Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.7.1.2 (nitrate reductase)
3,861 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In most cases the apparent target size obtained by radiation inactivation analysis corresponds to the subunit size or to the size of a multimeric complex. In this report, we examined whether the larger than expected target sizes of some enzymes could be due to secondary effects of free radicals. To test this proposal we carried out radiation inactivation analysis on Escherichia coli DNA polymerase I, Torula yeast glucose-6-phosphate dehydrogenase, Chlorella vulgaris nitrate reductase, and chicken liver sulfite oxidase in the presence and absence of free radical scavengers (benzoic acid and mannitol). In the presence of free radical scavengers, inactivation curves are shifted toward higher radiation doses. Plots of scavenger concentration versus enzyme activity showed that the protective effect of benzoic acid reached a maximum at 25 mM then declined. Mannitol alone had little effect, but appeared to broaden the maximum protective range of benzoic acid relative to concentration. The apparent target size of the polymerase activity of DNA polymerase I in the presence of free radical scavengers was about 40% of that observed in the absence of these agents. This is considerably less than the minimum polypeptide size and may reflect the actual size of the polymerase functional domain. Similar effects, but of lesser magnitude, were observed for glucose-6-phosphate dehydrogenase, nitrate reductase, and sulfite oxidase. These results suggest that secondary damage due to free radicals generated in the local environment as a result of ionizing radiation can influence the apparent target size obtained by this method.
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PMID:Radiation inactivation analysis of enzymes. Effect of free radical scavengers on apparent target sizes. 329 56

An assay for the simultaneous measurement of nitrite and nitrate, products of nitric oxide metabolism, is described. Others have reported pretreating sample by using nitrate reductase (NR) and NADPH to reduce endogenous NO3- before assaying the resultant NO2- using the Griess reaction. However, we found that the NADP+ formed during pretreatment interfered with the Griess reaction when NADPH was used at concentrations necessary to drive the NR reaction. For instance, 500 microM NADP+ in 100 microM NaNO3- (without NR) causes a 90% interference with the formation of Griess reaction product. To limit interference, we modified the method by decreasing the NADPH concentration to 1 microM. NADPH was regenerated by coupling the NR reaction with that catalyzed by glucose-6-phosphate dehydrogenase (GD). Using this method, NaNO3- standard curves were linear up to 100 microM and coincided with control curves obtained using NaNO2- incubated in parallel. Addition of urine up to a strength of 20% did not interfere with the assay. Comparison with an alternative assay based on cadmium reduction resulted in the following linear regression: [Cd method] = 0.915*[NR-GD method] + 0.37, r2 = 0.997. Coupling GD to NR to recycle NADPH allows this cofactor to be used at a low concentration so that interference with the Griess reaction is negligible.
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PMID:Sample pretreatment with nitrate reductase and glucose-6-phosphate dehydrogenase quantitatively reduces nitrate while avoiding interference by NADP+ when the Griess reaction is used to assay for nitrite. 773 51

Nitrate reductase (NaR) catalyses the reduction of nitrate to nitrite via a two-electron transfer. In fungi, the electron donor for NaR is NADPH whereas plants can have two enzymes, NADH:NaR and a bispecific NAD(P)H:NaR. PCR mutagenesis was employed to introduce mutations into the niaD gene of Aspergillus nidulans in order to identify residues involved in co-enzyme specificity. The niaD3000 mutation (NiaD T813D, K814Q) altered co-enzyme specificity: the new enzyme had high levels of NADH:NaR activity in vitro, whilst all NADPH-associated activity was lost. However, strains carrying this mutation did not grow on nitrate. Enzyme assays suggested that this was not due to inhibition of the mutant enzyme by NADPH. All revertants of the niaD3000 mutants had restored NADPH activity and lost NADH activity. Sequence analysis of these revertants showed that they all contained a single amino acid change at Asp-813, suggesting that this position is crucial to co-enzyme specificity. Further studies have shown that the mutant enzyme was not protected from deactivation by either co-factor in cell-free extracts (unlike the wild-type), and that induction of the glucose-6-phosphate dehydrogenase occurred independently of NADPH levels. These data highlight the importance of functional tests in vivo under physiological conditions.
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PMID:Structure-function analysis of NADPH:nitrate reductase from Aspergillus nidulans: analysis of altered pyridine nucleotide specificity in vivo. 1084 18

