(/sup 11/C)clorgyline and (/sup 11/C)-L-deprenyl and their use in measuring functional monoamine oxidase activity in the brain using positron emission tomography

DOEpatents

Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.

1986-04-17

This invention involves a new strategy for imaging the activity of the enzyme monoamine oxidase in the living body by using /sup 11/C-labeled enzyme inhibitors which bind irreversibly to an enzyme as a result of catalysis. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography. 2 figs.

Characterization of polyphenol oxidase from Cape gooseberry (Physalis peruviana L.) fruit.

PubMed

Bravo, Karent; Osorio, Edison

2016-04-15

Cape gooseberry (Physalis peruviana) is an exotic fruit highly valued, however it is a very rich source of polyphenol oxidase (PPO). In this study, Cape gooseberry PPO was isolated and biochemically characterized. The enzyme was extracted and purified using acetone and aqueous two-phase systems. The data indicated that PPO had the highest substrate affinity for chlorogenic acid, 4-methylcatechol and catechol. Chlorogenic acid was the most suitable substrate (Km=0.56±0.07 mM and Vmax=53.15±2.03 UPPO mL(-1) min(-1)). The optimal pH values were 5.5 for catechol and 4-methylcatechol and 5.0 for chlorogenic acid. Optimal temperatures were 40°C for catechol, 25°C for 4-methylcatechol and 20°C for chlorogenic acid. In inhibition tests, the most potent inhibitor was found to be ascorbic acid followed by L-cysteine and quercetin. This study shows possible treatments that can be implemented during the processing of Cape gooseberry fruits to prevent browning. Copyright © 2015 Elsevier Ltd. All rights reserved.

African Swine Fever Virus pB119L Protein Is a Flavin Adenine Dinucleotide-Linked Sulfhydryl Oxidase

PubMed Central

Rodríguez, Irene; Redrejo-Rodríguez, Modesto; Rodríguez, Javier M.; Alejo, Alí; Salas, José; Salas, María L.

2006-01-01

Protein pB119L of African swine fever virus belongs to the Erv1p/Alrp family of sulfhydryl oxidases and has been described as a late nonstructural protein required for correct virus assembly. To further our knowledge of the function of protein pB119L during the virus life cycle, we have investigated whether this protein possesses sulfhydryl oxidase activity, using a purified recombinant protein. We show that the purified protein contains bound flavin adenine dinucleotide and is capable of catalyzing the formation of disulfide bonds both in a protein substrate and in the small molecule dithiothreitol, the catalytic activity being comparable to that of the Erv1p protein. Furthermore, protein pB119L contains the cysteines of its active-site motif CXXC, predominantly in an oxidized state, and forms noncovalently bound dimers in infected cells. We also show in coimmunoprecipitation experiments that protein pB119L interacts with the viral protein pA151R, which contains a CXXC motif similar to that present in thioredoxins. Protein pA151R, in turn, was found to interact with the viral structural protein pE248R, which contains disulfide bridges and belongs to a class of myristoylated proteins related to vaccinia virus L1R, one of the substrates of the redox pathway encoded by this virus. These results suggest the existence in African swine fever virus of a system for the formation of disulfide bonds constituted at least by proteins pB119L and pA151R and identify protein pE248R as a possible final substrate of this pathway. PMID:16537584

African swine fever virus pB119L protein is a flavin adenine dinucleotide-linked sulfhydryl oxidase.

PubMed

Rodríguez, Irene; Redrejo-Rodríguez, Modesto; Rodríguez, Javier M; Alejo, Alí; Salas, José; Salas, María L

2006-04-01

Protein pB119L of African swine fever virus belongs to the Erv1p/Alrp family of sulfhydryl oxidases and has been described as a late nonstructural protein required for correct virus assembly. To further our knowledge of the function of protein pB119L during the virus life cycle, we have investigated whether this protein possesses sulfhydryl oxidase activity, using a purified recombinant protein. We show that the purified protein contains bound flavin adenine dinucleotide and is capable of catalyzing the formation of disulfide bonds both in a protein substrate and in the small molecule dithiothreitol, the catalytic activity being comparable to that of the Erv1p protein. Furthermore, protein pB119L contains the cysteines of its active-site motif CXXC, predominantly in an oxidized state, and forms noncovalently bound dimers in infected cells. We also show in coimmunoprecipitation experiments that protein pB119L interacts with the viral protein pA151R, which contains a CXXC motif similar to that present in thioredoxins. Protein pA151R, in turn, was found to interact with the viral structural protein pE248R, which contains disulfide bridges and belongs to a class of myristoylated proteins related to vaccinia virus L1R, one of the substrates of the redox pathway encoded by this virus. These results suggest the existence in African swine fever virus of a system for the formation of disulfide bonds constituted at least by proteins pB119L and pA151R and identify protein pE248R as a possible final substrate of this pathway.

Characterization of polyphenol oxidase from blueberry (Vaccinium corymbosum L.).

PubMed

Siddiq, M; Dolan, K D

2017-03-01

Polyphenol oxidase (PPO) was extracted and characterized from high-bush blueberries. PPO showed an optimum activity at pH 6.1-6.3 and 35°C, with the enzyme showing significant activity over a wide temperature range (25-60°C). Catechol was the most readily oxidized substrate followed by 4-methylcatechol, DL-DOPA, and dopamine. Blueberry PPO showed a K m of 15mM and V max of 2.57 ΔA 420 nm/min×10 -1 , determined with catechol. PPO was completely inactivated in 20min at 85°C, however, after 30minat 75°C it showed about 10% residual activity. Thermal treatment at 55 and 65°C for 30min resulted in the partial inactivation of PPO. Ascorbic acid, sodium diethyldithiocarbamic acid, L-cysteine, and sodium metabisulfite were effective inhibitors of PPO at 1.0mM. Benzoic acid and cinnamic acid series inhibitors showed relatively weak inhibition of PPO (21.8-27.6%), even at as high as 2.0mM concentration. Copyright © 2016 Elsevier Ltd. All rights reserved.

Localization of ascorbic acid, ascorbic acid oxidase, and glutathione in roots of Cucurbita maxima L.

PubMed

Liso, Rosalia; De Tullio, Mario C; Ciraci, Samantha; Balestrini, Raffaella; La Rocca, Nicoletta; Bruno, Leonardo; Chiappetta, Adriana; Bitonti, Maria Beatrice; Bonfante, Paola; Arrigoni, Oreste

2004-12-01

To understand the function of ascorbic acid (ASC) in root development, the distribution of ASC, ASC oxidase, and glutathione (GSH) were investigated in cells and tissues of the root apex of Cucubita maxima. ASC was regularly distributed in the cytosol of almost all root cells, with the exception of quiescent centre (QC) cells. ASC also occurred at the surface of the nuclear membrane and correspondingly in the nucleoli. No ASC could be observed in vacuoles. ASC oxidase was detected by immunolocalization mainly in cell walls and vacuoles. This enzyme was particularly abundant in the QC and in differentiating vascular tissues and was absent in lateral root primordia. Administration of the ASC precursor L-galactono-gamma-lactone markedly increased ASC content in all root cells, including the QC. Root treatment with the ASC oxidized product, dehydroascorbic acid (DHA), also increased ASC content, but caused ASC accumulation only in peripheral tissues, where DHA was apparently reduced at the expense of GSH. The different pattern of distribution of ASC in different tissues and cell compartments reflects its possible role in cell metabolism and root morphogenesis.

Recent advances in Parkinson's disease therapy: use of monoamine oxidase inhibitors.

