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Biochemistry. Biokhimiia Oct 2007Mitochondrial cytochrome c oxidase is able to oxidize various aromatic compounds like o-dianisidine, benzidine and its derivatives (diaminobenzidine, etc.),...
Mitochondrial cytochrome c oxidase is able to oxidize various aromatic compounds like o-dianisidine, benzidine and its derivatives (diaminobenzidine, etc.), p-phenylenediamine, as well as amidopyrine, melatonin, and some other pharmacologically and physiologically active substances via the peroxidase, but not the oxidase mechanism. Although specific peroxidase activity of cytochrome c oxidase is low compared with classical peroxidases, its activity may be of physiological or pathophysiological significance due to the presence of rather high concentrations of this enzyme in all tissues, as well as specific localization of the enzyme in the mitochondrial membrane favoring accumulation of hydrophobic aromatic substances.
Topics: Animals; Biochemistry; Catalysis; Cattle; Dose-Response Relationship, Drug; Electron Transport Complex IV; Kinetics; Mitochondria; Models, Chemical; Oxygenases; Peroxidases; Peroxides; Spectrophotometry
PubMed: 18021064
DOI: 10.1134/s0006297907100045 -
The New Phytologist 2007The respective distribution of superoxide (O(2) (.-)) and hydrogen peroxide (H(2)O(2)), two reactive oxygen species (ROS) involved in root growth and differentiation,...
The respective distribution of superoxide (O(2) (.-)) and hydrogen peroxide (H(2)O(2)), two reactive oxygen species (ROS) involved in root growth and differentiation, was determined within the Arabidopsis root tip. We investigated the effect of changing the levels of these ROS on root development and the possible interactions with peroxidases. H(2)O(2) was detected by confocal laser-scanning microscopy using hydroxyphenyl fluorescein (HPF). Both O(2) (.-) accumulation and peroxidase distribution were assessed by light microscopy, using nitroblue tetrazolium (NBT) and o-dianisidine, respectively. Root length and root hair length and density were also quantified following ROS scavenging. O(2) (.-) was predominantly located in the apoplast of cell elongation zone, whereas H(2)O(2) accumulated in the differentiation zone and the cell wall of root hairs in formation. Treatments that decrease O(2) (.-) concentration reduced root elongation and root hair formation, while scavenging H(2)O(2) promoted root elongation and suppressed root hair formation. The results allow to precise the respective role of O(2) (.-) and H(2)O(2) in root growth and development. The consequences of their distinct accumulation sites within the root tip are discussed, especially in relation to peroxidases.
Topics: Arabidopsis; Hydrogen Peroxide; Peroxidases; Plant Roots; Seedlings; Superoxides
PubMed: 17388896
DOI: 10.1111/j.1469-8137.2007.01995.x -
FEMS Immunology and Medical Microbiology Jun 2007Isolates of Burkholderia cenocepacia express a putative haem-binding protein (molecular mass 97 kDa) that displays intrinsic peroxidase activity. Its role has been...
Isolates of Burkholderia cenocepacia express a putative haem-binding protein (molecular mass 97 kDa) that displays intrinsic peroxidase activity. Its role has been re-evaluated, and we now show that it is a bifunctional catalase-peroxidase, with activity against tetramethylbenzidine (TMB), o-dianisidine, pyrogallol, and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic) acid (ABTS). Both peroxidase and catalase activities are optimal at pH 5.5-6.0. The gene encoding this enzyme was cloned and expressed in Escherichia coli. We have named it katG because of its similarity to other katGs, including that from Burkholderia pseudomallei. It is substantially similar to a previously described catalase-peroxidase of B. cenocepacia (katA). MS analysis indicated that the initial katG translation product may be post-translationally modified in B. cenocepacia to give rise to the mature 97-kDa catalase-peroxidase.
Topics: Amino Acid Sequence; Bacterial Proteins; Burkholderia; Cloning, Molecular; Molecular Sequence Data; Peroxidases
PubMed: 17371508
DOI: 10.1111/j.1574-695X.2007.00224.x -
Developmental Dynamics : An Official... Mar 2007Hematopoiesis in vertebrate development involves an embryonic, primitive wave and a later, definitive wave in which embryonic blood cells are replaced with adult blood...
