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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)

Intercellular distribution of enzymes involved in amino nitrogen synthesis was studied in leaves of species representing three C(4) groups, i.e. Sorghum bicolor, Zea mays, Digitaria sanguinalis (NADP malic enzyme type); Panicum miliaceum (NAD malic enzyme type); and Panicum maximum (phosphoenolpyruvate carboxykinase type). Nitrate reductase, nitrite reductase, glutamine synthetase, and glutamate synthase were predominantly localized in mesophyll cells of all the species, except in P. maximum where nitrite reductase had similar activity on a chlorophyll basis, in both mesophyll and bundle sheath cells. NADH-glutamate dehydrogenase was concentrated in the bundle sheath cells, while NADPH-glutamate dehydrogenase was localized in both mesophyll and bundle sheath cells. The activities of nitrate-assimilating enzymes, except for nitrate reductase, were high enough to account for the proposed in vivo rates of nitrate assimilation.Based on the differential centrifugation of cell homogenates of P. miliaceum, mesophyll chloroplasts appear to be the major site of nitrate assimilation since nitrite reductase, glutamine synthetase, glutamate synthase, and NADPH-glutamate dehydrogenase were primarily localized in the chloroplast fraction. Both the glutamine synthetase-glutamate synthase and glutamate dehydrogenase pathways were considered as alternative routes of amino nitrogen synthesis.
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PMID:Distribution of Nitrate-assimilating Enzymes between Mesophyll Protoplasts and Bundle Sheath Cells in Leaves of Three Groups of C(4) Plants. 1665 90

The effect of various day temperatures on NADH-nitrate reductase, NADH- and NADPH-glutamate dehydrogenases, nitrate, protein and leaf area, measured at intervals during the ontogeny of the first trifoliolate soybean leaf, was determined. At 32.5 C and 25 C, nitrate concentration, nitrate reductase, and NADPH-glutamate dehydrogenase activities increased concurrently with leaf development and then decreased as leaf maturation progressed. At 40 C, these three components showed no initial increase and the concentration or activities decreased throughout the development of the leaf. The effects of temperature on NADH-glutamate dehydrogenase were the reverse. Rates of protein accumulation were higher at 40 C during the first 2 days of leaf development while higher rates were measured the first 5 days of leaf growth at 32.5 C. At 25 C, protein accumulation was low during the first 3 days of leaf growth, increased in the period of 3 to 5 days, and then declined up to 8 days of leaf development. Leaf expansion progressed at faster rates at 32.5 C and 25 C and at a much slower rate at 40 C. Leaf growth was essentially complete after the fifth day regardless of temperature.In crude leaf homogenates, apparent irreversible inactivation temperatures were 36 C for nitrate reductase and 65 C for NADPH-glutamate dehydrogenase. In vivo studies indicated a lower inactivation temperature for NADPH-glutamate dehydrogenase; however, it was still more heat-tolerant than nitrate reductase.We envisaged that reduced nitrogen supplied by NO(3) (-) assimilation is a factor in leaf expansion.
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PMID:Influence of Temperature on Nitrate Metabolism and Leaf Expansion in Soybean (Glycine max L. Merr.) Seedlings. 1665 11

Nitrate reductase of the salt-tolerant alga Dunaliella parva could utilize NADPH as well as NADH as an electron donor. The two pyridine nucleotide-dependent activities could not be separated by either ion exchange chromatography on DEAE-cellulose or gel filtration on Sepharose 4B. The NADPH-dependent activity was not inhibited by phosphatase inhibitors. NADPH was not hydrolyzed to NADH and inorganic phosphate in the course of nitrate reduction. Reduction of nitrate in vitro could be coupled to a NADPH-regenerating system of glycerol and NADP-dependent glycerol dehydrogenase. It is concluded that the nitrate reductase of D. parva will function with NADPH as well as NADH. This is a unique characteristic not common to most algae.
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PMID:Specificity for Nicotinamide Adenine Dinucleotide and Nicotinamide Adenine Dinucleotide Phosphate of Nitrate Reductase from the Salt-tolerant Alga Dunaliella parva. 1665 20

By use of affinity chromatography on blue dextran-Sepharose, two nitrate reductases from rice (Oryza sativa L.) seedlings, specifically, NADH:nitrate oxidoreductase (EC 1.6.6.1) and NAD(P)-H:nitrate oxidoreductase (EC 1.6.6.2), have been partially separated. Nitrate-induced seedlings contained more NADH-nitrate reductase than NAD(P)H-nitrate reductase, whereas chloramphenicol-induced seedlings contained primarily NAD(P)H-nitrate reductase. NAD(P)H-nitrate reductase was shown to utilize NADPH directly as reductant. This enzyme has a preference for NADPH, but reacts about half as well with NADH.
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PMID:NADH- and NAD(P)H-Nitrate Reductases in Rice Seedlings. 1665 65