PubMed

Henchcliffe, Claire; Schumacher, H Christian; Burgut, F Tuna

2005-11-01

Monoamine oxidase inhibitors inhibit dopamine metabolism and are therefore effective in treating Parkinson's disease, a condition associated with progressive striatal dopamine deficiency secondary to degeneration of dopaminergic neurons in the substantia nigra. Selegiline is currently the most widely used monoamine oxidase-B inhibitor for Parkinson's disease, but has a low and variable bioavailability, and is metabolized to L-methamphetamine and L-amphetamine that carry a risk for potential neurotoxicity. There are two new approaches that circumvent these potential disadvantages. First, selegiline orally disintegrating tablets provide a novel delivery form of selegiline, avoiding first pass metabolism by rapid absorption through the oral mucosa, thus leading to significantly lower plasma concentrations of L-metamphetamine and L-amphetamine. Selegiline orally disintegrating tablets prove to be clinically effective and safe in patients with moderately advanced Parkinson's disease. Second, rasagiline is a new monoamine oxidase inhibitor, without known neurotoxic metabolites. In large clinical trials, rasagiline proves effective as monotherapy in early Parkinson's disease, as well as adjunctive therapy to levodopa in advanced disease. Clinical data suggest, in addition, a disease-modifying effect of rasagiline that may correlate with neuroprotective activity of monoamine oxidase-B inhibitors in animal models of Parkinson's disease.

Novel strategy for phenyllactic acid biosynthesis from phenylalanine by whole cell recombinant Escherichia coli coexpressing L-phenylalanine oxidase and L-lactate dehydrogenase.

PubMed

Zhang, Jianzhi; Li, Xi

2018-01-01

To enhance the efficiency of phenyllactic acid (PLA) production from L-phenylalanine (L-Phe) by introducing a novel artificial pathway into Escherichia coli RESULTS: The production of PLA from L-Phe by recombinant E. coli (ldh-lpox) coexpressing L-phenylalanine oxidase and L-lactate dehydrogenase was studied. The new PLA synthesis pathway was confirmed to be efficient in recombinant E. coli. Subsequently, two different biocatalyst processes were carried out and optimized for PLA production. In the whole cell biosynthesis process at high cell density using collected recombinant cells as catalyst, at optimal conditions (L-Phe 6 g/l, pH 7.5, 35 °C, CDW 24.5 g/l and 200 rpm), the recombinant E. coli (ldh-lpox) produced 1.62 g PLA/l with a conversion of 28% from L-Phe. Similarly, during the two-temperature-stage fermentation process in flasks using IPTG-induced cells, the temperature in the second stage was increased to 35 °C to benefit the biocatalyst process, and comparable phenyllactic acid production of 1.47 g/l was obtained from 12 g L-Phe/l. Recombinant E. coli (ldh-lpox) was efficient in PLA production realizing a high titer of several folds compared with studies using L-Phe as substrate.

Purification and characterization of polyphenol oxidase from banana (Musa sapientum L.) pulp.

PubMed

Yang, C P; Fujita, S; Ashrafuzzaman, M; Nakamura, N; Hayashi, N

2000-07-01

Polyphenol oxidase (EC 1.10.3.1, PPO) in the pulp of banana (Musa sapientum L.) was purified to 636-fold with a recovery of 3.0%, using dopamine as substrate. The purified enzyme exhibited a clear single band on polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS)-PAGE. The molecular weight of the enzyme was estimated to be about 41000 and 42000 by gel filtration and SDS-PAGE, respectively. The enzyme quickly oxidized dopamine, and its K(m) value for dopamine was 2.8 mM. The optimum pH was at 6.5, and the enzyme activity was stable in the range of pH 5-11 at 5 degrees C for 48 h. The enzyme had an optimum temperature of 30 degrees C and was stable even after a heat treatment at 70 degrees C for 30 min. The enzyme activity was completely inhibited by L-ascorbic acid, cysteine, sodium diethyldithiocarbamate, and potassium cyanide. Under a low buffer capacity, the enzyme was also strongly inhibited by citric acid and acetic acid at 10 mM.

Adipogenesis-related increase of semicarbazide-sensitive amine oxidase and monoamine oxidase in human adipocytes.

PubMed

Bour, Sandy; Daviaud, Danièle; Gres, Sandra; Lefort, Corinne; Prévot, Danielle; Zorzano, Antonio; Wabitsch, Martin; Saulnier-Blache, Jean-Sébastien; Valet, Philippe; Carpéné, Christian

2007-08-01

A strong induction of semicarbazide-sensitive amine oxidase (SSAO) has previously been reported during murine preadipocyte lineage differentiation but it remains unknown whether this emergence also occurs during adipogenesis in man. Our aim was to compare SSAO and monoamine oxidase (MAO) expression during in vitro differentiation of human preadipocytes and in adipose and stroma-vascular fractions of human fat depots. A human preadipocyte cell strain from a patient with Simpson-Golabi-Behmel syndrome was first used to follow amine oxidase expression during in vitro differentiation. Then, human preadipocytes isolated from subcutaneous adipose tissues were cultured under conditions promoting ex vivo adipose differentiation and tested for MAO and SSAO expression. Lastly, human adipose tissue was separated into mature adipocyte and stroma-vascular fractions for analyses of MAO and SSAO at mRNA, protein and activity levels. Both SSAO and MAO were increased from undifferentiated preadipocytes to lipid-laden cells in all the models: 3T3-F442A and 3T3-L1 murine lineages, human SGBS cell strain or human preadipocytes in primary culture. In human subcutaneous adipose tissue, the adipocyte-enriched fraction exhibited seven-fold higher amine oxidase activity and contained three- to seven-fold higher levels of mRNAs encoded by MAO-A, MAO-B, AOC3 and AOC2 genes than the stroma-vascular fraction. MAO-A and AOC3 genes accounted for the majority of their respective MAO and SSAO activities in human adipose tissue. Most of the SSAO and MAO found in adipose tissue originated from mature adipocytes. Although the mechanism and role of adipogenesis-related increase in amine oxidase expression remain to be established, the resulting elevated levels of amine oxidase activities found in human adipocytes may be of potential interest for therapeutic intervention in obesity.

Size-selective QD@MOF core-shell nanocomposites for the highly sensitive monitoring of oxidase activities.

PubMed

Wang, Ke; Li, Nan; Zhang, Jing; Zhang, Zhiqi; Dang, Fuquan

2017-01-15

In this work, we proposed a novel and facile method to monitor oxidase activities based on size-selective fluorescent quantum dot (QD)@metal-organic framework (MOF) core-shell nanocomposites (CSNCPs). The CSNCPs were synthesized from ZIF-8 and CdTe QDs in aqueous solution in 40min at room temperature with stirring. The prepared CdTe@ZIF-8 CSNCPs , which have excellent water dispersibility and stability, displays distinct fluorescence responses to hole scavengers of different molecular sizes (e.g., H 2 O 2 , substrate, and oxidase) due to the aperture limitation of the ZIF-8 shell. H 2 O 2 can efficiently quench the fluorescence of CdTe@ZIF-8 CSNCPs over a linearity range of 1-100nM with a detection limit of 0.29nM, whereas large molecules such as substrate and oxidase have very little effect on its fluorescence. Therefore, the highly sensitive detection of oxidase activities was achieved by monitoring the fluorescence quenching of CdTe@ZIF-8 CSNCPs by H 2 O 2 produced in the presence of substrate and oxidase, which is proportional to the oxidase activities. The linearity ranges of the uricase and glucose oxidase activity are 0.1-50U/L and 1-100U/L, respectively, and their detection limits are 0.024U/L and 0.26U/L, respectively. Therefore, the current QD@MOF CSNCPs based sensing system is a promising, widely applicable means of monitoring oxidase activities in biochemical research. Copyright © 2016 Elsevier B.V. All rights reserved.

