Nitrate reductase inhibition

It is not clear why some organisms have two 14-3-3 isoforms while others have up to 12. Binding 14-3-3 inhibits the plant enzyme nitrate reductase and there appears to be no selectivity between plant 14-3-3 isoforms in fact yeast and human isoforms appear to work equally as well in vitro. The best example where selectivity has been demonstrated is human 14-3-3o. 14-3-3o Preferential homodimerizes with itself and crystallization revealed a structural basis for this isoform s dimerization properties as well as for its specific selectivity for target binding proteins. Here partner specificity is the result of amino acid differences outside of the phosphopeptide-binding cleft. 

Ward, M.R., Tischner, R. Huffaker, R.C. (1988). Inhibition of nitrate transport by anti-nitrate reductase IgG fragments and the identification of plasma membrane associated nitrate reductase in roots of barley seedlings. Plant Physiology 88, 1141-5. 

Laboratory work suggests, however, that Fe limitation probably does not directly inhibit N03 uptake by compromising nitrate reductase activity (Milligan and Harrison, 2000). Instead, Fe limitation may block the N03 reduction pathway further downstream, by preventing nitrite (N02 ) reduction to NH4+ due to insufficient supplies of photosyntheticaUy-produced reductant. This study showed that Fe-deficient diatom cells continue to take up and reduce N03 to nitrite. 

The laser photolysis results on the ET behavior of these mutants have been confirmed by steady-state kinetic measurements [58, 59]. Interestingly, the latter experiments have shown that nonconservative mutations at F65 and E94 not only severely inhibit reactivity with FNR, but also with two other ferredoxin-dependent enzymes, nitrite reductase and nitrate reductase [58]. Apparently, similar structural constraints in their interactions with Fd are also operative in these other enzymes. 

Impact of iron on silicon pumping. Iron limitation of diatoms increases the pumping of silicon (relative to nitrogen and carbon) to deeper waters. Iron-limited diatoms are inhibited in their utihzation of nitrogen as a result of the iron requirement of nitrate reductase (Geider and LaRoche, 1994). However, iron-limited diatoms continue to take up silicic acid, although at lowered rates (De La Rocha et al., 2000). As a result, the Si N ratios of iron-limited diatoms... 

Tungsten inhibits molybdenum utilization, which is essential for the induction of nitrate reductase. 

Many metabolic processes such as glycolysis, Krebs cycle reactions, photosynthesis, protein synthesis, and lipid metabolism are affected by exposure to F. Much of the action of F on these processes can be attributed to F-dependent inhibition of enzymes. Examples of enzymes shown to be inhibited by F include enolase, phosphoglucomutase, phosphatase, hexokinase, PEP carboxylase, pyruvate kinase, succinic dehydrogenase, malic dehydrogenase, pyrophosphatase, phytase, nitrate reductase, mitochondrial ATPase, urease (Miller et al. 1983), lipase (Yu et al. 1987), amylase (Yu et al. 1988), invertase (Yu 1996 Ouchi et al. 1999), and superoxide dismutase (SOD) (Wilde and Yu 1998). 

Reduction of Cu and Fe organic complexes is saturable, and is inhibited by cell-impermeable probes, including polyclonal antibodies raised to Chlorella nitrate reductase (Jones and Morel, 1987). The model proposed suggests that a fraction of a membrane-bound nitrate reductase (the so-called diaphorase component that catalyzes NADH reduction), spans the cell membrane (Fig. 4). Organic metal complexes intercept electrons (destined for nitrate reduction intracellularly) at the outer cell surface. 

Ward, M. R., R. Tischncr, and R (. 11nflakcr (1988), Inhibition of Nitrate Transport by Anti nitrate Reductase IgG Fragments and the Identification of Plasma Membrane Associated Nitrate Reductase in Roots of Hailey Seedlings, Plant Physiol. 88, 114 1145. 

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. 

Electrons are transferred by the respiratory electron chain to a cytochrome b which then donates electrons to the Mo-protein (nitrate reductase). By using reduced viologens it should be possible to determine the kinetics of nitrate reduction by the nitrate reductase however, no information on this aspect is currently available. The sensitivity of nitrate reduction to inhibition by cyanide is considered to be due to association of the inhibitor with molybdenum residues in the nitrate reductase (Enoch and Lester, 1975). Cytochrome b involved in nitrate reduction does not bind cyanide. 

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. 

Although there are reports of inhibition of nitrate reductase by chelating agents which might chelate nonheme iron, analyses of the purified enzymes from eukaryotes have failed to reveal the Fe-sulfur complexes detected in the dissimilatory nitrate reductases from prokaryotes (Garrett and Nason, 1969 Ahmed and Spiller, 1976). The extensively purified nitrate reductases should be amenable for study with epr in order to determine the involvement of molybdenum in the reduction process. 

The mechanism of inhibition by oxygen of induction of dissimilatory nitrate reductase is not understood. However, the inhibition is not specific for nitrate reductase since a transitioi to anaerobic conditions results in the... 

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