Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Menadione is a synthetic derivative of the natural vitamins K with antiinflammatory activity among its potentially significant clinical properties. We have found this agent to stimulate the production of superoxide anion (O2-) in human polymorphonuclear leukocytes (PMN) and dimethylsulfoxide-differentiated HL-60 cells in a time-, cell number-, and drug concentration-dependent manner. Conversely, menadione attenuates both O2- production and lysozyme release in cells stimulated by phorbol myristate acetate (PMA), fMet-Leu-Phe, or Ca2+ ionophore. 4-Acetamido-4'-isothiocyano-2-2'-disulfonic acid stilbene and 4,4'-diisothiocyano-2-2'disulfonic acid stilbene, agents which inhibit transmembrane O2-) flux, do not alter menadione's effects on superoxide dismutase (SOD) inhibitable cytochrome c reduction in resting or PMA-stimulated PMN. Likewise, quinone reductase inhibitors, warfarin and dicumarol, known to attenuate vitamin K-dependent responses and enhance quinone-mediated oxidative stress, have no effect upon menadione-stimulated O2- production. Furthermore, menadione-induced suppression of stimulus-mediated lysozyme release is not reversed by cotreatment with oxygen metabolite scavenging enzymes SOD and catalase. Nevertheless, under conditions of restricted oxygen supply, the suppressive effect of menadione on stimulant-induced lysozyme release is greatly diminished. Thus, although pharmacological manipulation suggests otherwise, there appears to exist at least a component of the inhibitory activity of menadione that is oxygen dependent, and may be oxidative stress-related.
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PMID:Alteration of human granulocyte functional responses by menadione. 170 Jun 67

A simple, rapid, accurate, and precise colorimetric assay for the determination of L-phenylalanine in plasma samples using L-phenylalanine dehydrogenase [L-phenylalanine:NAD+-oxidoreductase (deaminating)] from Rhodococcus sp. M 4 is described. The enzyme catalyzes the NAD-dependent oxidative deamination of L-phenylalanine. However, the equilibrium of reaction favors L-phenylalanine formation. By stoichiometric coupling of this reaction with diaphorase/iodonitro tetrazolium chloride (INT) the formed NADH converts INT to a formazan whereby the reaction is displaced in favor of phenylpyruvate. Using a kinetic approach the increase in absorbance at 492 nm shows linearity over more than 30 min. Deproteinized standard solutions of L-phenylalanine in the range from 30 to 1200 mumol/liter show a linearity between the dAformazan/30 min and the substrate concentration. In phenylketonuria (PKU) plasma samples no interferences caused by L-tyrosine or phenylpyruvic acid are seen. Applicability is demonstrated by comparative determination of plasma L-phenylalanine of treated PKU patients by the colorimetric method and automated amino acid analysis.
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PMID:Monitoring of phenylketonuria: a colorimetric method for the determination of plasma phenylalanine using L-phenylalanine dehydrogenase. 281 48

Vitamin K-dependent carboxylase activity has been demonstrated in the crude microsomal fraction of the intima of bovine aortae. The procedure for the isolation of vessel wall carboxylase is a slight modification of the general preparation procedure for tissue microsomes. The highest activity of the non-hepatic enzyme was observed at 25 degrees C and hardly any NADH-dependent vitamin K reductase could be demonstrated. The optimal reaction conditions for both vessel wall as well as liver carboxylase were similar: 0.1 M-NaCl/0.05 M-Tris/HCl, pH 7.4, containing 8 mM-dithiothreitol, 0.4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonic acid (CHAPS), 0.4 mM-vitamin K hydroquinone and 2 M-(NH4)2SO4. Warfarin inhibits the hepatic and non-hepatic carboxylase/reductase enzyme complex more or less to a similar degree. We have measured the apparent Km values for the following substrates: Phe-Leu-Glu-Glu-Leu ('FLEEL'), decarboxylated osteocalcin, decarboxylated fragment 13-29 from descarboxyprothrombin and decarboxylated sperm 4-carboxyglutamic acid-containing (Gla-)protein. The results obtained demonstrated that liver and vessel wall carboxylase may be regarded as isoenzymes with different substrate specificities. The newly discovered enzyme is the first vitamin K-dependent carboxylase which shows an absolute substrate specificity: FLEEL and decarboxylated osteocalcin were good substrates for vessel wall carboxylase, but decarboxylated fragment 13-29 and decarboxylated sperm Gla-protein were not carboxylated at all.
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PMID:Isolation and partial characterization of a vitamin K-dependent carboxylase from bovine aortae. 349 40