Nitrogen assimilation in crabgrass Digitaria sanguinalis (L.) Scop., was studied by comparing leaf extracts with isolated mesophyll cell and bundle sheath strand extracts. The results show that both nitrate and nitrate reductase are localized in mesophyll cells; glutamine synthetase is nearly equally distributed in the mesophyll and bundle sheath; approximately 67% of the glutamate synthase activity is in the bundle sheath and 33% is in the mesophyll; and 80% of the glutamate dehydrogenase activity is in the bundle sheath, with the NADH-dependent form exhibiting a 2.5-fold higher activity than the NADPH-dependent form.Isolated crabgrass mesophyll cells reduce NO(2) (-) coupled to the photochemical production of O(2) but are inactive with NO(3) (-). The NO(2) (-) -dependent O(2) evolution is light-dependent; inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea; stimulated by photophosphorylation uncouplers; and exhibits a stoichiometry of O(2) evolved to NO(2) (-) reduced of 1.45 and 0.67 in coupled and uncoupled experiments, respectively. Isolated bundle sheath strands are inactive in O(2) evolution with NO(3) (-) or NO(2) (-).Based on these results, plus literature data, two schemes for crabgrass leaf nitrogen assimilation are presented, depending on whether the plant is using ammonium or nitrate as its nitrogen source. It is proposed that the increased nitrogen use efficiency in crabgrass and other C(4) plants is due partially to a "division of labor" between mesophyll and bundle sheath cells, where NO(3) (-) and NO(2) (-) reductase in mesophyll cells act as nitrogen reduction traps in an analogous fashion to phosphoenolpyruvate carboxylase acting as a CO(2) trap during C(4) photosynthesis.
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PMID:Nitrogen Assimilation Pathways in Leaf Mesophyll and Bundle Sheath Cells of C(4) Photosynthesis Plants Formulated from Comparative Studies with Digitaria sanguinalis (L.) Scop. 1666 Sep 55

The cotyledons of soybean begin to develop photosynthetic capacity shortly after emergence. The cotyledons develop nitrate reductase (NR) activity in parallel with an increase in chlorophyll and a decrease in protein. In crude extracts of 5- to 8-day-old cotyledons, NR activity is greatest with NADH as electron donor. In extracts of older cotyledons, NR activity is greatest with NADPH. Blue-Sepharose was used to purify and separate the NR activities into two fractions. When the blue-Sepharose was eluted with NADPH, NR activity was obtained which was most active with NADPH as electron donor. Assays of the NADPH-eluted NR with different concentrations of nitrate revealed that the highest activity was obtained in 80 millimolar KNO(3). Thus, this fraction has properties similar to the low nitrate affinity NAD(P)H:NR of soybean leaves. When 5- to 8-day-old cotyledons were extracted and purified, further elution of the blue-Sepharose with KNO(3), subsequent to the NADPH elution, yielded an NR fraction most active with NADH. Assays of this fraction with different nitrate concentrations revealed that this NR had a higher nitrate affinity and was similar to the NADH:NR of soybean leaves. The KNO(3)-eluted NR fraction which was purified from the extracts of 9- to 14-day-old cotyledons, was most active with NADPH. The analysis of these fractions prepared from the extracts of older cotyledons indicated that residual NAD(P)H:NR contaminated the NADH:NR. Despite this complication, the pattern of development of the purified NR fractions was consistent with the changes observed in the crude extract NR activities. It was concluded that NADH:NR was most active in young cotyledons and that as the cotyledons aged the NAD(P)H:NR became more active.
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PMID:Development of NAD(P)H: and NADH:Nitrate Reductase Activities in Soybean Cotyledons. 1666 Dec 45

The nitrate reductase activity of 5-day-old whole corn roots was isolated using phosphate buffer. The relatively stable nitrate reductase extract can be separated into three fractions using affinity chromatography on blue-Sepharose. The first fraction, eluted with NADPH, reduces nearly equal amounts of nitrate with either NADPH or NADH. A subsequent elution with NADH yields a nitrate reductase which is more active with NADH as electron donor. Further elution with salt gives a nitrate reductase fraction which is active with both NADH and NADPH, but is more active with NADH. All three nitrate reductase fractions have pH optima of 7.5 and Stokes radii of about 6.0 nanometers. The NADPH-eluted enzyme has a nitrate K(m) of 0.3 millimolar in the presence of NADPH, whereas the NADH-eluted enzyme has a nitrate K(m) of 0.07 millimolar in the presence of NADH. The NADPH-eluted fraction appears to be similar to the NAD(P)H:nitrate reductase isolated from corn scutellum and the NADH-eluted fraction is similar to the NADH:nitrate reductases isolated from corn leaf and scutellum. The salt-eluted fraction appears to be a mixture of NAD(P)H: and NADH:nitrate reductases.
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PMID:Purification and Characterization of NAD(P)H:Nitrate Reductase and NADH:Nitrate Reductase from Corn Roots. 1666 53

