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 amino acid modifier phenylglyoxal (PG) gradually inactivated the methyl viologen-coupled nitrate reductase activity of the anoxically grown whole cells of Paracoccus denitrificans. A double log plot of the pseudo-first-order inactivation rate constant versus PG concentration was linear with a mean slope of 1.4 (0.1M sodium phosphate) or 0.87 (0.1M sodium borate). Phenylglyoxalation of cells lowered the limiting velocity (V), while hardly affecting the apparent half-saturation concentration (K(m)) of nitrate. Nitrate afforded no protection against inactivation. The inhibition by PG could be removed by the detergent Triton X-100 or by the lipid-soluble tetraphenylphosphonium countercation, suggesting that PG exerts its effect at the level of nitrate transport. Based on studies with membrane potential- and pH-sensitive fluorescent probes, the inhibition was shown not to be due to changes in the electrochemical gradient of hydrogen ions. Both K(m) and V values for nitrate uptake increased in a hyperbolic fashion in response to exogenously added nitrite. Nitrite promoted a bypass of the inhibition caused by low concentrations of the proton-conducting agent carbonyl cyanide m-chlorophenylhydrazone (CCCP), but was almost ineffective in the case of the PG block. These results are rationalized in terms of two nitrate import pathways that are comparably inhibited by PG and differ in their sensitivities to CCCP. A simplified kinetic model for phenylglyoxalation is proposed to account for the observed nonintegral reaction orders.
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PMID:Inhibition by phenylglyoxal of nitrate transport in Paracoccus denitrificans: a comparison with the effect of a protonophorous uncoupler. 1250 99

Previously, it has been shown that treatment of Paracoccus denitrificans cells with phenylglyoxal inhibits the methyl-viologen-linked nitrate reductase activity by blocking the nitrate transporter. This inhibition disappears if tetraphenylphosphonium cation (TPP(+)) is added to the assay medium. In the present paper, the following evidence suggests that the effect of TPP(+) results from an increased transmembrane anion permeability and not from transporter reactivation or cell lysis. (1) Beside nitrate, TPP(+) also mediated the utilisation of chlorate, which normally lacks access to the cytoplasm. (2) The TPP(+) pathway had about hundred-times higher K(m) values for nitrate and chlorate than nitrate reductase in Triton X-100 permeabilised cells. (3) Although the uncoupler CCCP alone failed to overcome the PG block, it stimulated the operation of the TPP(+) pathway. (4) The method of continuous variations allowed the transport stoichiometry TPP(+)/NO(3)(-) to be determined as 3, indicating charge compensation for nitrate movement and the subsequent transmembrane two-electron redox reaction. Anion uptake was also measured independently from passive swelling of uncoupled spheroplasts in iso-osmotic solutions of ammonium salts. The permeability to nitrate lay in the permeability sequence Cl(-)<NO(3)(-)<ClO(4)(-)<SCN(-) and was further enhanced by TPP(+).
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PMID:Passive penetration of nitrate through the plasma membrane of Paracoccus denitrificans and its potentiation by the lipophilic tetraphenylphosphonium cation. 1261 55

A nitrate reductase was solubilized with Triton X-100 from the membranes of Pseudomonas chlororaphis DSM 50135 grown microaerobically in the presence of nitrate. Like other membrane-bound nitrate reductases, it contains three subunits, of 129, 66 (64) and 24 kDa, referred to in the literature as alpha, beta and gamma, respectively. Electrocatalytic studies revealed that only the membrane-bound, not the solubilized form of the enzyme, can accept electrons from a menaquinone analog, menadione, whereas both forms can accept electrons from methylviologen. The isolated enzyme possesses several iron-sulfur clusters and a molybdopterin guanine dinucleotide active center. The iron-sulfur clusters can be grouped in two classes according to their redox properties, the high-potential and low-potential clusters. In the as-isolated enzyme, two forms of the molybdenum center, high- and low-pH, are detectable by electron paramagnetic resonance spectroscopy. The low-pH form shows a hyperfine splitting due to a proton, suggesting the presence of an -OHx ligand. Dithionite reduces the Mo(V) center to Mo(IV) and subsequent reoxidization with nitrate originates a new Mo(V) signal, identical to the oxidized low-pH form but lacking its characteristic hyperfine splitting. The isolated preparation also contains heme c (in a sub-stoichiometric amount) with the ability to relay electrons to the molybdenum center, suggesting that this nitrate reductase may contain heme c instead of the heme b usually found in this class of enzymes.
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PMID:Isolation and spectroscopic characterization of the membrane-bound nitrate reductase from Pseudomonas chlororaphis DSM 50135. 1580 88

Xanthomonas maltophilia ATCC 17666 is an obligate aerobe that accumulates nitrite when grown on nitrate. Spectra of membranes from nitrate-grown cells exhibited b-type cytochrome peaks and A(615-630) indicative of d-type cytochrome but no absorption peaks corresponding to c-type cytochromes. The nitrate reductase (NR) activity was located in the membrane fraction. Triton X-100-extracted reduced methyl viologen-NRs were purified on DE-52, hydroxylapatite, and Sephacryl S-300 columns to specific activities of 52 to 67 mumol of nitrite formed per min per mg of protein. The cytochrome-containing NR(I) separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis into a 135-kDa alpha-subunit, a 64-kDa beta-subunit, and a 23-kDa gamma-subunit with relative band intensities indicative of a 1:1:1 alpha/beta/gamma subunit ratio and a M(r) of 222,000. The electronic spectrum of dithionite-reduced purified NR displayed peaks at 425, 528, and 558 nm, indicative of the presence of a cytochrome b, an interpretation consistent with the pyridine hemochrome spectrum formed. The cytochrome b of the NR was reduced under anaerobic conditions by menadiol and oxidized by nitrate with the production of nitrite. This NR contained 0.96 Mo, 12.5 nonheme iron, and 1 heme per 222 kDa: molybdopterin was detected with the Neurospora crassa nit-1 assay. A smaller reduced methyl viologen-NR (169 kDa), present in various concentrations in the Triton X-100 preparations, lacked a cytochrome spectrum and did not oxidize menadiol. The characteristics of the NRs and the absence of c-type cytochromes provide insights into why X. maltophilia accumulates nitrite.
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PMID:Purification of Two Nitrate Reductases from Xanthomonas maltophilia Grown in Aerobic Cultures. 1634 5

Latent nitrate reductase activity (NRA) was detected in corn (Zea mays L., Golden Jubilee) root microsome fractions. Microsome-associated NRA was stimulated up to 20-fold by Triton X-100 (octylphenoxy polyethoxyethanol) whereas soluble NRA was only increased up to 1.2-fold. Microsome-associated NRA represented up to 19% of the total root NRA. Analysis of microsomal fractions by aqueous two-phase partitioning showed that the membrane-associated NRA was localized in the second upper phase (U2). Analysis with marker enzymes indicated that the U2 fraction was plasma membrane (PM). The PM-associated NRA was not removed by washing vesicles with up to 1.0 M NACl but was solubilized from the PM with 0.05% Triton X-100. In contrast, vanadate-sensitive ATPase activity was not solubilized from the PM by treatment with 0.1% Triton X-100. The results show that a protein capable of reducing nitrate is embedded in the hydrophobic region of the PM of corn roots.
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PMID:Latent nitrate reductase activity is associated with the plasma membrane of corn roots. 2421 88


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