The vitamin K-dependent carboxylase extracted from rat liver microsomes by 3-([3-cholamidopropyl] dimethylammoniol)-1-propane sulfonate detergent solution has been partially purified by chromatography on Ultrogel AcA-34 followed by carboxymethyl-Sepharose chromatography and pentapeptide affinity chromatography. The carboxylase appears to be composed of two proteins, the enzyme and endogenous substrate as judged by the incorporation of 14CO2 into trichloroacetic acid insoluble protein. The apparent Km for Phe-Leu-Glu-Glu-Leu as carboxylation substrate is approximately 3 mM. 2,3,5,6-Tetrachloro-4-pyridinol at 10 microM inhibits 90% of the enzyme activity, whereas maximal stimulation (1.7-fold) by pyridoxal 5'-phosphate is obtained at 1 mM and by Mn2+ at 5 mM. The stimulation by pyridoxal 5'-phosphate and by Mn2+ are not additive. The carboxylation of Phe-Leu-Glu-Glu-Leu at 20 degrees C is linear for 90 min. Vitamin K1 plus NADH do not replace vitamin K1 hydroquinone, indicating that vitamin K reductase is not part of this purified carboxylase-substrate complex. Vitamin K epoxidase activity co-elutes with the carboxylase complex. Some 400-fold purification from microsomes has been obtained to yield enzyme preparations with a specific activity of approximately 17,000 pmol of CO2 fixed into peptide/mg of enzyme protein, which is some 15-fold greater than any previously reported enzyme preparation from rat liver microsomes.
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PMID:Vitamin K-dependent carboxylase. Partial purification and properties of the enzyme-substrate complex. 717 81

The carboxyl-terminal region of plant ferredoxin-NADP+ reductases is formed by an invariant alpha-helix/loop/beta-strand, culminating in a conserved tyrosine that displays extensive interaction with the prosthetic group FAD. We have investigated the potential role of the terminal region in reductase function, by introducing mutations and deletions on pea ferredoxin-NADP+ reductase overexpressed in Escherichia coli. Replacement of the terminal tyrosine by tryptophan, phenylalanine, serine, and glycine resulted in a 2.2-, 2.0-, 22-, and 302-fold reduction, respectively, in kcat for the diaphorase reaction, whereas elimination of the tyrosine caused a 846-fold decrease in kcat. Km values were largely unaffected by the substitutions. Similar results were obtained when the mutants were assayed for cytochrome c reduction, indicating that aromaticity is the most important factor to the function of the tyrosine in catalysis. The presence of the phenol ring at the carboxyl-terminal position of wild-type reductase is important, but not an absolute requirement for enzyme function or FAD assembly. Deletion of the alpha-helix/beta-strand region prevented reductase proper folding in the bacterial host, while shortening of the terminal region by splicing 3 amino acids at the beginning of the alpha-helix produced a moderately soluble reductase, devoid of FAD and enzymatic activity.
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PMID:Probing the role of the carboxyl-terminal region of ferredoxin-NADP+ reductase by site-directed mutagenesis and deletion analysis. 836 77