The nature of the association between nitrate reductase (NR) and membranes was examined. Nitrate reductase activity (NRA) associated with the microsomal fraction of barley (Hordeum vulgare L.) roots amounted to 0.6 to 0.8% of soluble NRA following sonication in the presence of 250 mM KI and repeated osmotic shock. This treatment removed all contaminating soluble NRA from microsomes of uninduced barley roots that had been homogenized in a soluble extract from roots of NO3(-)-induced plants. On continuous sucrose gradients, NRA co-migrated specifically with VO4(-)-sensitive ATPase activity, a plasma membrane (PM) marker; activity of glucose-6-phosphate dehydrogenase, assayed as cytosolic marker, co-migrated with NRA. Microsomal NRA was absent in barley deficient in soluble NR. Perturbation and trypsinolysis experiments with PM vesicles isolated by aqueous two-phase partitioning indicated that NR is associated with the periphery of the cytoplasmic face of the bilayer. These results demonstrate that PM and soluble NRs are essentially the same protein but that the membrane-associated form is tightly bound. Although it is possible that PM-associated NR exists in vivo, unequivocal evidence for this has yet to be shown. However, PM NR is definitely present in vitro.
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PMID:Characterization of the association of nitrate reductase with barley (Hordeum vulgare L.) root membranes. 1153

Accumulation of proline in response to toxic heavy metal exposure seems to be wide-spread among plants. To elucidate the role for proline in plant responses to heavy metal stress, we studied the effect of proline on Cd-induced and Zn-induced inhibition of glucose-6-phosphate dehydrogenase (G-6-PDH; EC 1.1.1.49) and nitrate reductase (NR; EC 1.6.6.2) in vitro. Proline appeared to protect both enzymes against Zn and, though less effectively, against Cd. Measurements with a Cd(2+)-specific electrode strongly suggested that this protection was based on a reduction of the free metal ion activity in the assay buffer, due to the formation of metal-proline complexes. There were no indications of any significant role for proline-water or proline-protein interactions. The significance of these findings with regard to heavy metal-induced proline accumulation in vivo is discussed.
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PMID:In vitro alleviation of heavy metal-induced enzyme inhibition by proline. 1171 Oct 61

The Al-tolerant cultivar TAM202 and the Al-sensitive cultivar TAM 105 of winter wheat (Triticum aestivum L.) were exposed to 0, 50, 75, 100 or 150 microM of Al. The absorption of Al by wheat, the growth of root, several key enzymes concerned with C, N and P metabolism, as well as key constituents of antioxidant system, were investigated. The results showed that TAM105 absorbed more Al than TAM202 and its root growth (presented by the length) was inhibited more severely. The root growth was most closely related to mononuclear Al (Ala) activity. The metabolic enzymes (presented by glucose-6-phosphate dehydrogenase, nitrate reductase and acid phosphatase) in TAM202 were Al-tolerant. Presented by superoxide dimutase (SOD) and the content of reduced glutathione (GSH) and malondialdehyde (MDA), antioxidant system in TAM202 indicated lower oxidative stress and greater ability to protect the cultivar.
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PMID:Effects of aluminum on physiological metabolism and antioxidant system of wheat (Triticum aestivum L.). 1199 40

Physiological alterations and regulation of heterocyst and nitrogenase formation have been studied in Het(-) Fix(-) mutant strain of diazotrophic cyanobacterium Anabaena variabilis. Het(-) Fix(-) mutant strain of A. variabilis has been isolated by N-methyl-N'-nitro-N"-nitrosoguanidine (NTG) mutagenesis and was screened with the penicillin enrichment (500 microg ml(-1)). Growth, heterocyst differentiation, nitrogenase and glutamine synthetase (biosynthetic and transferase), (14)CO(2)-fixation, nitrate reductase (NR), nitrite reductase (NiR), glucose-6-phosphate dehydrogenase (G6PDH), and isocitrate dehydrogenase (IDH) activities, and NO(3)(-), NO(2)(-), and NH(4)(+) uptake and whole cell protein profile in different metabolic conditions were studied in the Het(-) Fix(-) mutant strain taking wild-type A. variabilis as reference. Het(-) Fix(-) mutant strain was incapable of assimilating elemental nitrogen (N(2)) due to its inability to form heterocysts and nitrogenase and this was the reason for its inability to grow in BG-11(0) medium (free from combined nitrogen). In contrast, wild-type strain grew reasonably well in the absence of combined nitrogen sources and also showed heterocyst differentiation (8.5%) and nitrogenase activity (10.8 etamol C(2)H(4) formed microg(-1) Chl a h(-1)) in N(2)-medium. Wild-type strain also exhibited higher NR, NiR, and GS activities compared to its Het(-) Fix(-) mutant strain, which may presumably be due to acquisition of high uptake of NO(3)(-), NO(2)(-), and NH(2)(+). Wild-type strain in contrast to its Het(-) Fix(-) mutant strain also exhibited high level of G6PDH, IDH, and (14)CO(2) fixation activities. Low levels of G6PDH and IDH activities in Het(-) Fix(-) mutant strain further confirmed the lack of heterocyst differentiation and nitrogenase activity in the Het(-) Fix(-) mutant strain.NR, NiR, and GS activities in both the strains were energy-dependent and the energy required is mainly derived from photophosphorylation. Furthermore, it was found that de novo protein synthesis is necessarily required for the activities of NR, NiR, and GS in both wild-type and its Het(-) Fix(-) mutant strain.
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PMID:Physiological alterations and regulation of heterocyst and nitrogenase formation in Het(-) Fix(-) mutant strain of Anabaena variabilis. 1223 60