Polyphenol oxidase activity from three sicilian artichoke [ Cynara cardunculus L. Var. scolymus L. (Fiori)] cultivars: studies and technological application on minimally processed production.

PubMed

Todaro, Aldo; Peluso, Orazio; Catalano, Anna Eghle; Mauromicale, Giovanni; Spagna, Giovanni

2010-02-10

Several papers helped with the development of more methods to control browning, or study thermal polyphenol oxidase (PPO) inactivation, but did not provide any solutions to technological process problems and food process improvement. Artichokes [ Cynara cardunculus L. var. scolymus L. (Fiori)] are susceptible to browning; this alteration could affect and reduce the suitability for its use, fresh or processed. Within this study, the catecholase and cresolase activities of PPO from three different Sicilian artichokes cultivar were characterized with regard to substrate specificity and enzyme kinetics, optimum pH and temperature, temperature and pH stability, and inhibitor test; all of the results were used for technological purposes, particularly to optimize minimally processed productions (ready-to-eat and cook-chilled artichokes).

Inheritance of polyphenol oxidase activity in wheat breeding lines derived from matings of low polyphenol oxidase parents

USDA-ARS?s Scientific Manuscript database

Polyphenol oxidase (PPO) in grain plays a major role in time-dependent discoloration of wheat (Triticum aestivum L.) products, especially fresh noodles. Breeding wheat cultivars with low or nil PPO activity can reduce the undesirable product darkening. The low PPO line PI 117635 was crossed to two...

Minimizing the effects of oxygen interference on l-lactate sensors by a single amino acid mutation in Aerococcus viridansl-lactate oxidase.

PubMed

Hiraka, Kentaro; Kojima, Katsuhiro; Lin, Chi-En; Tsugawa, Wakako; Asano, Ryutaro; La Belle, Jeffrey T; Sode, Koji

2018-04-30

l-lactate biosensors employing l-lactate oxidase (LOx) have been developed mainly to measure l-lactate concentration for clinical diagnostics, sports medicine, and the food industry. Some l-lactate biosensors employ artificial electron mediators, but these can negatively impact the detection of l-lactate by competing with the primary electron acceptor: molecular oxygen. In this paper, a strategic approach to engineering an AvLOx that minimizes the effects of oxygen interference on sensor strips was reported. First, we predicted an oxygen access pathway in Aerococcus viridans LOx (AvLOx) based on its crystal structure. This was subsequently blocked by a bulky amino acid substitution. The resulting Ala96Leu mutant showed a drastic reduction in oxidase activity using molecular oxygen as the electron acceptor and a small increase in dehydrogenase activity employing an artificial electron acceptor. Secondly, the Ala96Leu mutant was immobilized on a screen-printed carbon electrode using glutaraldehyde cross-linking method. Amperometric analysis was performed with potassium ferricyanide as an electron mediator under argon or atmospheric conditions. Under argon condition, the response current increased linearly from 0.05 to 0.5mM l-lactate for both wild-type and Ala96Leu. However, under atmospheric conditions, the response of wild-type AvLOx electrode was suppressed by 9-12% due to oxygen interference. The Ala96Leu mutant maintained 56-69% of the response current at the same l-lactate level and minimized the relative bias error to -19% from -49% of wild-type. This study provided significant insight into the enzymatic reaction mechanism of AvLOx and presented a novel approach to minimize oxygen interference in sensor applications, which will enable accurate detection of l-lactate concentrations. Copyright © 2017 Elsevier B.V. All rights reserved.

Partial purification and characterization of polyphenol oxidase from banana (Musa sapientum L.) peel.

PubMed

Yang, C P; Fujita, S; Kohno, K; Kusubayashi, A; Ashrafuzzaman, M; Hayashi, N

2001-03-01

Polyphenol oxidase (EC 1.10.3.1, o-diphenol: oxygen oxidoreductase, PPO) of banana (Musa sapientum L.) peel was partially purified about 460-fold with a recovery of 2.2% using dopamine as substrate. The enzyme showed a single peak on Toyopearl HW55-S chromatography. However, two bands were detected by staining with Coomassie brilliant blue on PAGE: one was very clear, and the other was faint. Molecular weight for purified PPO was estimated to be about 41 000 by gel filtration. The enzyme quickly oxidized dopamine, and its Km value (Michaelis constant) for dopamine was 3.9 mM. Optimum pH was 6.5 and the PPO activity was quite stable in the range of pH 5-11 for 48 h. The enzyme had an optimum temperature at 30 degrees C and was stable up to 60 degrees C after heat treatment for 30 min. The enzyme activity was strongly inhibited by sodium diethyldithiocarbamate, potassium cyanide, L-ascorbic acid, and cysteine at 1 mM. Under a low buffer capacity, the enzyme was also strongly inhibited by citric acid and acetic acid at 10 mM.

MipLAAO, a new L-amino acid oxidase from the redtail coral snake Micrurus mipartitus

PubMed Central

2018-01-01

L-amino acid oxidases (LAAOs) are ubiquitous enzymes in nature. Bioactivities described for these enzymes include apoptosis induction, edema formation, induction or inhibition of platelet aggregation, as well as antiviral, antiparasite, and antibacterial actions. With over 80 species, Micrurus snakes are the representatives of the Elapidae family in the New World. Although LAAOs in Micrurus venoms have been predicted by venom gland transcriptomic studies and detected in proteomic studies, no enzymes of this kind have been previously purified from their venoms. Earlier proteomic studies revealed that the venom of M. mipartitus from Colombia contains ∼4% of LAAO. This enzyme, here named MipLAAO, was isolated and biochemically and functionally characterized. The enzyme is found in monomeric form, with an isotope-averaged molecular mass of 59,100.6 Da, as determined by MALDI-TOF. Its oxidase activity shows substrate preference for hydrophobic amino acids, being optimal at pH 8.0. By nucleotide sequencing of venom gland cDNA of mRNA transcripts obtained from a single snake, six isoforms of MipLAAO with minor variations among them were retrieved. The deduced sequences present a mature chain of 483 amino acids, with a predicted pI of 8.9, and theoretical masses between 55,010.9 and 55,121.0 Da. The difference with experimentally observed mass is likely due to glycosylation, in agreement with the finding of three putative N-glycosylation sites in its amino acid sequence. A phylogenetic analysis of MmipLAAO placed this new enzyme within the clade of homologous proteins from elapid snakes, characterized by the conserved Serine at position 223, in contrast to LAAOs from viperids. MmipLAAO showed a potent bactericidal effect on S. aureus (MIC: 2 µg/mL), but not on E. coli. The former activity could be of interest to future studies assessing its potential as antimicrobial agent. PMID:29900074

MipLAAO, a new L-amino acid oxidase from the redtail coral snake Micrurus mipartitus.