Escherichia coli fumarate reductase (FRD) is a four-subunit enzyme that catalyzes the terminal step in anaerobic respiration to fumarate. The hydrophobic FrdC and FrdD subunits anchor the FrdA and FrdB catalytic subunits to the inner surface of the cytoplasmic membrane and are required for the enzyme to interact with quinones. Thirty-five single-site mutations were constructed in the FrdC and FrdD polypeptides by site-directed mutagenesis. Each mutant enzyme was characterized for its ability to catalyze quinone oxidation and reduction and to support growth of E. coli DW35 (delta frdABCD sdhC::kan) under selective conditions requiring functional enzyme. Replacement of FrdCE29 with Asp, Leu, Lys, or Phe had a deleterious effect both on quinol oxidase and quinone reductase activities. Substitution of FrdCH82 with Arg, Leu, Tyr, or Glu also decreased menaquinol oxidase activity, but had variable effects on the reverse reaction, the reduction of ubiquinone. Data are presented to support the hypothesis that the positive charge at FrdCH82 is required for stabilization of the quinone radical intermediate and the negative charge at FrdCE29 for deprotonation of menaquinol. Other critical amino acids identified in FrdC included Ala-32, Phe-38, Trp-86, Phe-87, and in FrdD residues Phe-57, Gln-59, Ser-60, and His-80. The established roles of such residues in the QA and QB sites of the photosynthetic reaction center would suggest a similar type of structure operative in the FRD complex. In such a model, Glu-29, Ala-32, His-82, Trp-86 of FrdC and His-80 of FrdD are considered participants in a QB-type site, and FrdD Phe-57, Gln-59, and Ser-60 components in an apolar QA-type site.
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PMID:Escherichia coli fumarate reductase frdC and frdD mutants. Identification of amino acid residues involved in catalytic activity with quinones. 841 59

The Saccharomyces cerevisiae succinate dehydrogenase (SDH) of the mitochondrial electron transport chain oxidizes succinate and reduces ubiquinone. Using a random mutagenesis approach, we identified functionally important amino acid residues in one of the anchor subunits, Sdh4p. We analyzed three point mutations (F69V, S71A, and H99L) and one nonsense mutation (Y89OCH) that truncates the Sdh4p subunit at the third predicted transmembrane segment. The F69V and the S71A mutations result in greatly impaired respiratory growth in vivo and quinone reductase activities in vitro, with negligible effects on enzyme stability. In contrast, the Y89OCH and the H99L mutations elicit large structural perturbations that impair assembly as evidenced by reduced covalent FAD levels, membrane-associated succinate-phenazine methosulfate reductase activities, and thermal stability. We propose that the Phe-69 and the Ser-71 residues are involved in the formation of a quinone-binding site, whereas the His-99 residue is at the interface of the peripheral and the membrane domains. In addition, the properties of the Y89OCH mutation are consistent with the interpretation that the third transmembrane segment is not involved in catalysis but rather plays an important structural role. The mutant enzymes are differentially sensitive to a quinone analog inhibitor, providing further evidence for a two-quinone binding model in the yeast SDH.
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PMID:The Quinone-binding sites of the Saccharomyces cerevisiae succinate-ubiquinone oxidoreductase. 1127 23

The retinopetal neurons of Crocodylus niloticus were visualized by retrograde transport of rhodamine beta-isothiocyanate or Fast Blue administered by intraocular injection. Approximately 6,000 in number, these neurons are distributed in seven regions extending from the mesencephalic tegmentum to the rostral rhombencephalon, approximately 70% being located contralaterally to the injected eye. None of the centrifugal neurons projects to both retinae. The retinopetal neurons are located in rostrocaudal sequence in seven regions: the formatio reticularis lateralis mesencephali, the substantia nigra, the griseum centralis tectalis, the nucleus subcoeruleus dorsalis, the nucleus isthmi parvocellularis, the locus coeruleus, and the commissura nervi trochlearis. The greatest number of cells (approximately 93%) is found in the nucleus subcoeruleus dorsalis. The majority are multipolar or bipolar in shape and resemble the ectopic centrifugal visual neurons of birds, although a small number of monopolar neurons resembling those of the avian isthmo-optic nucleus may also be observed. A few retinopetal neurons in the griseum centralis tectalis were tyrosine hydroxylase (TH) immunoreactive. Moreover, in the nuclei subcoeruleus dorsalis and isthmi parvocellularis, both ipsilaterally and contralaterally, approximately one retinopetal neuron in three (35%) was immunoreactive to nitric oxide synthase (NOS), and a slightly higher proportion (38%) of retinopetal neurons were immunoreactive for choline acetyltransferase (ChAT). Some of them contained colocalized ChAT and NOS/reduced nicotinamide adenine dinucleotide phosphate-diaphorase. Fibers immunoreactive to TH, serotonin (5-HT), neuropeptide Y (NPY), or Phe-Met-Arg-Phe-amide (FMRF-amide) were frequently observed to make intimate contact with rhodamine-labeled retinopetal neurons. These findings are discussed in relation to previous results obtained in other reptilian species and in birds.
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PMID:Centrifugal visual system of Crocodylus niloticus: a hodological, histochemical, and immunocytochemical study. 1464 91