Total pyridine nucleotide concentration of root tissue for young soybean (Glycine max var. Bansei) and sunflower (Helianthus annuus L. var. Mammoth Russian) plants is the same with either ammonium or nitrate, but nitrate results in an increased proportion of total oxidized plus reduced NADP (NADP[H]) seemingly at the expense of NAD. The activity of NADH- and NADPH-dependent forms of glutamic acid dehydrogenase is correlated with the ratio of total oxidized plus reduced NAD to NADP(H). The low NAD: NADH ratio maintained in nitrate roots despite active NADH utilization via nitrate reductase and glutamic acid dehydrogenase may be the result of nitrate-stimulated glycolysis. Nitrate roots also maintain a high level of NADPH, presumably by the stimulatory effect of nitrate utilization on glucose-6-phosphate dehydrogenase activity. In the presence of nitrate rather than ammonium, the highly active nitrate-reducing leaves of soybean show a greater proportion of total pyridine nucleotide in the form of NADP(H) than do the inactive leaves of sunflower.For all tissues examined, ammonium nutrition yields a higher concentration of total adenine nucleotide than is found with nitrate. The data indicate the production of a higher level of metabolites that enter into purine synthesis with ammonium than with nitrate. Glutamine synthetase activity can be correlated with the concept that enzymes utilizing ATP for biosynthetic purposes increase in activity in accordance with the energy level of the cell.
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PMID:Influence of ammonium and nitrate nutrition on the pyridine and adenine nucleotides of soybean and sunflower. 1665 13

Nitric oxide (NO) produced from NO synthase(s) (NOS) is an important cell signaling molecule in physiology and pathophysiology. It remains challenging, however, to measure NO accurately and reproducibly in many cell types producing relatively low levels of NO from the enzymes such as endothelial NO synthase (eNOS). In the present study, we describe a very sensitive and convenient analytical method that affords measurement of 1 to 2 nM concentration of NO(x) (nitrite plus nitrate) in culture media. In the present study, we used an ultra-sensitive NO-selective electrochemical sensor (AmiNO700) in combination with a highly efficient nitrate conversion method, which coupled the nitrate reductase step with the glucose-6-phosphate dehydrogenase system. An aliquot of conditioned culture media was first treated with nitrate reductase, NADPH, glucose-6-phosphate dehydrogenase and glucose-6-phosphate to convert nitrate to nitrite quantitatively. The nitrite (that is present originally plus the reduced nitrate) was then reduced to equimolar NO in an acidic iodide bath while NO was being detected by the sensor. With this analytical method, we can quantitatively and reliably measure basal and stimulated NO release from cultured endothelial cells. We believe this improved assay should be useful in measuring a wide range of NO levels, especially the low but physiologically relevant levels, in many cell types.
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PMID:An improved method to measure nitrate/nitrite with an NO-selective electrochemical sensor. 1705 88

The pivotal role of glucose-6-phosphate dehydrogenase (G-6-PDH)-mediated nitric oxide (NO) production in the tolerance to oxidative stress induced by 100 mM NaCl in red kidney bean (Phaseolus vulgaris) roots was investigated. The results show that the G-6-PDH activity was enhanced rapidly in the presence of NaCl and reached a maximum at 100 mM. Western blot analysis indicated that the increase of G-6-PDH activity in the red kidney bean roots under 100 mM NaCl was mainly due to the increased content of the G-6-PDH protein. NO production and nitrate reductase (NR) activity were also induced by 100 mM NaCl. The NO production was reduced by NaN(3) (an NR inhibitor), but not affected by N(omega)-nitro-L-arginine (L-NNA) (an NOS inhibitor). Application of 2.5 mM Na(3)PO(4), an inhibitor of G-6-PDH, blocked the increase of G-6-PDH and NR activity, as well as NO production in red kidney bean roots under 100 mM NaCl. The activities of antioxidant enzymes in red kidney bean roots increased in the presence of 100 mM NaCl or sodium nitroprusside (SNP), an NO donor. The increased activities of all antioxidant enzymes tested at 100 mM NaCl were completely inhibited by 2.5 mM Na(3)PO(4). Based on these results, we conclude that G-6-PDH plays a pivotal role in NR-dependent NO production, and in establishing tolerance of red kidney bean roots to salt stress.
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PMID:Glucose-6-phosphate dehydrogenase plays a pivotal role in nitric oxide-involved defense against oxidative stress under salt stress in red kidney bean roots. 1728 95


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