PubMed

Rey-Suárez, Paola; Acosta, Cristian; Torres, Uday; Saldarriaga-Córdoba, Mónica; Lomonte, Bruno; Núñez, Vitelbina

2018-01-01

L-amino acid oxidases (LAAOs) are ubiquitous enzymes in nature. Bioactivities described for these enzymes include apoptosis induction, edema formation, induction or inhibition of platelet aggregation, as well as antiviral, antiparasite, and antibacterial actions. With over 80 species, Micrurus snakes are the representatives of the Elapidae family in the New World. Although LAAOs in Micrurus venoms have been predicted by venom gland transcriptomic studies and detected in proteomic studies, no enzymes of this kind have been previously purified from their venoms. Earlier proteomic studies revealed that the venom of M. mipartitus from Colombia contains ∼4% of LAAO. This enzyme, here named MipLAAO, was isolated and biochemically and functionally characterized. The enzyme is found in monomeric form, with an isotope-averaged molecular mass of 59,100.6 Da, as determined by MALDI-TOF. Its oxidase activity shows substrate preference for hydrophobic amino acids, being optimal at pH 8.0. By nucleotide sequencing of venom gland cDNA of mRNA transcripts obtained from a single snake, six isoforms of MipLAAO with minor variations among them were retrieved. The deduced sequences present a mature chain of 483 amino acids, with a predicted pI of 8.9, and theoretical masses between 55,010.9 and 55,121.0 Da. The difference with experimentally observed mass is likely due to glycosylation, in agreement with the finding of three putative N-glycosylation sites in its amino acid sequence. A phylogenetic analysis of MmipLAAO placed this new enzyme within the clade of homologous proteins from elapid snakes, characterized by the conserved Serine at position 223, in contrast to LAAOs from viperids. MmipLAAO showed a potent bactericidal effect on S. aureus (MIC: 2 µg/mL), but not on E. coli . The former activity could be of interest to future studies assessing its potential as antimicrobial agent.

Kinetic mechanism of L-α-glycerophosphate oxidase from Mycoplasma pneumoniae.

PubMed

Maenpuen, Somchart; Watthaisong, Pratchaya; Supon, Pacharee; Sucharitakul, Jeerus; Parsonage, Derek; Karplus, P Andrew; Claiborne, Al; Chaiyen, Pimchai

2015-08-01

L-α-glycerophosphate oxidase is an FAD-dependent enzyme that catalyzes the oxidation of L-α-glycerophosphate (Glp) by molecular oxygen to generate dihydroxyacetone phosphate (DHAP) and hydrogen peroxide (H2O2). The catalytic properties of recombinant His6-GlpO from Mycoplasma pneumoniae (His6-MpGlpO) were investigated through transient and steady-state kinetics and ligand binding studies. The results indicate that the reaction mechanism of His6-MpGlpO follows a ping-pong model. Double-mixing mode stopped-flow experiments show that, after flavin-mediated substrate oxidation, DHAP leaves rapidly prior to the oxygen reaction. The values determined for the individual rate constants and kcat (4.2 s(-1) at 4 °C), in addition to the finding that H2 O2 binds to the oxidized enzyme, suggest that H2O2 release is the rate-limiting step for the overall reaction. The results indicate that His6 -MpGlpO contains mixed populations of fast- and slow-reacting species. It is predominantly the fast-reacting species that participates in turnover. In contrast to other GlpO enzymes previously described, His6-MpGlpO is able to catalyze the reverse reaction of reduced enzyme and DHAP. This result may be explained by the standard reduction potential value of His6-MpGlpO (-167 ± 1 mV), which is lower than those of GlpO from other species. We found that D,L-glyceraldehyde 3-phosphate (GAP) may be used as a substrate in the His6-MpGlpO reaction, although it exhibited an approximately 100-fold lower kcat value in comparison with the reaction of Glp. These results also imply involvement of GlpO in glycolysis, as well as in lipid and glycerol metabolism. The kinetic models and distinctive properties of His6-MpGlpO reported here should be useful for future drug development against Mycoplasma pneumoniae infection. © 2015 FEBS.

Polyphenol oxidase activity and differential accumulation of polyphenolics in seed coats of pinto bean (Phaseolus vulgaris L.) characterize postharvest color changes.

PubMed

Marles, M A Susan; Vandenberg, Albert; Bett, Kirstin E

2008-08-27

Postharvest darkening of pinto bean (Phaseolus vulgaris L.) was evaluated in a population of recombinant inbred lines derived from a cross between CDC Pintium (a regular-darkening line) and 1533-15 (a slow-darkening line). Flavonoid metabolite concentrations, polyphenol oxidase activity, lignin concentration, and seed coat anatomy characteristics were assessed for cosegregation with the darkening phenotype. Significantly lower kaempferol concentrations (p = 0.00001) together with differences in polyphenol oxidase activity (p = 0.0045) were two of the key findings associated with these recombinant inbred lines. In addition, two different assays (thioglycolic acid and Klason lignin) to quantify lignin together with an assessment of extractable condensed tannin were used to estimate the contribution of these polymers to changes in the seed coat tissue. This is the first report of precise biochemical characterization of polyphenolics that associate with postharvest darkening in legumes.

Identification and statistical optimization of fermentation conditions for a newly isolated extracellular cholesterol oxidase-producing Streptomyces cavourensis strain NEAE-42.

PubMed

El-Naggar, Noura El-Ahmady; El-Shweihy, Nancy M; El-Ewasy, Sara M

2016-09-20

Due to broad range of clinical and industrial applications of cholesterol oxidase, isolation and screening of bacterial strains producing extracellular form of cholesterol oxidase is of great importance. One hundred and thirty actinomycete isolates were screened for their cholesterol oxidase activity. Among them, a potential culture, strain NEAE-42 is displayed the highest extracellular cholesterol oxidase activity. It was selected and identified as Streptomyces cavourensis strain NEAE-42. The optimization of different process parameters for cholesterol oxidase production by Streptomyces cavourensis strain NEAE-42 using Plackett-Burman experimental design and response surface methodology was carried out. Fifteen variables were screened using Plackett-Burman experimental design. Cholesterol, initial pH and (NH4)2SO4 were the most significant positive independent variables affecting cholesterol oxidase production. Central composite design was chosen to elucidate the optimal concentrations of the selected process variables on cholesterol oxidase production. It was found that, cholesterol oxidase production by Streptomyces cavourensis strain NEAE-42 after optimization process was 20.521U/mL which is higher than result obtained from the basal medium before screening process using Plackett-Burman (3.31 U/mL) with a fold of increase 6.19. The cholesterol oxidase level production obtained in this study (20.521U/mL) by the statistical method is higher than many of the reported values.

Identification of a Catalase-Phenol Oxidase in Betalain Biosynthesis in Red Amaranth (Amaranthus cruentus)

PubMed Central

Teng, Xiao-Lu; Chen, Ning; Xiao, Xing-Guo

2016-01-01

Betalains are a group of nitrogen-containing pigments that color plants in most families of Caryophyllales. Their biosynthesis has long been proposed to begin with hydroxylation of L-tyrosine to L-DOPA through monophenolase activity of tyrosinase, but biochemical evidence in vivo remains lacking. Here we report that a Group 4 catalase, catalase-phenol oxidase (named as AcCATPO), was identified, purified and characterized from leaves of Amaranthus cruentus, a betalain plant. The purified enzyme appeared to be a homotrimeric protein composed of subunits of about 58 kDa, and demonstrated not only the catalase activity toward H2O2, but also the monophenolase activity toward L-tyrosine and diphenolase activity toward L-DOPA. Its catalase and phenol oxidase activities were inhibited by common classic catalase and tyrosinase inhibitors, respectively. All its peptide fragments identified by nano-LC-MS/MS were targeted to catalases, and matched with a cDNA-encoded polypeptide which contains both classic catalase and phenol oxidase active sites. These sites were also present in catalases of non-betalain plants analyzed. AcCATPO transcript abundance was positively correlated with the ratio of betaxanthin to betacyanin in both green and red leaf sectors of A. tricolor. These data shows that the fourth group catalase, catalase-phenol oxidase, is present in plant, and might be involved in betaxanthin biosynthesis. PMID:26779247

Cytochrome c oxidase inhibition in the rice weevil Sitophilus oryzae (L.) by formate, the toxic metabolite of volatile alkyl formates.