NAD(P)H quinone oxidoreductase 1 (NQO1) is a ubiquitous flavoenzyme that catalyzes two-electron reduction of quinones to hydroquinones utilizing NAD(P)H as an electron donor. NQO1 binds and stabilizes several short-lived proteins including the tumor suppressors p53 and p73 and the enzyme ornithine decarboxylase (ODC). Dicoumarol is a widely used potent competitive inhibitor of NQO1 enzymatic activity, which competes with NAD(P)H for binding to NQO1. Dicoumarol also disrupts the binding of NQO1 to p53, p73, and ODC and induces their ubiquitin-independent proteasomal degradation. We report here the crystal structure of human NQO1 in complex with dicoumarol at 2.75 A resolution. We have identified the interactions of dicoumarol with the different residues of NQO1 and the conformational changes imposed upon dicoumarol binding. The most prominent conformational changes that occur in the presence of dicoumarol involve Tyr 128 and Phe 232 that are present on the surface of the NQO1 catalytic pocket. On the basis of the comparison of the NQO1 structure in complex with different NQO1 inhibitors and our previous analysis of NQO1 mutants, we propose that the specific conformation of Tyr 128 and Phe 232 is important for NQO1 interaction with p53 and other client proteins.
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PMID:The crystal structure of NAD(P)H quinone oxidoreductase 1 in complex with its potent inhibitor dicoumarol. 1670 May 48

We describe here a new microquantification method of l-phenylalanine concentration in an extract from a dried blood spot by using the diaphorase-resazurin system. To miniaturize the fluorometric enzymatic microplate assay for the diagnosis of phenylketonuria, an enzyme chip immobilized with His-tag fused phenylalanine dehydrogenase (PheDH) was developed. His-tag fused PheDH was immobilized on the surface of nickel-coated slide glass. A microarray sheet (8 x 30 well) was fabricated with poly(dimethylsiloxane) (PDMS) using the photolithographic technique. An enzyme reaction chamber in a double-layered structure was constructed with different types of microarray PDMS sheets on the surface of Ni-coated slide glass immobilized with His-tagged PheDH. To evaluate the affinity toward the Ni-chelating ligand, eight kinds of His-tagged PheDH variants were constructed and expressed. (His)(6)- and (His)(9)-PheDH variants at the N terminus showed high adsorption ratio to Ni-chelating ligand. The V(max) and k(cat) values of the (His)(6)-PheDH variant at the N terminus for l-phenylalanine were higher than those of the (His)(9)-PheDH variant, and the (His)(6)-PheDH variant was found to be most suitable for immobilization onto nickel-coated slide glass. Fluorescence formed by resazurin-coupled enzymatic reaction (in a 0.2-microl reaction mixture) on the enzyme chip exhibited good linearity and a correlation coefficient up to 12.8 mg/dl of the l-phenylalanine-containing sample extracted from a dried blood spot on filter paper.
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PMID:Application of an enzyme chip to the microquantification of l-phenylalanine. 1704 6


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