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)

The NAD(P)H-nitrate reductase complex (overall-NR) of Chlamydomonas reinhardii exhibits two partial activities: NAD(P)H-cytochrome c reductase (diaphorase) and reduced benzyl viologen-NR (terminal-NR). Mild tryptic digestion of the enzyme complex resulted in the loss of both overall and terminal-NR activities, whereas diaphorase activity remained unaltered. The diaphorase activity of mutant 104 and the terminal-NR activity of mutant 305 of C. reinhardii, which are the sole activities related to NR present in these mutants, responded to tryptic treatment to the same extent as the corresponding activities of the wild enzyme complex. Trypsin disassembled the 220-kd NR native complex by destroying the aggregation capability of the diaphorase subunits without affecting their activity nor molecular size (45 kd). A 67-kd thermostable protein, containing molybdenum co-factor, was also released from trypsin-treated NR. This protein lacked diaphorase and NR activities but was able to reconstitute the overall-NR complex by complementation with untreated diaphorase subunit of mutant 104. Our results support a tetrameric structure for the C. reinhardii NR complex, containing two kinds of subunits.
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PMID:Heteromultimeric structure of the nitrate reductase complex of Chlamydomonas reinhardii. 1645 30

Chlamydomonas reinhardii cells, growing photoautotrophically under air, excreted to the culture medium much higher amounts of NO(2) (-) and NH(4) (+) under blue than under red light. Under similar conditions, but with NO(2) (-) as the only nitrogen source, the cells consumed NO(2) (-) and excreted NH(4) (+) at similar rates under blue and red light. In the presence of NO(3) (-) and air with 2% CO(2) (v/v), no excretion of NO(2) (-) and NH(4) (+) occurred and, moreover, if the bubbling air of the cells that were currently excreting NO(2) (-) and NH(4) (+) was enriched with 2% CO(2) (v/v), the previously excreted reduced nitrogen ions were rapidly reassimilated. The levels of total nitrate reductase and active nitrate reductase increased several times in the blue-light-irradiated cells growing on NO(3) (-) under air. When tungstate replaced molybdate in the medium (conditions that do not allow the formation of functional nitrate reductase), blue light activated most of the preformed inactive enzyme of the cells. Furthermore, nitrate reductase extracted from the cells in its inactive form was readily activated in vitro by blue light. It appears that under high irradiance (90 w m(-2)) and low CO(2) tensions, cells growing on NO(3) (-) or NO(2) (-) may not have sufficient carbon skeletons to incorporate all the photogenerated NH(4) (+). Because these cells should have high levels of reducing power, they might use NO(3) (-) or, in its absence, NO(2) (-) as terminal electron acceptors. The excretion of the products of NO(2) (-) and NH(4) (+) to the medium may provide a mechanism to control reductant level in the cells. Blue light is suggested as an important regulatory factor of this photorespiratory consumption of NO(3) (-) and possibly of the whole nitrogen metabolism in green algae.
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PMID:In Vivo Blue-Light Activation of Chlamydomonas reinhardii Nitrate Reductase. 1666 18

The utilization of NO(3) (-) by green algae growing photoautotrophically under air, which are growth conditions close to their more habitual situations in nature, is associated with the excretion of NO(2) (-) and NH(4) (+) to the culture medium. The entire process is promoted by blue light and depends on photosynthetically active radiation for the required reducing equivalents. The stimulation of NO(3) (-) utilization and of its associated NO(2) (-) and NH(4) (+) excretions saturated at very low quantum fluxes of blue light (15 microequivalents per square meter per second) in Chlamydomonas reinhardii cells sparged with CO(2)-free air and irradiated with 50 microequivalents per square meter per second background red light. The wavelength dependence data of this stimulation correlated closely with the in situ photoactivation of nitrate reductase and also with the light induced increase in its biosynthesis and/or assembly.These results indicate that the photoregulation of inorganic N metabolism in C. reinhardii is mainly due to the blue light modulation of nitrate reductase. Although flavins are the most suitable candidates to act as physiological photoreceptors, the wavelength dependence data only show a major peak in the blue region between 400 and 500 nanometers.
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PMID:Spectral Dependence of Photoregulation of Inorganic Nitrogen Metabolism in Chlamydomonas reinhardii. 1666 36