PubMed

Haritos, V S; Dojchinov, G

2003-10-01

Volatile alkyl formates are potential replacements for the ozone-depleting fumigant, methyl bromide, as postharvest insecticides and here we have investigated their mode of insecticidal action. Firstly, a range of alkyl esters, ethanol and formic acid were tested in mortality bioassays with adults of the rice weevil, Sitophilus oryzae (L.) and the grain borer, Rhyzopertha dominica (F.) to determine whether the intact ester or one of its components was the toxic moiety. Volatile alkyl formates and formic acid caused similar levels of mortality (LC(50) 131-165 micromol l(-1)) to S. oryzae and were more potent than non-formate containing alkyl esters and ethanol (LC(50)>275 micromol l(-1)). The order of potency was the same in R. dominica. Ethyl formate was rapidly metabolised in vitro to formic acid when incubated with insect homogenates, presumably through the action of esterases. S. oryzae and R. dominica fumigated with a lethal dose of ethyl formate had eight and 17-fold higher concentrations of formic acid, respectively, in their bodies than untreated controls. When tested against isolated mitochondria from S. oryzae, alkyl esters, alcohols, acetate and propionate salts were not inhibitory towards cytochrome c oxidase (EC 1.9.3.1), but sodium cyanide and sodium formate were inhibitory with IC(50) values of 0.0015 mM and 59 mM, respectively. Volatile formate esters were more toxic than other alkyl esters, and this was found to be due, at least in part, to their hydrolysis to formic acid and its inhibition of cytochrome c oxidase.

Lower growth temperature increases alternative pathway capacity and alternative oxidase protein in tobacco.

PubMed

Vanlerberghe, G C; McIntosh, L

1992-09-01

Suspension cells of NT1 tobacco (Nicotiana tabacum L. cv bright yellow) have been used to study the effect of growth temperature on the CN-resistant, salicylhydroxamic acid-sensitive alternative pathway of respiration. Mitochondria isolated from cells maintained at 30 degrees C had a low capacity to oxidize succinate via the alternative pathway, whereas mitochondria isolated from cells 24 h after transfer to 18 degrees C displayed, on average, a 5-fold increase in this capacity (from 7 to 32 nanoatoms oxygen per milligram protein per minute). This represented an increase in alternative pathway capacity from 18 to 45% of the total capacity of electron transport. This increased capacity was lost upon transfer of cells back to 30 degrees C. A monoclonal antibody to the terminal oxidase of the alternative pathway (the alternative oxidase) from Sauromatum guttatum (T.E. Elthon, R.L. Nickels, L. McIntosh [1989] Plant Physiology 89: 1311-1317) recognized a 35-kilodalton mitochondrial protein in tobacco. There was an excellent correlation between the capacity of the alternative path in isolated tobacco mitochondria and the levels of this 35-kilodalton alternative oxidase protein. Cycloheximide could inhibit both the increased level of the 35-kilodalton alternative oxidase protein and the increased alternative pathway capacity normally seen upon transfer to 18 degrees C. We conclude that transfer of tobacco cells to the lower temperature increases the capacity of the alternative pathway due, at least in part, to de novo synthesis of the 35-kilodalton alternative oxidase protein.

Characterization and purification of polyphenol oxidase from artichoke (Cynara scolymus L.).

PubMed

Dogan, Serap; Turan, Yusuf; Ertürk, Hatibe; Arslan, Oktay

2005-02-09

In this study, the polyphenol oxidase (PPO) of artichoke (Cynara scolymus L.) was first purified by a combination of (NH(4))(2)SO(4) precipitation, dialysis, and a Sepharose 4B-L-tyrosine-p-aminobenzoic acid affinity column. At the end of purification, 43-fold purification was achieved. The purified enzyme migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis indicated that PPO had a 57 kDa molecular mass. Second, the contents of total phenolic and protein of artichoke head extracts were determined. The total phenolic content of artichoke head was determined spectrophotometrically according to the Folin-Ciocalteu procedure and was found to be 425 mg 100 g(-1) on a fresh weight basis. Protein content was determined according to Bradford method. Third, the effects of substrate specificity, pH, temperature, and heat inactivation were investigated on the activity of PPO purified from artichoke. The enzyme showed activity to 4-methylcatechol, pyrogallol, catechol, and L-dopa. No activity was detected toward L-tyrosine, resorsinol, and p-cresol. According to V(max)/K(m) values, 4-methylcatechol (1393 EU min(-1) mM(-1)) was the best substrate, followed by pyrogallol (1220 EU min(-1) mM(-1)), catechol (697 EU min(-1) mM(-1)), and L-dopa (102 EU min(-1) mM(-1)). The optimum pH values for PPO were 5.0, 8.0, and 7.0 using 4-methylcatechol, pyrogallol, and catechol as substrate, respectively. It was found that optimum temperatures were dependent on the substrates studied. The enzyme activity decreased due to heat denaturation of the enzyme with increasing temperature and inactivation time for 4-methylcatechol and pyrogallol substrates. However, all inactivation experiments for catechol showed that the activity of artichoke PPO increased with mild heating, reached a maximum, and then decreased with time. Finally, inhibition of artichoke PPO was investigated with inhibitors such as L-cysteine, EDTA, ascorbic

In-Gel Determination of L-Amino Acid Oxidase Activity Based on the Visualization of Prussian Blue-Forming Reaction

PubMed Central

Zhou, Ning; Zhao, Chuntian

2013-01-01

L-amino acid oxidase (LAAO) is attracting increasing attention due to its important functions. Diverse detection methods with their own properties have been developed for characterization of LAAO. In the present study, a simple, rapid, sensitive, cost-effective and reproducible method for quantitative in-gel determination of LAAO activity based on the visualization of Prussian blue-forming reaction is described. Coupled with SDS-PAGE, this Prussian blue agar assay can be directly used to determine the numbers and approximate molecular weights of LAAO in one step, allowing straightforward application for purification and sequence identification of LAAO from diverse samples. PMID:23383337

Identification in Marinomonas mediterranea of a novel quinoprotein with glycine oxidase activity.

PubMed

Campillo-Brocal, Jonatan Cristian; Lucas-Elio, Patricia; Sanchez-Amat, Antonio

2013-08-01

A novel enzyme with lysine-epsilon oxidase activity was previously described in the marine bacterium Marinomonas mediterranea. This enzyme differs from other l-amino acid oxidases in not being a flavoprotein but containing a quinone cofactor. It is encoded by an operon with two genes lodA and lodB. The first one codes for the oxidase, while the second one encodes a protein required for the expression of the former. Genome sequencing of M. mediterranea has revealed that it contains two additional operons encoding proteins with sequence similarity to LodA. In this study, it is shown that the product of one of such genes, Marme_1655, encodes a protein with glycine oxidase activity. This activity shows important differences in terms of substrate range and sensitivity to inhibitors to other glycine oxidases previously described which are flavoproteins synthesized by Bacillus. The results presented in this study indicate that the products of the genes with different degrees of similarity to lodA detected in bacterial genomes could constitute a reservoir of different oxidases. © 2013 The Authors. Microbiology Open published by John Wiley & Sons Ltd.

Antiproliferative activity of king cobra (Ophiophagus hannah) venom L-amino acid oxidase.

