after the onset of the stationary phase of growth.
Possible roles for P5C dehydrogenase isozymes in proline and arginine metabolism are discussed.
Received: 23 May 1996 / Accepted: 18 December 1996 相似文献
11.
Pooja Bhatnagar-Mathur Vincent Vadez M. Jyostna Devi M. Lavanya G. Vani Kiran K. Sharma 《Molecular breeding : new strategies in plant improvement》2009,23(4):591-606
Abiotic stresses including water deficit severely limits crop yields in the semi-arid tropics. In chickpea, annual losses
of over 3.7 million tones have been estimated to be due to water deficit conditions alone. Therefore, major efforts are needed
to improve its tolerance to water deficit, and genetic engineering approaches provide an increasing hope for this possibility.
We have used transgenic technology for the introduction of an osmoregulatory gene P5CSF129A encoding the mutagenized Δ1-pyrroline-5-carboxylate synthetase (P5CS) for the overproduction of proline. A total of 49 transgenic events of chickpea were produced with the 35S:P5CSF129A gene
through Agrobacterium tumefaciens-mediated gene transfer through the use of axillary meristem explants. Eleven transgenic events that accumulated high proline
(2–6 folds) were further evaluated in greenhouse experiments based on their transpiration efficiency (TE), photosynthetic
activity, stomatal conductance, and root length under water stress. Almost all the transgenic events showed a decline in transpiration
at lower values of the fraction of transpirable soil water (dryer soil), and extracted more water than their untransformed
parents. The accumulation of proline in the selected events was more pronounced that increased significantly in the leaves
when exposed to water stress. However, the overexpression of P5CSF129A gene resulted only in a modest increase in TE, thereby indicating that the enhanced proline had little bearing on the components
of yield architecture that are significant in overcoming the negative effects of drought stress in chickpea. 相似文献
12.
Rajendra P. Kandpal C. S. Vaidyanathan M. Udaya Kumar K. S. Krishna Sastry N. Appaji Rao 《Journal of biosciences》1981,3(4):361-370
Free proline content in Ragi (Eleusine coracana) leaves increased markedly (6 to 85 fold) as the degree of water stress, created by polyethylene gylcol treatment, was prolonged
There was also a marginal increase in soluble proteins in the stressed leaves as compared to that in the controls. Water stress
stimulated the activities of ornithine aminotransferase and pyrroline-5-carboxylate reductase, the enzymes of proline biosynthesis
and markedly inhibited the enzymes involved in proline degradation viz., proline oxidase and pyrroline-5-carboxylate dehydrogenase.
These results suggest that increase in free proline content of Ragi leaves could be due to enhanced activities of the enzymes
synthesizing proline but more importantly due to severe inhibition of the enzymes degrading proline. These observations establish
for the first time, the pathway of proline metabolism in plants by way of detection of the activities of all the enzymes involved
and also highlight the role of these enzymes in proline accumulation during water stress. 相似文献
13.
14.
The role of ornithine aminotransferase in fruiting body formation of the mushroom Agaricus bisporus 总被引:1,自引:1,他引:0
Matthijs J.M. Wagemaker Daniel C. Eastwood Jelle Welagen Chris van der Drift Mike S.M. Jetten Kerry Burton Leo J.L.D. Van Griensven Huub J.M. Op den Camp 《Mycological Research》2007,111(8):909-918
15.
《Bioscience, biotechnology, and biochemistry》2013,77(3):622-627
The enzyme involved in the reduction of Δ 1-piperideine-6-carboxylate (P6C) to L-pipecolic acid (L-PA) has never been identified. We found that Escherichia coli JM109 transformed with the lat gene encoding L-lysine 6-aminotransferase (LAT) converted L-lysine (L-Lys) to L-PA. This suggested that there is a gene encoding “P6C reductase” that catalyzes the reduction of P6C to L-PA in the genome of E. coli. The complementation experiment of proC32 in E. coli RK4904 for L-PA production clearly shows that the expression of both lat and proC is essential for the biotransformation of L-Lys to L-PA. Further, We showed that both LAT and pyrroline-5-carboxylate (P5C) reductase, the product of proC, were needed to convert L-Lys to L-PA in vitro. These results demonstrate that P5C reductase catalyzes the reduction of P6C to L-PA. Biotransformation of L-Lys to L-PA using lat-expressing E. coli BL21 was done and L-PA was accumulated in the medium to reach at an amount of 3.9 g/l after 159 h of cultivation. It is noteworthy that the ee-value of the produced pipecolic acid was 100%. 相似文献
16.
