Nitrate reductase azide

Plant use of iron depends on the plant s ability to respond chemically to iron stress. This response causes the roots to release H+ and deduct ants, to reduce Fe3+, and to accumulate citrate, making iron available to the plant. Reduction sites are principally in the young lateral roots. Azide, arsenate, zinc, copper, and chelating agents may interfere with use of iron. Chemical reactions induced by iron stress affect nitrate reductase activity, use of iron from Fe3+ phosphate and Fe3+ chelate, and tolerance of plants to heavy metals. The iron stress-response mechanism is adaptive and genetically controlled, making it possible to tailor plants to grow under conditions of iron stress. 

Dissimilatory nitrate reductases (Pichinoty type A) in membrane fractions from bacteria have been shown capable of utilizing a variety of respiratory Intermediates and reduced pyridine nucleotides for nitrate reduction (Cole and Wimpeny, 1968 Knook et ai, 1973 Burke and Lascelles, 1975 Enoch and Lester, 1975). Reduction of nitrate by the membrane fractions, when respiratory substrates or pyridine nucleotides serve as reduc-tant, is generally inhibited by azide, cyanide and p-chloromercuribenzoate. Nitrate reduction, mediated by respiratory substrates, could be inhibited by n-heptylhydroxyquinoline-N oxide (HONO) or dicoumoral (Ruiz-Herrera and DeMoss, 1%9 Knook et al., 1973 Burke and Lascelles, 1975). However, in Micrococcus denitrificans (Lam and Nicholas, 1969) and in Bacillus stearothermophilus (Downey, 1%6) nitrate reduction is not inhibited by... 

Dissimilatory nitrate reduction by particulate preparations from bacteria can utilize reduced viologen dyes as electron donors. With this reductant, nitrate reduction is insensitive to inhibition byp-chloromercuribenzoate and HONO, but is prevented by cyanide and azide (Burke and Lascelles, 1975 Ruiz-Herrera and DeMoss, 1969). Respiratory intermediates or reduced pyridine nucleotides cannot serve as electron donors for the nitrate reductase solubilized from membrane complexes and the assay must be performed with reduced viologen dyes. 

Pyridine nucleotide mediated nitrate reductase and NADPH cytochrome c reductase activities in the purified preparations are inhibited by p-hydroxymercuribenzoate (Garrett and Nason, 1969 McDonald and Coddington, 1974 Guerrero and Gutierrez, 1977). This inhibition could be overcome with cysteine or dithiothreitol. Cyanide and azide inhibit pyridine nucleotide and reduced viologen dye mediated nitrate reduction but do not affect NADPH cytochrome c reductase activity. 

Sulfhydryl reagents and heat will inhibit nitrate and cytochrome c reduction, but do not interfere with nitrate reduction mediated by FMNH2, etc. Azide and cyanide inhibit nitrate reduction but do not interfere with the NAD(P)H-mediated cytochrome c reduction. From work with barley (Wray and Filner, 1970) and spinach (Rucklidge et al., 1976) it can be inferred that NADH nitrate reductase is composed of a NADH-cytochrome c dehydrogenase and a Mo-protein. Nitrate induces the apoprotein (dehydrogenase) in molybdenum-deficient spinach leaves. This apoprotein mixed with an acid-dissociated product of spinach nitrate reductase can form an active nitrate reductase. 

In bacteria, with Pichinoty type A dissimilatory nitrate reductase, induction of the enzyme occurs only under anaerobic conditions, and occasionally anaerobiosis is sufficient to trigger formation in the absence of nitrate (Schulp Md Stouthamer, 1970 Sinclair and White, 1970). However, in these cases of induction by anaerobiosis, the level of enzyme production is greatly enhanced by the presence of nitrate. Dissimilatory nitrate reductase may also be induced under anaerobic conditions by nitrite and azide. 

Apparent dissociation constants for nitrate complexes of nitrate reductase and of the two xanthine oxidase forms are in the range 4-20 mM. For nitrate reductase some evidence for a complex with azide was also obtained, but no other ions tested had significant effects on the spectra (Vincent and Bray, 1978). Clearly further work on the anion complexes is required. Ideally, one would like to see parameters for a range of anions all occupying the same enzyme site. From these, hopefully, some structural information might be deducible. 

Nitrate can also be converted to N02 using cadmium reduction (columns or spongy cadmium) as described above. Once the NOa" is in the form of N02, the N02 can be isolated via organic extraction (e.g., Olson, 1981) or with SPE after the N02 is converted to an azo dye (Kator et al, 1992). Nitrate can be isolated by conversion to N2O via sodium azide in an acetic acid buffer solution (Mcllvin and Altabet, 2005). Another approaches uses a genetically engineered denitrifier to convert N03 to N2O (Sigman et al, 2001) the bacteria wiU denitrify NOa" in a sample to N2O, but lacking nitrous oxide reductase the bacteria cannot take the reaction to completion and form N2. The N2O produced by either approach can then be analyzed on a mass spectrometer. A more detailed discussion of these methods is presented in Chapter 31 by Lipschultz, this volume. 

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