PubMed

Li Lee, Mui; Chung, Ivy; Yee Fung, Shin; Kanthimathi, M S; Hong Tan, Nget

2014-04-01

King cobra (Ophiophagus hannah) venom L-amino acid oxidase (LAAO), a heat-stable enzyme, is an extremely potent antiproliferative agent against cancer cells when compared with LAAO isolated from other snake venoms. King cobra venom LAAO was shown to exhibit very strong antiproliferative activities against MCF-7 (human breast adenocarcinoma) and A549 (human lung adenocarcinoma) cells, with an IC50 value of 0.04±0.00 and 0.05±0.00 μg/mL, respectively, after 72-hr treatment. In comparison, its cytotoxicity was about 3-4 times lower when tested against human non-tumourigenic breast (184B5) and lung (NL 20) cells, suggesting selective antitumour activity. Furthermore, its potency in MCF-7 and A549 cell lines was greater than the effects of doxorubicin, a clinically established cancer chemotherapeutic agent, which showed an IC50 value of 0.18±0.03 and 0.63±0.21 μg/mL, respectively, against the two cell lines. The selective cytotoxic action of the LAAO was confirmed by phycoerythrin (PE) annexin V/7-amino-actinomycin (AAD) apoptotic assay, in which a significant increase in apoptotic cells was observed in LAAO-treated tumour cells than in their non-tumourigenic counterparts. The ability of LAAO to induce apoptosis in tumour cells was further demonstrated using caspase-3/7 and DNA fragmentation assays. We also determined that this enzyme may target oxidative stress in its killing of tumour cells, as its cytotoxicity was significantly reduced in the presence of catalase (a H2O2 scavenger). In view of its heat stability and selective and potent cytotoxic action on cancer cells, king cobra venom LAAO can be potentially developed for treating solid tumours. © 2013 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

Variations of L- and D-amino acid levels in the brain of wild-type and mutant mice lacking D-amino acid oxidase activity.

PubMed

Du, Siqi; Wang, Yadi; Weatherly, Choyce A; Holden, Kylie; Armstrong, Daniel W

2018-05-01

D-amino acids are now recognized to be widely present in organisms and play essential roles in biological processes. Some D-amino acids are metabolized by D-amino acid oxidase (DAO), while D-Asp and D-Glu are metabolized by D-aspartate oxidase (DDO). In this study, levels of 22 amino acids and the enantiomeric compositions of the 19 chiral proteogenic entities have been determined in the whole brain of wild-type ddY mice (ddY/DAO +/+ ), mutant mice lacking DAO activity (ddY/DAO -/- ), and the heterozygous mice (ddY/DAO +/- ) using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). No significant differences were observed for L-amino acid levels among the three strains except for L-Trp which was markedly elevated in the DAO +/- and DAO -/- mice. The question arises as to whether this is an unknown effect of DAO inactivity. The three highest levels of L-amino acids were L-Glu, L-Asp, and L-Gln in all the three strains. The lowest L-amino acid level was L-Cys in ddY/DAO +/- and ddY/DAO -/- mice, while L-Trp showed the lowest level in ddY/DAO +/+ mice. The highest concentration of D-amino acid was found to be D-Ser, which also had the highest % D value (~ 25%). D-Glu had the lowest % D value (~ 0.01%) in all the three strains. Significant differences of D-Leu, D-Ala, D-Ser, D-Arg, and D-Ile were observed in ddY/DAO +/- and ddY/DAO -/- mice compared to ddY/DAO +/+ mice. This work provides the most complete baseline analysis of L- and D-amino acids in the brains of ddY/DAO +/+ , ddY/DAO +/- , and ddY/DAO -/- mice yet reported. It also provides the most effective and efficient analytical approach for measuring these analytes in biological samples. This study provides fundamental information on the role of DAO in the brain and may be relevant for future development involving novel drugs for DAO regulation.

Lower Growth Temperature Increases Alternative Pathway Capacity and Alternative Oxidase Protein in Tobacco 1

PubMed Central

Vanlerberghe, Greg C.; McIntosh, Lee

1992-01-01

Suspension cells of NT1 tobacco (Nicotiana tabacum L. cv bright yellow) have been used to study the effect of growth temperature on the CN-resistant, salicylhydroxamic acid-sensitive alternative pathway of respiration. Mitochondria isolated from cells maintained at 30°C had a low capacity to oxidize succinate via the alternative pathway, whereas mitochondria isolated from cells 24 h after transfer to 18°C displayed, on average, a 5-fold increase in this capacity (from 7 to 32 nanoatoms oxygen per milligram protein per minute). This represented an increase in alternative pathway capacity from 18 to 45% of the total capacity of electron transport. This increased capacity was lost upon transfer of cells back to 30°C. A monoclonal antibody to the terminal oxidase of the alternative pathway (the alternative oxidase) from Sauromatum guttatum (T.E. Elthon, R.L. Nickels, L. McIntosh [1989] Plant Physiology 89: 1311-1317) recognized a 35-kilodalton mitochondrial protein in tobacco. There was an excellent correlation between the capacity of the alternative path in isolated tobacco mitochondria and the levels of this 35-kilodalton alternative oxidase protein. Cycloheximide could inhibit both the increased level of the 35-kilodalton alternative oxidase protein and the increased alternative pathway capacity normally seen upon transfer to 18°C. We conclude that transfer of tobacco cells to the lower temperature increases the capacity of the alternative pathway due, at least in part, to de novo synthesis of the 35-kilodalton alternative oxidase protein. Images Figure 3 Figure 4 PMID:16652932

On the use of L-012, a luminol-based chemiluminescent probe, for detecting superoxide and identifying inhibitors of NADPH oxidase: A re-evaluation

PubMed Central

Zielonka, Jacek; Lambeth, J. David; Kalyanaraman, Balaraman

2014-01-01

L-012, a luminol-based chemiluminescent (CL) probe, is widely used in vitro and in vivo to detect NADPH oxidase (Nox)-derived superoxide (O2·−) and identify Nox inhibitors. Yet understanding of the free radical chemistry of L-012 probe is still lacking. We report that peroxidase and H2O2 induce superoxide dismutase (SOD)-sensitive, L-012-derived CL in the presence of oxygen. O2·− alone does not react with L-012 to emit luminescence. Self-generated O2·− during oxidation of L-012 and luminol-analogs artifactually induce CL inhibitable by SOD. These aspects make assays based on luminol analogs less than ideal for specific detection and identification of O2·− and NOX inhibitors. PMID:24080119

Polyphenol oxidase activity and antioxidant properties of Yomra apple (Malus communis L.) from Turkey.

PubMed

Can, Zehra; Dincer, Barbaros; Sahin, Huseyin; Baltas, Nimet; Yildiz, Oktay; Kolayli, Sevgi

2014-12-01

In this study, firstly, antioxidant and polyphenol oxidase (PPO) properties of Yomra apple were investigated. Seventeen phenolic constituents were measured by reverse phase-high-performance liquid chromatography (RP-HPLC). Total phenolic compounds (TPCs), ferric reducing antioxidant power (FRAP) and 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH) scavenging activities were performed to measure antioxidant capacity. Some kinetic parameters (Km, Vmax), and inhibition behaviors against five different substrates were measured in the crude extract. Catechin and chlorogenic acid were found as the major components in the methanolic extract, while ferulic acid, caffeic acid, p-hydroxybenzoic acid, quercetin and p-coumaric acid were small quantities. Km values ranged from 0.70 to 10.10 mM in the substrates, and also 3-(4-hydroxyphenyl) propanoic acid (HPPA) and L-DOPA showed the highest affinity. The inhibition constant of Ki were ranged from 0.05 to 14.90 mM against sodium metabisulphite, ascorbic acid, sodium azide and benzoic acid, while ascorbic acid and sodium metabisulphite were the best inhibitors.