Esteban Snchez Luis Ramn Lpez-Lefebre Pablo Carlos García Rosa María Rivero Juan Manuel Ruiz Luis Romero 《Journal of plant physiology》2001,158(5)
The objective of the present work was to determine what impact extremely high nitrogen dosages would have on proline metabolism in order to use this amino acid as a bioindicator of N status of green bean plants (Phaseolus vulgaris L. cv. Strike). In this effort, we identified the most favourable pathway of proline synthesis under our experimental conditions. The N was applied to the nutrient solution in the form of NH4NO3 at 5.4 mmol/L (N1, optimal level), 11.6 mmol/L (N2), 17.4 mmol/L (N3), and 23.2 mmol/L (N4). Our results indicate that the application of high N dosages inPhaseolus is characterized by the accumulation of NO3−, NH4+ and proline in root and foliar organs. However, although the enzymes in charge of proline biosynthesis, ornithine-δ-aminotransferase (OAT, EC 2.6.1.13) and Δ1-pyrroline-5-carboxylate synthetase (P5CS, EC 2.7.2.11/1.2.2.41) vary in behaviour depending on the N status, in our experiment, this amino acid appears to be synthesized mainly by the enzyme ornithine-δ-aminotransferase. This suggests predominance of the ornithine pathway over the glutamine pathway. Finally, under our experimental conditions, proline can be defined as a good indicator of N excess of green bean plants. 相似文献
17.
S. G. Thompson P. T.-H. Wong S. F. Leong† E. G. McGeer 《Journal of neurochemistry》1985,45(6):1791-1796
A possible alternative route for production of a small glutamate pool in brain is from proline or ornithine to 1-pyrroline-5-carboxylate (P5C) and thence to glutamate. The conversion from ornithine to P5C is catalyzed by ornithine delta-aminotransferase (OrnT) whereas that from proline is catalyzed by proline oxidase (PrO). The conversion of P5C to glutamate is catalyzed by 1-pyrroline-5-carboxylate dehydrogenase (PDH). Biochemical assays of PDH and PrO in various rat brain regions indicate no positive correlation between the two enzymes nor between either activity and high-affinity glutamate uptake or the regional distribution of OrnT. We have localized PDH and PrO histochemically by modifications of the Van Gelder [J. Neurochem. 12, 231-237, (1965)] method for gamma-aminobutyric acid (GABA) transaminase. The enzymes were found only in certain types of glial cells; the best stained were the Bergmann glial cells of the cerebellum but, for PDH, there was also good staining of astrocytes in the dentate area of the hippocampus. Since both these areas are believed to have heavy glutamate innervation and numerous GABA interneurons, these findings may reflect an alternative route of glutamate production in glial cells near some glutamate and/or GABA tracts but they do not support this as a possible route for glutamate formation in most brain regions. The findings do, however, provide further evidence for chemical specialization of glial cells. 相似文献
18.
Inagaki E Ohshima N Takahashi H Kuroishi C Yokoyama S Tahirov TH 《Journal of molecular biology》2006,362(3):490-501
Delta(1)-pyrroline-5-carboxylate dehydrogenase (P5CDh) plays an important role in the metabolic pathway from proline to glutamate. It irreversibly catalyzes the oxidation of glutamate-gamma-semialdehyde, the product of the non-enzymatic hydrolysis of Delta(1)-pyrroline-5-carboxylate, into glutamate with the reduction of NAD(+) into NADH. We have confirmed the P5CDh activity of the Thermus thermophilus protein TT0033 (TtP5CDh), and determined the crystal structure of the enzyme in the ligand-free form at 1.4 A resolution. To investigate the structural basis of TtP5CDh function, the TtP5CDh structures with NAD(+), with NADH, and with its product glutamate were determined at 1.8 A, 1.9 A, and 1.4 A resolution, respectively. The solved structures suggest an overall view of the P5CDh catalytic mechanism and provide insights into the P5CDh deficiencies in the case of the human type II hyperprolinemia. 相似文献
19.
20.
Summary In this article we review recent work on the physiology of proline and 1-pyrroline-5-carboxylate (P5C) in living organisms and consider recent progress in our understanding of the role of P5C synthetase in collagen metabolism and the regulation of urea cycle in vertebrates. Much of this recent progress has been made possible by advances in our knowledge of the enzymes and genes involved in proline biosynthesis in man. The availability of well characterized P5C synthetase deficiency in man has been an impetus for the cloning of the cDNA encoding for this enzyme from man and facilitated the establishment of the phenotype-genotype relationships in P5C synthetase deficiency in higher vertebrates.Abbreviations GK
-glutamyl kinase
- GPR
-glutamyl phosphate reductase
- P5CR
1-pyrroline-5-carboxylate reductase
- GSA
glutamic--semialdehyde
- P5C
1-pyrroline-5-carboxylate
- P1
Inorganic phosphate
- AMP, ADP, ATP
Adenosine 5-mono-, di-, triphosphate
- NAD+, NADH
nicotinamide adenine dinucleotide, and its reduced form
- NADP+, NADPH
nicotinamide adenine dinucleotide phosphate, and its reduced form; DEAF: diethylaminoethyle
- OAT
ornithine amino transferase; CHO: Chinese hamster ovary
- IGF-1
insulin-like growth factor-1
- P5CDH
pyrroline 5carboxylate dehydrogenase
- IMP
inosine 5-monophosphate 相似文献