Hematopoiesis in vertebrate development involves an embryonic, primitive wave and a later, definitive wave in which embryonic blood cells are replaced with adult blood cells. We here show that zebrafish fgf1 is involved in vivo in primitive hematopoiesis. Fibroblast growth factor-1 (FGF1) morpholino knockdown leads to abnormal accumulation of blood cells in the posterior intermediate cell mass at 32 hr postfertilization. Expression of the erythroid markers gata1 and ika, normally diminishing in differentiating erythrocytes at this stage, is maintained at abnormally high levels in primitive blood cells. The onset of erythrocyte differentiation as assessed by o-dianisidine staining is severely delayed. Most fgf1 morphants later recover to wild-type appearance, and primitive erythrocytes eventually differentiate. Zebrafish fgf1 is syntenic to human FGF1, which maps to a critically deleted region in human del(5q) syndrome posing an increased risk of leukemia to patients. As its knockdown in zebrafish changes expression of gata1, a gene involved in hematopoietic stem cell decisions, FGF1 should be considered to play a role in the pathogenesis of del(5q) syndrome.
Topics: Animals; Cell Differentiation; Erythrocytes; Erythropoiesis; Fibroblast Growth Factor 1; GATA1 Transcription Factor; Gene Expression Regulation, Developmental; Ikaros Transcription Factor; Phylogeny; Reverse Transcriptase Polymerase Chain Reaction; Zebrafish; Zebrafish Proteins
PubMed: 17219402
DOI: 10.1002/dvdy.21056 -
Journal of Insect Science (Online) Dec 2005Many researchers investigating plant-insect interactions maintain their insect colonies on artificial diet and assume that salivary enzymes and elicitors remain...
Many researchers investigating plant-insect interactions maintain their insect colonies on artificial diet and assume that salivary enzymes and elicitors remain representative of natural situations. These salivary elicitors, such as the enzyme glucose oxidase (GOX), play important roles in influencing plant defense responses. In fact, GOX has been implicated in suppressing induced nicotine-production in tobacco plants (Musser et al., 2002). In this study, we investigated the effect of artificial or plant diet on the GOX activity in caterpillars of the beet armyworm, Spodoptera exigua. In the later developmental stages, whole body GOX of S. exigua caterpillars reared on a wheat germ-based artificial diet is over ten times higher than when insects were fed plants of the legume, Medicago trunctula. Labial salivary GOX accounted for most of this whole body activity in 4th instar caterpillars (57.8%), with the remaining activity present in the carcass. Hemolymph GOX levels were below the detection limits of the o-dianisidine-peroxidase assay used to measure activity. Labial salivary GOX activity was significantly higher in 4th larval instars reared on artificial diet compared with plant-fed caterpillars (U/mg per pair labial salivary gland, p = 0.0062), suggesting that diet effects GOX activity. When 4th instar plant-fed caterpillars were transferred to artificial diet, increased labial salivary GOX activity is closely correlated with the amount of time spent feeding on artificial diet. This study shows that the labial salivary GOX activity of S. exigua caterpillars is dependent on diet and developmental stage and that caution must be exercised in the design of plant-insect experiments.
Topics: Animals; Diet; Glucose Oxidase; Larva; Medicago truncatula; Saliva; Spodoptera
PubMed: 17119630
DOI: 10.1093/jis/5.1.48 -
The FEBS Journal Jul 2006Candida albicans is the most prevalent yeast pathogen in humans, and recently it has become increasingly resistant to the current antifungal agents. In this study we... (Comparative Study)
Comparative Study
Candida albicans is the most prevalent yeast pathogen in humans, and recently it has become increasingly resistant to the current antifungal agents. In this study we investigated C. albicans dihydroorotate dehydrogenase (DHODH, EC 1.3.99.11), which catalyzes the fourth step of de novo pyrimidine synthesis, as a new target for controlling infection. We propose that the enzyme is a member of the DHODH family 2, which comprises mitochondrially bound enzymes, with quinone as the direct electron acceptor and oxygen as the final electron acceptor. Full-length DHODH and N-terminally truncated DHODH, which lacks the targeting sequence and the transmembrane domain, were subcloned from C. albicans, recombinantly expressed in Escherichia coli, purified, and characterized for their kinetics and substrate specificity. An inhibitor screening with 28 selected compounds was performed. Only the dianisidine derivative, redoxal, and the biphenyl quinoline-carboxylic acid derivative, brequinar sodium, which are known to be potent inhibitors of mammalian DHODH, markedly reduced C. albicans DHODH activity. This study provides a background for the development of antipyrimidines with high efficacy for decreasing in situ pyrimidine nucleotide pools in C. albicans.