Homogeneous squash cotyledon reduced nicotinamide-adenine dinucleotide (NADH):nitrate reductase (NR) was isolated using blue-Sepharose and polyacrylamide gel electrophoresis. Gel slices containing NR were pulverized and injected into a previously unimmunized rabbit. This process was repeated weekly and antiserum to NR was obtained after four weeks. Analysis of the antiserum by Ouchterlony double diffusion using a blue-Sepharose preparation of NR resulted in a single precipitin band while immunoelectrophoresis revealed two minor contaminants. The antiserum was found to inhibit the NR reaction and the partial reactions to different degrees. When the NADH:NR and the reduced methyl viologen:NR activities were inhibited 90% by specifically diluted antiserum, the reduction of cytochrome c was inhibited 50%, and the reduction of ferricyanide was inhibited only 30%. Antiserum was also used to compare the cross reactivities of NR from squash cotyledons, spinach, corn, and soybean leaves, Chlorella vulgaris, and Neurospora crassa. These tests revealed a high degree of similarity between NADH:NR from the squash and spinach, while NADH:NR from corn and soybean and the NAD(P)H:NR from soybean were less closely related to the squash NADH:NR. The green algal (C. vulgaris) NADH:NR and the fungal (N. crassa) NADPH:NR were very low in cross reactivity and are apparently quite different from squash NADH:NR in antigenicity. Antiserum to N. crassa NADPH:NR failed to give a positive Ouchterlony result with higher plant or C. vulgaris NADH:NR, but this antiserum did inhibit the activity of squash NR. Thus, it can be concluded from these immunological comparisons that all seven forms of assimilatory NR studied here have antigenic determinants in common and are probably derived from a common ancestor. Although these assimilatory NR have similar catalytic characteristics, they appear to have diverged to a great degree in their structural features.
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PMID:Immunological approach to structural comparisons of assimilatory nitrate reductases. 1666 83

NADPH nitrate reductase activity in higher plants has been attributed to the presence of NAD(P)H bispecific nitrate reductases and to the presence of phosphatases capable of hydrolyzing NADPH to NADH. To determine which of these conditions exist in barley (Hordeum vulgare L. cv. Steptoe), we characterized the NADH and NADPH nitrate reductase activities in crude and affinity-chromatography-purified enzyme preparations. The pH optima were 7.5 for NADH and 6 to 6.5 for the NADPH nitrate reductase activities. The ratio of NADPH to NADH nitrate reductase activities was much greater in crude extracts than it was in a purified enzyme preparation. However, this difference was eliminated when the NADPH assays were conducted in the presence of lactate dehydrogenase and pyruvate to eliminate NADH competitively. The addition of lactate dehydrogenase and pyruvate to NADPH nitrate reductase assay media eliminated 80 to 95% of the NADPH nitrate reductase activity in crude extracts. These results suggest that a substantial portion of the NADPH nitrate reductase activity in barley crude extracts results from enzyme(s) capable of converting NADPH to NADH. This conversion may be due to a phosphatase, since phosphate and fluoride inhibited NADPH nitrate reductase activity to a greater extent than the NADH activity. The NADPH activity of the purified nitrate reductase appears to be an inherent property of the barley enzyme, because it was not affected by lactate dehydrogenase and pyruvate. Furthermore, inorganic phosphate did not accumulate in the assay media, indicating that NADPH was not converted to NADH. The wild type barley nitrate reductase is a NADH-specific enzyme with a slight capacity to use NADPH.
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PMID:Pyridine nucleotide specificity of barley nitrate reductase. 1666 69

A barley (Hordeum vulgare L.) mutant, nar1a (formerly Az12), deficient in NADH nitrate reductase activity is, nevertheless, capable of growth with nitrate as the sole nitrogen source. In an attempt to identify the mechanism(s) of nitrate reduction in the mutant, nitrate reductase from nar1a was characterized to determine whether the residual activity is due to a leaky mutation or to the presence of a second nitrate reductase. The results obtained indicate that the nitrate reductase in nar1a differs from the wild-type enzyme in several important aspects. The pH optima for both the NADH and the NADPH nitrate reductase activities from nar1a were approximately pH 7.7, which is slightly greater than the pH 7.5 optimum for the NADH activity and considerably greater than the pH 6.0 to 6.5 optimum for the NADPH activity of the wild-type enzyme. The nitrate reductase from nar1a exhibits greater NADPH than NADH activity and has apparent K(m) values for nitrate and NADH that are approximately 10 times greater than those of the wild-type enzyme. The nar1a nitrate reductase has apparent K(m) values of 170 micromolar for NADPH and 110 micromolar for NADH. NADPH, but not NADH, inhibited the enzyme at concentrations greater than 50 micromolar.Unlike that of the wild-type, the nitrate reductase from nar1a did not bind to blue dextran-Sepharose. The nar1a enzyme did bind to Affi Gel Blue, but recoveries were low. The NADH and NADPH nitrate reductase activities of nar1a were not separated by affinity chromatography. The nitrate reductase in nar1a is a different enzyme than the wild-type NADH nitrate reductase and appears to be a NAD(P)H-bispecific enzyme.
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PMID:Characteristics of a Nitrate Reductase in a Barley Mutant Deficient in NADH Nitrate Reductase. 1666 70


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