Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.6.5.2 (NQO1)
 6,196 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. NADPH-dependent nitrite reductase from the leaves of higher plants was purified at least 70-fold and separated into two enzyme fractions. The first enzyme, a diaphorase with ferredoxin-NADP-reductase activity, is required only to transfer electrons from NADPH to a suitable electron acceptor, which then donates electrons to nitrite reductase proper. 2. Purified nitrite reductase accepted electrons from ferredoxin (the natural donor) or from reduced dyes. Ferredoxin was reduced by illuminated chloroplasts or dithionite, or by NADPH when diaphorase was present. The purified enzyme did not accept electrons directly from NADPH. 3. Ferredoxins purified from maize, spinach or Clostridium were interchangeable in the nitrite-reductase system. 4. Nitrite reductase had K(m) 0.15mm for nitrite. The pH optimum varied with plant and method of assay. The preparation had low sulphite-reductase activity. Ammonia was the product of nitrite reduction. 5. For some plants, the assay of crude preparations with NADPH was limited by diaphorase and the addition of diaphorase gave a better estimate of nitrite-reductase activity. A simple method of assay is described that uses dithionite with benzyl viologen as electron donor.
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PMID:The purification and properties of nitrite reductase from higher plants, and its dependence on ferredoxin. 438 17

Experiments were performed to determine whether conditions which cause the rapid loss of nitrate reductase activity in Neurospora crassa mycelia were accompanied by the loss of antigenically detectable nitrate reductase protein. When mycelia with nitrate reductase activity were transferred to ammonia media, there was a rapid loss in the reduced nicotinamide adenine dinucleotide-nitrate reductase activity plus the parallel loss of the reduced nicotinamide adenine dinucleotide-diaphorase and the reduced methyl viologen-nitrate reductase activities associated with the nitrate reductase. In addition, there was the loss of cross-reacting material to anti-nitrate reductase antisera that was concomitant with the loss of nitrate reductase activity. When mycelia were exposed to either ammonia plus cycloheximide, nitrate plus cycloheximide, or nitrogen-free media, or to media which lacked an assimilable carbon source, the amount of cross-reacting material declined in concert with the nitrate reductase activity. The mutant nit-6, which lacks nitrite reductase activity, was exposed to ammonia or nitrate plus cycloheximide media. The nitrate reductase and the amount of cross-reacting material declined together as in the wild-type mycelia. We conclude that the loss of nitrate reductase activity was accompanied by the specific loss of this protein and that no pool of inactivated nitrate reductase molecules existed.
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PMID:Repression of nitrate reductase activity and loss of antigenically detectable protein in Neurospora crassa. 644 48

1. Enzyme systems from Cucurbita pepo have been shown to catalyse the reduction of nitrite and hydroxylamine to ammonia in yields about 90-100%. 2. Reduced benzyl viologen serves as an efficient electron donor for both systems. Activity of the nitrite-reductase system is directly related to degree of dye reduction when expressed in terms of the function for oxidation-reduction potentials, but appears to decrease to negligible activity below about 9% dye reduction. 3. NADH and NADPH alone produce negligible nitrite loss, but NADPH can be linked to an endogenous diaphorase system to reduce nitrite to ammonia in the presence of catalytic amounts of benzyl viologen. 4. The NADH- or NADPH-nitrate-reductase system that is also present can accept electrons from reduced benzyl viologen, but shows relationships opposite to that for the nitrite-reductase system with regard to effect of degree of dye reduction on activity. The product of nitrate reduction may be nitrite alone, or nitrite and ammonia, or ammonia alone, according only to the degree of dye reduction. 5. The relative activities of nitrite-reductase and hydroxylamine-reductase systems show different relationships with degree of dye reduction and may become reversed in magnitude when effects of degree of dye reduction are tested over a suitable range. 6. Nitrite severely inhibits the rate of reduction of hydroxylamine without affecting the yield of ammonia as a percentage of total substrate loss, but hydroxylamine has a negligible effect on the activity of the nitrite-reductase system. 7. The apparent K(m) for nitrite (1 mum) is substantially less than that for hydroxylamine, for which variable values between 0.05 and 0.9mm (mean 0.51 mm) have been observed. 8. The apparent K(m) values for reduced benzyl viologen differ for the nitrite-reductase and hydroxylamine-reductase systems: 60 and 7.5 mum respectively. 9. It is concluded that free hydroxylamine may not be an intermediate in the reduction of nitrite to ammonia by plants, and a possible mechanism for reduction of both compounds by the same enzyme system is discussed in the light of current ideas relating to other organisms.
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PMID:THE REDUCTION OF NITRATE, NITRITE AND HYDROXYLAMINE TO AMMONIA BY ENZYMES FROM CUCURBITA PEPO L. IN THE PRESENCE OF REDUCED BENZYL VIOLOGEN AS ELECTRON DONOR. 1434 47