Topics: Amino Acid Motifs; Amino Acid Sequence; Aminobiphenyl Compounds; Biphenyl Compounds; Candida albicans; Conserved Sequence; Dihydroorotate Dehydrogenase; Enzyme Inhibitors; Escherichia coli; Glutathione Transferase; Humans; Hydrogen-Ion Concentration; Inhibitory Concentration 50; Kinetics; Molecular Sequence Data; Molecular Structure; Mutation; Oxidoreductases Acting on CH-CH Group Donors; Protein Structure, Tertiary; Recombinant Fusion Proteins; Sequence Homology, Amino Acid; Substrate Specificity
PubMed: 16774642
DOI: 10.1111/j.1742-4658.2006.05327.x -
Applied and Environmental Microbiology Sep 2005A multicopper oxidase gene from Staphylococcus aureus was cloned and overexpressed. Purified recombinant multicopper oxidase oxidized the substrate...
A multicopper oxidase gene from Staphylococcus aureus was cloned and overexpressed. Purified recombinant multicopper oxidase oxidized the substrate 3,3'-dimethoxybenzidine in the presence of copper. Disruption of mco showed copper sensitivity and H(2)O(2) resistance, suggesting roles for mco in copper homeostasis and oxidative stress response. Northern blot analysis showed copper-induced mco transcription.
Topics: Cloning, Molecular; Copper; Dianisidine; Gene Expression Regulation, Bacterial; Heat-Shock Response; Hydrogen Peroxide; Molecular Sequence Data; Oxidation-Reduction; Oxidative Stress; Oxidoreductases; Sequence Analysis, DNA; Staphylococcus aureus
PubMed: 16151171
DOI: 10.1128/AEM.71.9.5650-5653.2005 -
Journal of Clinical Laboratory Analysis 2004A spectrophotometric method based on myeloperoxidase activity for the determination of leukocytes in urine is described. Red cells that may be found in urine samples...
A spectrophotometric method based on myeloperoxidase activity for the determination of leukocytes in urine is described. Red cells that may be found in urine samples were lysed by an ammonium chloride method. Leukocytes were then sedimented by centrifugation and lysed using Triton X-100 (Sigma Chemicals Co., St. Louis, MO). Myeloperoxidase-catalyzed oxidation of o-dianisidine was carried out at 37 degrees C, pH 7. The reaction was stopped with the addition of 2 M H2SO4, and a stable form of oxidized o-dianisidine in acidic solution was obtained. Solid particles that may be found in urine samples were removed by centrifugation to avoid turbidity, and absorbance values of the supernatants were recorded at 400 nm. An Average number of leukocytes were noted per number of fields by microscopic examination and were related with the absorbance values of the supernatants at 400 nm. Pearson correlation (r) between our presented spectrophotometric analysis results and visual microscopic analysis was 0.877. Roche Combur 10-test M strips (Roche, Mannheim, Germany) and Multistix 10 SG Bayer test strips (Bayer Diagnostics, UK) were 0.645 and 0.648, respectively (P < 0.0001).
Topics: Dianisidine; Humans; Leukocyte Count; Leukocytes; Peroxidase; Reagent Strips; Spectrophotometry; Urine
PubMed: 15202119
DOI: 10.1002/jcla.20032 -
The Journal of Biological Chemistry May 2003Catalase-peroxidases (KatGs) are unique peroxidases exhibiting a high catalase activity and a peroxidase activity with a wide range of artificial electron donors....
