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Query: EC:1.7.1.1 (nitrate reductase)
 3,728 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

Soybean (Glycine max [L.] Merr.) seeds were imbibed and germinated with or without NO(3) (-), tungstate, and norflurazon (San 9789). Norflurazon is a herbicide which causes photobleaching of chlorophyll by inhibiting carotenoid synthesis and which impairs normal chloroplast development. After 3 days in the dark, seedlings were placed in white light to induce extractable nitrate reductase activity. The induction of maximal nitrate reductase activity in greening cotyledons did not require NO(3) (-) and was not inhibited by tungstate. Induction of nitrate reductase activity in norflurazon-treated cotyledons had an absolute requirement for NO(3) (-) and was completely inhibited by tungstate. Nitrate was not detected in seeds or seedlings which had not been treated with NO(3) (-). The optimum pH for cotyledon nitrate reductase activity from norflurazon-treated seedlings was at pH 7.5, and near that for root nitrate reductase activity, whereas the optimum pH for nitrate reductase activity from greening cotyledons was pH 6.5. Induction of root nitrate reductase activity was also inhibited by tungstate and was dependent on the presence of NO(3) (-), further indicating that the isoform of nitrate reductase induced in norflurazon-treated cotyledons is the same or similar to that found in roots. Nitrate reductases with and without a NO(3) (-) requirement for light induction appear to be present in developing leaves. In vivo kinetics (light induction and dark decay rates) and in vitro kinetics (Arrhenius energies of activation and NADH:NADPH specificities) of nitrate reductases with and without a NO(3) (-) requirement for induction were quite different. K(m) values for NO(3) (-) were identical for both nitrate reductases.
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PMID:Differential light induction of nitrate reductases in greening and photobleached soybean seedlings. 1666 85

Chenopodium rubrum cells were grown in suspension as a photoautotrophic culture with a 16 hour day. Cell growth had three phases: a 3-day lag, a 3-week logarithmic phase, and a 10-day stationary phase. Chlorophyll content increased steadily during log phase and reached a level of 0.5 to 0.6 mg Chl g(-1) fresh weight. Soluble protein of the cells increased more rapidly from day 4 to day 12 than during midlog phase. Initially, ammonium was taken up in preference to nitrate. However, during the second two weeks of growth, ammonium and nitrate were taken up simultaneously; this period of growth was the time of highest rates of N uptake by the cultured cells. Glutamine synthetase had a high specific activity (17 mumol.hour(-1) mg(-1) protein) in day 1 cells, and this level was sustained until midlog phase when it increased by 20%. Methyl viologen-dependent glutamate synthase specific activity increased rapidly in lag phase cells (day 4 = 10 mumol.hour(-1) mg(-1) protein), but decreased by day 9 to about 50% of the peak and remained constant. NADH:nitrate reductase specific activity increased rapidly in lag phase cells and reached a plateau that lasted from day 4 to 14 (1 mumol.hour(-1) mg(-1) protein). Methyl viologen-dependent nitrite reductase specific activity was high when assayed on day 5 and increased to a maximum on day 15 to 16 (12 mumol.hour(-1) mg(-1) protein). NADPH- and NADH-dependent glutamate dehydrogenase specific activities remained rather constant throughout the growth cycle. The cells appeared to have developed photosynthetic competence and to have leaf-like activities of nitrogen assimilation enzymes.
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PMID:Development of Nitrogen Assimilation Enzymes during Photoautotrophic Growth of Chenopodium rubrum Suspension Cultures. 1666 39

NADH:nitrate reductase (EC 1.6.6.1) and NAD(P)H:nitrate reductase (EC 1.6.6.2) were purified from wild-type soybean (Glycine max [L.] Merr., cv Williams) and nr(1)-mutant soybean plants. Purification included Blue Sepharose- and hydroxylapatite-column chromatography using acetone powders from fully expanded unifoliolate leaves as the enzyme source.Two forms of constitutive nitrate reductase were sequentially eluted with NADPH and NADH from Blue Sepharose loaded with extract from wild-type plants grown on urea as sole nitrogen source. The form eluted with NADPH was designated c(1)NR, and the form eluted with NADH was designated c(2)NR. Nitrate-grown nr(1) mutant soybean plants yielded a NADH:nitrate reductase (designated iNR) when Blue Sepharose columns were eluted with NADH; NADPH failed to elute any NR form from Blue Sepharose loaded with this extract. Both c(1)NR and c(2)NR had similar pH optima of 6.5, sedimentation behavior (s(20,w) of 5.5-6.0), and electrophoretic mobility. However, c(1)NR was more active with NADPH than with NADH, while c(2)NR preferred NADH as electron donor. Apparent Michaelis constants for nitrate were 5 millimolar (c(1)NR) and 0.19 millimolar (c(2)NR). The iNR from the mutant had a pH optimum of 7.5, s(20,w) of 7.6, and was less mobile on polyacrylamide gels than c(1)NR and c(2)NR. The iNR preferred NADH over NADPH and had an apparent Michaelis constant of 0.13 millimolar for nitrate.Thus, wild-type soybean contains two forms of constitutive nitrate reductase, both differing in their physical properties from nitrate reductases common in higher plants. The inducible nitrate reductase form present in soybeans, however, appears to be similar to most substrateinduced nitrate reductases found in higher plants.
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PMID:Nitrate Reductases from Wild-Type and nr(1)-Mutant Soybean (Glycine max [L.] Merr.) Leaves : I. Purification, Kinetics, and Physical Properties. 1666 14


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