Antibodies against nitrate reductase from Monoraphidium braunii have been used to determine the antigenic relationships of nitrate reductases from different green algae. Nitrate reductases from Chlamydomonas reinhardii, Chlorella fusca, Dunaliella salina, and Scenedesmus obliquus, were inhibited by, and cross-reacted with, antibodies raised against the enzyme from Monoraphidium braunii.These antibodies were also used to determine, by immunoelectron microscopy, the intracellular location of nitrate reductase in the aforementioned green algae. In all cases, the enzyme was specifically located in the pyrenoid.
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PMID:Nitrate reductase of green algae is located in the pyrenoid. 1666 19

All nitrate reductase-related activities of Chlamydomonas reinhardtii wild-type and mutant 305 cells were degraded in vivo under conditions in which the reversible inactivation could take place. When the enzyme was in the inactive form, half-lives of all nitrate reductase-related activities in wild and mutant 305 strains decreased significantly. The only nitrate reductase-related activity present in mutant 104, nitrate reductase-diaphorase, was incapable of undergoing reversible inactivation and was not degraded under any of the conditions tested. Addition of nitrate to inactive nitrate reductase of mutant 305 caused the in vivo reactivation of the enzyme and halted its degradation. Our results indicate that reversibly inactivated nitrate reductase from C. reinhardtii is the main target for a degradation system, and that nitrate reductase related diaphorase must be integrated in a reversibly inactive nitrate reductase complex to undergo degradation. A physiological role for the interconversion process of nitrate reductase can be understood on the basis of these facts.
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PMID:Involvement of Reversible Inactivation in the Regulation of Nitrate Reductase Enzyme Levels in Chlamydomonas reinhardtii. 1666 99

The specific activities of nitrate reductase, nitrite reductase, glutamine synthetase, glutamate synthase, and glutamate dehydrogenase were determined in intact protoplasts and intact chloroplasts from Chlamydomonas reinhardtii. After correction for contamination, the data were used to calculate the portion of each enzyme in the algal chloroplast. The chloroplast of C. reinhardtii contained all enzyme activities for nitrogen assimilation, except nitrate reductase, which could not be detected in this organelle. Glutamate synthase (NADH- and ferredoxin-dependent) and glutamate dehydrogenase were located exclusively in the chloroplast, while for nitrite reductase and glutamine synthetase an extraplastidic activity of about 20 and 60%, respectively, was measured. Cells grown on ammonium, instead of nitrate as nitrogen source, had a higher total cellular activity of the NADH-dependent glutamate synthase (+95%) and glutamate dehydrogenase (+33%) but less activity of glutamine synthetase (-10%). No activity of nitrate reductase could be detected in ammonium-grown cells. The distribution of nitrogen-assimilating enzymes among the chloroplast and the rest of the cell did not differ significantly between nitrate-grown and ammonium-grown cells. Only the plastidic portion of the glutamine synthetase increased to about 80% in cells grown on ammonium (compared to about 40% in cells grown on nitrate).
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PMID:Localization of Nitrogen-Assimilating Enzymes in the Chloroplast of Chlamydomonas reinhardtii. 1666 9

Two new nitrate reductase-deficient mutants from Chlamydomonas reinhardtii have been genetically and biochemically characterized. Both H1 and F23 mutants carry single recessive allelic mutations that map at a new locus designated nit-7. This locus is unlinked to the other six nit loci related to the nitrate assimilation pathway in C. reinhardtii. Both mutant alleles H1 and F23 lack an active molybdopterin cofactor, the activity of which is restored neither in vitro nor in vivo by high concentrations of molybdate. Nitrate reductase subunits in these mutants seem to assemble, although not in a stable form, in a high molecular weight complex and, as in other molybdenum cofactor-defective mutants of C. reinhardtii, they cannot reconstitute nitrate reductase activity with an active molybdenum cofactor source from extracts of ammonium-grown cells. The results suggest that nit-7 mutants are defective in molybdopterin biosynthesis. They do produce some precursor(s) that are capable of binding to nitrate reductase subunits.
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PMID:nit 7: A New Locus for Molybdopterin Cofactor Biosynthesis in the Green Alga Chlamydomonas reinhardtii. 1666 43