Catalase-peroxidases (KatGs) are unique peroxidases exhibiting a high catalase activity and a peroxidase activity with a wide range of artificial electron donors. Exchange of tyrosine 249 in Synechocystis KatG, a distal side residue found in all as yet sequenced KatGs, had dramatic consequences on the bifunctional activity and the spectral features of the redox intermediate compound II. The Y249F variant lost catalase activity but retained a peroxidase activity (substrates o-dianisidine, pyrogallol, guaiacol, tyrosine, and ascorbate) similar to the wild-type protein. In contrast to wild-type KatG and similar to monofunctional peroxidases, the formation of the redox intermediate compound I could be followed spectroscopically even by addition of equimolar hydrogen peroxide to ferric Y249F. The corresponding bimolecular rate constant was determined to be (1.1 +/- 0.1) x 107 m-1 s-1 (pH 7 and 15 degrees C), which is typical for most peroxidases. Additionally, for the first time a clear transition of compound I to an oxoferryl-like compound II with peaks at 418, 530, and 558 nm was monitored when one-electron donors were added to compound I. Rate constants of reaction of compound I and compound II with tyrosine ((5.0 +/- 0.3) x 104 m-1 s-1 and (1.7 +/- 0.4) x 102 m-1 s-1) and ascorbate ((1.3 +/- 0.2) x 104 m-1 s-1 and (8.8 +/- 0.1) x 101 m-1 s-1 at pH 7 and 15 degrees C) were determined by using the sequential stopped-flow technique. The relevance of these findings is discussed with respect to the bifunctional activity of KatGs and the recently published first crystal structure.
Topics: Amino Acid Sequence; Bacterial Proteins; Base Sequence; Circular Dichroism; Cyanobacteria; DNA Primers; Electron Spin Resonance Spectroscopy; Kinetics; Molecular Sequence Data; Peroxidases; Sequence Homology, Amino Acid; Spectrophotometry, Ultraviolet; Tyrosine
PubMed: 12649295
DOI: 10.1074/jbc.M211625200 -
The Journal of Biological Chemistry Apr 2003Catalase-peroxidase (KatG) from Mycobacterium tuberculosis is responsible for the activation of the antitubercular drug isonicotinic acid hydrazide (INH) and is...
Catalase-peroxidase (KatG) from Mycobacterium tuberculosis is responsible for the activation of the antitubercular drug isonicotinic acid hydrazide (INH) and is important for survival of M. tuberculosis in macrophages. Characterization of the structure and catalytic mechanism of KatG is being pursued to provide insights into drug (INH) resistance in M. tuberculosis. Site-directed mutagenesis was used to prepare the INH-resistant mutant KatG[S315T], and the overexpressed enzyme was characterized and compared with wild-type KatG. KatG[S315T] exhibits a reduced tendency to form six-coordinate heme, because of coordination of water to iron during purification and storage, and also forms a highly unstable Compound III (oxyferrous enzyme). Catalase activity and peroxidase activity measured using t-butylhydroperoxide and o-dianisidine were moderately reduced in the mutant compared with wild-type KatG. Stopped-flow spectrophotometric experiments revealed a rate of Compound I formation similar to wild-type KatG using peroxyacetic acid to initiate the catalytic cycle, but no Compound I was detected when bulkier peroxides (chloroperoxybenzoic acid, t-butylhydroperoxide) were used. The affinity of resting (ferric) KatG[S315T] for INH, measured using isothermal titration calorimetry, was greatly reduced compared with wild-type KatG, as were rates of reaction of Compound I with the drug. These observations reveal that although KatG[S315T] maintains reasonably good steady state catalytic rates, poor binding of the drug to the enzyme limits drug activation and brings about INH resistance.
Topics: Antitubercular Agents; Bacterial Proteins; Catalase; Drug Resistance, Bacterial; Iron; Isoniazid; Kinetics; Mutagenesis, Site-Directed; Mutation, Missense; Mycobacterium tuberculosis; Peroxidases; Spectrum Analysis
PubMed: 12586821
DOI: 10.1074/jbc.M300326200