Molybdenum is absolutely required for the nitrate-reducing activity of the nicotinamide adenine dinucleotide nitrate reductase complex isolated from Chlorella fusca. The whole enzyme nicotinamide adenine dinucleotide nitrate reductase is formed by cells grown in the absence of added molybdate, but only its first activity (nicotinamide adenine dinucleotide diaphorase) is functional. The second activity of the complex, which subsequently participates also in the enzymatic transfer of electrons from nicotinamide adenine dinucleotide to nitrate (FNH(2)-nitrate reductase), depends on the presence of molybdenum. Neither molybdate nor nitrate is required for nitrate reductase synthesis de novo, but ammonia acts as a nutritional repressor of the complete enzyme complex. Under conditions which exclude de novo synthesis of nitrate reductase, the addition of molybdate to molybdenum-deficient cells clearly increases the activity level of this enzyme, thus suggesting in vivo incorporation of the trace metal into the pre-existing inactive apoenzyme.Competition studies with tungstate corroborate these conclusions and indicate that the only role played by molybdenum in Chlorella is connected with the reduction of nitrate to nitrite. Tungsten seems to act by replacing molybdenum in the nitrate reductase complex, thus rendering inactive the FNH(2)-nitrate reductase portion of the nicotinamide adenine dinucleotide nitrate reductase complex.
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PMID:Role of molybdenum in nitrate reduction by chlorella. 1665 84

Nicotinic acid adenine dinucleotide phosphate (NAADP) has been shown to mobilize Ca(2+) from intracellular stores in a wide variety of organisms, ranging from plants to humans. We have developed a novel enzyme cycling assay for NAADP that involves coupled reactions catalyzed by four enzymes. In this system, NAADP is first converted into nicotinic acid adenine dinucleotide (NAAD) by alkaline phosphatase, after which the NAAD is converted to NAD, AMP, and PPi by NAD synthetase (NADS) in the presence of ATP and ammonia. The NAD is then amplified using an enzyme cycling system driven by glucose dehydrogenase and diaphorase. The resultant formation of formazan dye is measured spectrophotometrically based on the increase in absorbance at 450 nm. Using this method, NAADP (20-400 nM) was assayed, and a highly linear correlation was obtained between the NAADP concentration and the increase in absorbance at 450 nm. The cycling rate was approximately 95 cycles/min. In addition, the within-run coefficients of variation (CVs) for 25, 50, and 100 nM NAADP solutions were 9.33, 4.86, and 3.13%, respectively. Interference by NAD analogs (e.g., NAAD, NADP) in the sample was eliminated prior to running the assay by treating the sample with NADS and NAD nucleosidase (NADase). In sum, our findings indicate this enzyme cycling assay to be readily applicable for determination for NAADP in a variety of biological samples and to be particularly appropriate for use with an autoanalyzer.
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PMID:An enzymatic cycling assay for nicotinic acid adenine dinucleotide phosphate using NAD synthetase. 1739 43

Current conventional measurement of allantoin levels in human serum uses an HPLC method. However, performing this assay is time-consuming and sample-intensive, and it requires expensive equipment. We have developed a novel enzyme cycling method for measuring allantoin concentrations in human serum. In the first step, serum allantoin is converted to allantoate by the action of allantoinase (EC 3.5.2.5), and endogenous ammonia is simultaneously removed by the action of glutamine synthetase II (EC 6.3.1.2). In the second step, l-methionine sulfoximine is used to inhibit glutamine synthetase II, and ammonia is liberated from allantoate by the activity of allantoate amidohydrolase (EC 3.5.3.9). In the final step, the ammonia is then converted to NAD by NAD synthetase (EC 6.3.1.5). Subsequent action of glucose dehydrogenase (EC 1.1.1.47) and diaphorase (EC 1.6.99.2) in the presence of glucose and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) acts to cycle the formed NAD between its oxidized and reduced forms, resulting in the production of WST-1 formazan, which is monitored at 450 nm. The assay standard curve is linear from 0 to 70 muM allantoin. The level of allantoin in healthy subjects was measured to be 8.2+/-3.1 microM (n=30).
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PMID:An enzyme cycling method for measurement of allantoin in human serum. 1844 70