Biocompatibility selenium nanoparticles with an intrinsic oxidase-like activity

NASA Astrophysics Data System (ADS)

Guo, Leilei; Huang, Kaixun; Liu, Hongmei

2016-03-01

Selenium nanoparticles (SeNPs) are considered to be the new selenium supplement forms with high biological activity and low toxicity; however, the molecular mechanism by which SeNPs exert the biological function is unclear. Here, we reported that biocompatibility SeNPs possessed intrinsic oxidase-like activity. Using Na2SeO3 as a precursor and glutathione as a reductant, biocompatibility SeNPs were synthesized by the wet chemical reduction method in the presence of bovine serum albumin (BSA). The results of structure characterization revealed that synthesized SeNPs were amorphous red elementary selenium with spherical morphology, and ranged in size from 25 to 70 nm size with a narrow distribution (41.4 ± 6.7 nm). The oxidase-like activity of the as-synthesized SeNPs was tested with 3,3',5,5'-tetramethylbenzidine (TMB) as a substrate. The results indicated that SeNPs could catalyze the oxidization of TMB by dissolved oxygen. These SeNPs showed an optimum catalytic activity at pH 4 and 30 °C, and the oxidase-like activity was higher as the concentration of SeNPs increased and the size of SeNPs decreased. The Michaelis constant ( K m) values and maximal reaction velocity ( V max) of the SeNPs for TMB oxidation were 0.0083 mol/L and 3.042 μmol/L min, respectively.

In vitro antioxidant, lipoxygenase and xanthine oxidase inhibitory activities of fractions from Cienfuegosia digitata Cav., Sida alba L. and Sida acuta Burn f. (Malvaceae).

PubMed

Konaté, K; Souza, A; Coulibaly, A Y; Meda, N T R; Kiendrebeogo, M; Lamien-Meda, A; Millogo-Rasolodimby, J; Lamidi, M; Nacoulma, O G

2010-11-15

In this study polyphenol content, antioxidant activity, lipoxygenase (LOX) and Xanthine Oxidase (XO) inhibitory effects of n-hexane, dichloromethane, ethyl acetate and n-butanol fractions of aqueous acetone extracts from S. alba L., S. acuta Burn f and Cienfuegosia digitata Cav. were investigated. The total phenolics, flavonoids, flavonols and total tannins were determined by spectrophotometric methods using Folin-ciocalteu, AlCl3 reagents and tannic acid, respectively. The antioxidant potential was evaluated using three methods: inhibition of free radical 2,2-diphenyl-1-picrylhydramzyl (DPPH), ABTS radical cation decolorization assay and Iron (III) to iron (II) reduction activity (FRAP). For enzymatic activity, lipoxygenase and xanthine oxidase inhibitory activities were used. This study shows a relationship between polyphenol contents, antioxidant and enzymatic activities. Present results showed that ethyl acetate and dichloromethane fractions elicit the highest polyphenol content, antioxidant and enzymatic activities.

Utilization of Diamine Oxidase Enzyme from Mung Bean Sprouts (Vigna radiata L) for Histamine biosensors

NASA Astrophysics Data System (ADS)

Karim, Abdul; Wahab, A. W.; Raya, I.; Natsir, H.; Arif, A. R.

2018-03-01

This research is aimed to utilize the diamine oxidase enzyme (DAO) which isolated from mung bean sprouts (Vigna radiata L) to develop histamine biosensors based on electode enzyme with the amperometric method (cyclic voltammetry).The DAO enzyme is trapped inside the membrane of chitin-cellulose acetate 2:1 and glutaraldehyde which super imposed on a Pt electrode. Histamine will be oxidized by DAO enzyme to produce aldehydes and H2O2 that acting as electron transfer mediators.The performance of biosensors will be measured at various concentrations of glutaraldehyde, temperature changes and different range of pH. Recently, it has been found that the optimal conditions obtained from the paramaters as follows; at 25% of glutaraldehyde, temperature of 37°C and pH of 7.4. Eventually, the results provided an expectation for applying histamine biosensors in determining the freshness and safety of fish specifically skombroidae families.

Extracellular cholesterol oxidase production by Streptomyces aegyptia, in vitro anticancer activities against rhabdomyosarcoma, breast cancer cell-lines and in vivo apoptosis.

PubMed

El-Naggar, Noura El-Ahmady; Soliman, Hoda M; El-Shweihy, Nancy M

2018-02-09

In recent years, microbial cholesterol oxidases have gained great attention due to its widespread use in medical applications for serum cholesterol determination. Streptomyces aegyptia strain NEAE-102 exhibited high level of extracellular cholesterol oxidase production using a minimum medium containing cholesterol as the sole source of carbon. Fifteen variables were screened using Plackett-Burman design for the enhanced cholesterol oxidase production. The most significant variables affecting enzyme production were further optimized by using the face-centered central composite design. The statistical optimization resulted in an overall 4.97-fold increase (15.631 UmL -1 ) in cholesterol oxidase production in the optimized medium as compared with the unoptimized medium before applying Plackett Burman design (3.1 UmL -1 ). The purified cholesterol oxidase was evaluated for its in vitro anticancer activities against five human cancer cell lines. The selectivity index values on rhabdomyosarcoma and breast cancer cell lines were 3.26 and 2.56; respectively. The in vivo anticancer activity of cholesterol oxidase was evaluated against Ehrlich solid tumor model. Compared with control mice, tumors growth was significantly inhibited in the mice injected with cholesterol oxidase alone, doxorubicin alone and cholesterol oxidase/doxorubicin combination by 60.97%, 72.99% and 97.04%; respectively. These results demonstrated that cholesterol oxidase can be used as a promising natural anticancer drug.

Purification and characterization of polyphenol oxidase from cauliflower (Brassica oleracea L.).

PubMed

Rahman, Andi Nur Faidah; Ohta, Mayumi; Nakatani, Kazuya; Hayashi, Nobuyuki; Fujita, Shuji

2012-04-11

Polyphenol oxidase (PPO) of cauliflower was purified to 282-fold with a recovery rate of 8.1%, using phloroglucinol as a substrate. The enzyme appeared as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The estimated molecular weight of the enzyme was 60 and 54 kDa by SDS-PAGE and gel filtration, respectively. The purified enzyme, called phloroglucinol oxidase (PhO), oxidized phloroglucinol (K(m) = 3.3 mM) and phloroglucinolcarboxylic acid. The enzyme also had peroxidase (POD) activity. At the final step, the activity of purified cauliflower POD was 110-fold with a recovery rate of 3.2%. The PhO and POD showed the highest activity at pH 8.0 and 4.0 and were stable in the pH range of 3.0-11.0 and 5.0-8.0 at 5 °C for 20 h, respectively. The optimum temperature was 55 °C for PhO and 20 °C for POD. The most effective inhibitor for PhO was sodium diethyldithiocarbamate at 10 mM (IC(50) = 0.64 and K(i) = 0.15 mM), and the most effective inhibitor for POD was potassium cyanide at 1.0 mM (IC(50) = 0.03 and K(i) = 29 μM).

Structure–function characterization reveals new catalytic diversity in the galactose oxidase and glyoxal oxidase family

PubMed Central

Yin, DeLu (Tyler); Urresti, Saioa; Lafond, Mickael; Johnston, Esther M.; Derikvand, Fatemeh; Ciano, Luisa; Berrin, Jean-Guy; Henrissat, Bernard; Walton, Paul H.; Davies, Gideon J.; Brumer, Harry

2015-01-01

Alcohol oxidases, including carbohydrate oxidases, have a long history of research that has generated fundamental biological understanding and biotechnological applications. Despite a long history of study, the galactose 6-oxidase/glyoxal oxidase family of mononuclear copper-radical oxidases, Auxiliary Activity Family 5 (AA5), is currently represented by only very few characterized members. Here we report the recombinant production and detailed structure–function analyses of two homologues from the phytopathogenic fungi Colletotrichum graminicola and C. gloeosporioides, CgrAlcOx and CglAlcOx, respectively, to explore the wider biocatalytic potential in AA5. EPR spectroscopy and crystallographic analysis confirm a common active-site structure vis-à-vis the archetypal galactose 6-oxidase from Fusarium graminearum. Strikingly, however, CgrAlcOx and CglAlcOx are essentially incapable of oxidizing galactose and galactosides, but instead efficiently catalyse the oxidation of diverse aliphatic alcohols. The results highlight the significant potential of prospecting the evolutionary diversity of AA5 to reveal novel enzyme specificities, thereby informing both biology and applications. PMID:26680532

Phenol oxidase activity in secondary transformed peat-moorsh soils

NASA Astrophysics Data System (ADS)

Styła, K.; Szajdak, L.