In Chlamydomonas reinhardtii mutants defective at the structural locus for nitrate reductase (nit-1) or at loci for biosynthesis of the molybdopterin cofactor (nit-3, nit-4, or nit-5 and nit-6), both nitrite uptake and nitrite reductase activities were repressed in ammonium-grown cells and expressed at high amounts in nitrogen-free media or in media containing nitrate or nitrite. In contrast, wild-type cells required nitrate induction for expression of high levels of both activities. In mutants defective at the regulatory locus for nitrate reductase (nit-2), very low levels of nitrite uptake and nitrite reductase activities were expressed even in the presence of nitrate or nitrite. Both restoration of nitrate reductase activity in mutants defective at nit-1, nit-3, and nit-4 by isolating diploid strains among them and transformation of a structural mutant upon integration of the wild-type nit-1 gene gave rise to the wild-type expression pattern for nitrite uptake and nitrite reductase activities. Conversely, inactivation of nitrate reductase by tungstate treatment in nitrate, nitrite, or nitrogen-free media made wild-type cells respond like nitrate reductase-deficient mutants with respect to the expression of nitrite uptake and nitrite reductase activities. Our results indicate that nit-2 is a regulatory locus for both the nitrite uptake system and nitrite reductase, and that the nitrate reductase enzyme plays an important role in the regulation of the expression of both enzyme activities.
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PMID:Nitrate Reductase Regulates Expression of Nitrite Uptake and Nitrite Reductase Activities in Chlamydomonas reinhardtii. 1666 56

Positive signaling by nitrate in its assimilation pathway has been studied in Chlamydomonas reinhardtii. Among >34,000 lines generated by plasmid insertion, 10 mutants were unable to activate nitrate reductase (NIA1) gene expression and had a Nit(-) (no growth in nitrate) phenotype. Each of these 10 lines was mutated in the nitrate assimilation-specific regulatory gene NIT2. The complete NIT2 cDNA sequence was obtained, and its deduced amino acid sequence revealed GAF, Gln-rich, Leu zipper, and RWP-RK domains typical of transcription factors and transcriptional coactivators associated with signaling pathways. The predicted Nit2 protein sequence is structurally related to the Nin (for nodule inception) proteins from plants but not to NirA/Nit4/Yna proteins from fungi and yeast. NIT2 expression is negatively regulated by ammonium and is optimal in N-free medium with no need for the presence of nitrate. However, intracellular nitrate is required to allow Nit2 to activate the NIA1 promoter activity. Nit2 protein was expressed in Escherichia coli and shown to bind to specific sequences at the NIA1 gene promoter. Our data indicate that NIT2 is a central regulatory gene required for nitrate signaling on the Chlamydomonas NIA1 gene promoter and that intracellular nitrate is needed for NIT2 function and to modulate NIA1 transcript levels.
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PMID:Nitrate signaling by the regulatory gene NIT2 in Chlamydomonas. 1802 71

Molybdenum is an essential element for almost all living beings, which, in the form of a molybdopterin-cofactor, participates in the active site of enzymes involved in key reactions of carbon, nitrogen, and sulfur metabolism. This metal is taken up by cells in form of the oxyanion molybdate. Bacteria acquire molybdate by an ATP-binding-cassette (ABC) transport system in a widely studied process, but how eukaryotic cells take up molybdenum is unknown because molybdate transporters have not been identified so far. Here, we report a eukaryotic high-affinity molybdate transporter, encoded by the green alga Chlamydomonas reinhardtii gene MoT1. An antisense RNA strategy over the MoT1 gene showed that interference of the expression of this gene leads to the inhibition of molybdate transport activity and, in turn, of the Mo-containing enzyme nitrate reductase, indicating a function of MoT1 in molybdate transport. MOT1 functionality was also shown by heterologous expression in Saccharomyces cerevisiae. Molybdate uptake mediated by MOT1 showed a K(m) of approximately 6 nM, which is the range of the lowest K(m) values reported and was activated in the presence of nitrate. Analysis of deduced sequence from the putative protein coded by MoT1 showed motifs specifically conserved in similar proteins present in the databases, and defines a family of membrane proteins in both eukaryotes and prokaryotes probably involved in molybdate transport and distantly related to plant sulfate transporters SULTR. These findings represent an important step in the understanding of molybdate transport, a crucial process in eukaryotic cells.
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PMID:A high-affinity molybdate transporter in eukaryotes. 1807 39


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