In the green alga Ankistrodesmus braunii, all the activities associated with the nitrate reductase complex (i.e., NAD(P)H-nitrate reductase, NAD(P)H-cytochrome c reductase and FMNH2-or MVH-nitrate reductase) are nutritionally repressed by ammonia or methylamine. Besides, ammonia or methylamine promote in vivo the reversible inactivation of nitrate reductase, but not of NAD(P)H-cytochrome c reductase. Subsequent removal of the inactivating agent from the medium causes reactivation of the inactive enzyme. Menadione has a striking stimulation on the in vivo reactivation of the inactive enzyme. The nitrate reductase activities, but not the diaphorase activity, can be inactivated in vitro by preincubating a partially purified enzyme preparation with NADH or NADPH. ADP, in the presence of Mg(2+), presents a cooperative effect with NADH in the in vitro inactivation of nitrate reductase. This effect appears to be maximum at a concentration of ADP equimolecular with that of NADH.
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PMID:Studies on the regulation of assimilatory nitrate reductase in Ankistrodesmus braunii. 2442 Jun 58

The fixation of atmospheric dinitrogen to ammonia by industrial technologies (such as the Haber Bosch process) has revolutionized humankind. In contrast to industrial technologies, a single enzyme is known for its ability to reduce or "fix" dinitrogen: nitrogenase. Nitrogenase is a complex oxidoreductase enzymatic system that includes a catalytic protein (where dinitrogen is reduced) and an electron-transferring reductase protein (termed the Fe protein) that delivers the electrons necessary for dinitrogen fixation. The catalytic protein most commonly contains a FeMo cofactor (called the MoFe protein), but it can also contain a VFe or FeFe cofactor. Besides their ability to fix dinitrogen to ammonia, these nitrogenases can also reduce substrates such as carbon dioxide to formate. Interestingly, the VFE nitrogenase can also form carbon-carbon bonds. The vast majority of research surrounding nitrogenase employs the Fe protein to transfer electrons, which is also associated with the rate-limiting step of nitrogenase catalysis and also requires the hydrolysis of adenosine triphosphate. Thus, there is significant interest in artificially transferring electrons to the catalytic nitrogenase proteins. In this Account, we review nitrogenase electrocatalysis whereby electrons are delivered to nitrogenase from electrodes. We first describe the use of an electron mediator (cobaltocene) to transfer electrons from electrodes to the MoFe protein. The reduction of protons to molecular hydrogen was realized, in addition to azide and nitrite reduction to ammonia. Bypassing the rate-limiting step within the Fe protein, we also describe how this approach was used to interrogate the rate-limiting step of the MoFe protein: metal-hydride protonolysis at the FeMo-co. This Account next reviews the use of cobaltocene to mediate electron transfer to the VFe protein, where the reduction of carbon dioxide and the formation of carbon-carbon bonds (yielding the formation of ethene and propene) was realized. This approach also found success in mediating electron transfer to the FeFe catalytic protein, which exhibited improved carbon dioxide reduction in comparison to the MoFe protein. In the final example of mediated electron transfer to the catalytic protein, this Account also reviews recent work where the coupling of infrared spectroscopy with electrochemistry enabled the potential-dependent binding of carbon monoxide to the FeMo-co to be studied. As an alternative to mediated electron transfer, recent work that has sought to transfer electrons to the catalytic proteins in the absence of electron mediators (by direct electron transfer) is also reviewed. This approach has subsequently enabled a thermodynamic landscape to be proposed for the cofactors of the catalytic proteins. Finally, this Account also describes nitrogenase electrocatalysis whereby electrons are first transferred from an electrode to the Fe protein, before being transferred to the MoFe protein alongside the hydrolysis of adenosine triphosphate. In this way, increased quantities of ammonia can be electrocatalytically produced from dinitrogen fixation. We discuss how this has led to the further upgrade of electrocatalytically produced ammonia, in combination with additional enzymes (diaphorase, alanine dehydrogenase, and transaminase), to selective production of chiral amine intermediates for pharmaceuticals. This Account concludes by discussing current and future research challenges in the field of electrocatalytic nitrogen fixation by nitrogenase.
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PMID:Nitrogenase Bioelectrochemistry for Synthesis Applications. 3180 Feb 7