2009-04-01

et al. (2000). In peat the highest activities of phenol oxidase was observed in the combinations marked as Shelterbelt and whereas the lowest - in Zbechy, Bridge and Hirudo. Activities of this enzyme in peat ranged from 15.35 to 38.33 μmol h-1g d.m soil. Increased activities of phenol oxidase have been recorded on the depth 50-100cm - catotelm (21.74-38.33 μmol h-1g d.m soil) in comparison with the depth 0-50cm - acrotelm (15.35-28.32 μmol h-1g d.m soil). References Freeman, C., Ostle N.J., Fener, N., Kang H. 2004. A regulatory role for phenol oxidase during decomposition in peatlands. Soil Biology and Biochemistry, 36, 1663-1667. Matocha Ch.J., Haszler G.R., Grove J.H. 2004. Nitrogen fertilization suppresses soil phenol oxidase enzyme activity in no-tillage systems. Soil Science, 169/10, 708-714. Perucci P., Casucci C., Dumontet S. 2000. An improved method to evaluate the o-diphenol oxidase activity of soil. Soil Biology and Biochemistry, 32, 1927-1933. Sokolowska Z., Szajdak L., Matyka-Sarzyńska D. 2005. Impact of the degree of secondary transformation on amid-base properties of organic compounds in mucks. Geoderma, 127, 80-90. Szajdak L., Szczepański M., Bogacz A. 2007. Impact of secondary transformation of peat-moorsh soils on the decrease of nitrogen and carbon compounds in ground water. Agronomy Research, 5/2, 189-200.

NADPH oxidase inhibitors: a patent review.

PubMed

Kim, Jung-Ae; Neupane, Ganesh Prasad; Lee, Eung Seok; Jeong, Byeong-Seon; Park, Byung Chul; Thapa, Pritam

2011-08-01

NADPH oxidases, a family of multi-subunit enzyme complexes, catalyze the production of reactive oxygen species (ROS), which may contribute to the pathogenesis of a variety of diseases. In addition to the first NADPH oxidase found in phagocytes, four non-phagocytic NADPH oxidase isoforms have been identified, which all differ in their catalytic subunit (Nox1-5) and tissue distribution. This paper provides a comprehensive review of the patent literature on NADPH oxidase inhibitors, small molecule Nox inhibitors, peptides and siRNAs. Since each member of the NADPH oxidase family has great potential as a therapeutic target, several different compounds have been registered as NADPH oxidase inhibitors in the patent literature. As yet, none have gone through clinical trials, and some have not completed preclinical trials, including safety and specificity evaluation. Recently, small molecule pyrazolopyridine and triazolopyrimidine derivatives have been submitted as potent NADPH oxidase inhibitors and reported as first-in-class inhibitors for idiopathic pulmonary fibrosis and acute stroke, respectively. Further clinical efficacy and safety data are warranted to prove their actual clinical utility.

Grouping of multicopper oxidases in Lentinula edodes by sequence similarities and expression patterns.

PubMed

Sakamoto, Yuichi; Nakade, Keiko; Yoshida, Kentaro; Natsume, Satoshi; Miyazaki, Kazuhiro; Sato, Shiho; van Peer, Arend F; Konno, Naotake

2015-12-01

The edible white rot fungus Lentinula edodes possesses a variety of lignin degrading enzymes such as manganese peroxidases and laccases. Laccases belong to the multicopper oxidases, which have a wide range of catalytic activities including polyphenol degradation and synthesis, lignin degradation, and melanin formation. The exact number of laccases in L. edodes is unknown, as are their complete properties and biological functions. We analyzed the draft genome sequence of L. edodes D703PP-9 and identified 13 multicopper oxidase-encoding genes; 11 laccases in sensu stricto, of which three are new, and two ferroxidases. lcc8, a laccase previously reported in L. edodes, was not identified in D703PP-9 genome. Phylogenetic analysis showed that the 13 multicopper oxidases can be classified into laccase sensu stricto subfamily 1, laccase sensu stricto subfamily 2 and ferroxidases. From sequence similarities and expression patterns, laccase sensu stricto subfamily 1 can be divided into two subgroups. Laccase sensu stricto subfamily 1 group A members are mainly secreted from mycelia, while laccase sensu stricto subfamily 1 group B members are expressed mainly in fruiting bodies during growth or after harvesting but are lowly expressed in mycelia. Laccase sensu stricto subfamily 2 members are mainly expressed in mycelia, and two ferroxidases are mainly expressed in the fruiting body during growth or after harvesting, and are expressed at very low levels in mycelium. Our data suggests that L. edodes laccases in same group share expression patterns and would have common biological functions.

Characterization and expression analysis of a banana gene encoding 1-aminocyclopropane-1-carboxylate oxidase.

PubMed

Huang, P L; Do, Y Y; Huang, F C; Thay, T S; Chang, T W

1997-04-01

A cDNA encoding the banana 1-aminocyclopropane-1-carboxylate (ACC) oxidase has previously been isolated from a cDNA library that was constructed by extracting poly(A)+ RNA from peels of ripening banana. This cDNA, designated as pMAO2, has 1,199 bp and contains an open reading frame of 318 amino acids. In order to identify ripening-related promoters of the banana ACC oxidase gene, pMAO2 was used as a probe to screen a banana genomic library constructed in the lambda EMBL3 vector. The banana ACC oxidase MAO2 gene has four exons and three introns, with all of the boundaries between these introns and exons sharing a consensus dinucleotide sequence of GT-AG. The expression of MAO2 gene in banana begins after the onset of ripening (stage 2) and continuous into later stages of the ripening process. The accumulation of MAO2 mRNA can be induced by 1 microliter/l exogenous ethylene, and it reached steady state level when 100 microliters/l exogenous ethylene was present.

Thermal stability of L-ascorbic acid and ascorbic acid oxidase in broccoli (Brassica oleracea var. italica).

PubMed

Munyaka, Ann Wambui; Makule, Edna Edward; Oey, Indrawati; Van Loey, Ann; Hendrickx, Marc

2010-05-01

The thermal stability of vitamin C (including l-ascorbic acid [l-AA] and dehydroascorbic acid [DHAA]) in crushed broccoli was evaluated in the temperature range of 30 to 90 degrees C whereas that of ascorbic acid oxidase (AAO) was evaluated in the temperature range of 20 to 95 degrees C. Thermal treatments (for 15 min) of crushed broccoli at 30 to 60 degrees C resulted in conversion of l-AA to DHAA whereas treatments at 70 to 90 degrees C retained vitamin C as l-AA. These observations indicated that enzymes (for example, AAO) could play a major role in the initial phase (that is, oxidation of l-AA to DHAA) of vitamin C degradation in broccoli. Consequently, a study to evaluate the temperature-time conditions that could result in AAO inactivation in broccoli was carried out. In this study, higher AAO activity was observed in broccoli florets than stalks. During thermal treatments for 10 min, AAO in broccoli florets and stalks was stable until around 50 degrees C. A 10-min thermal treatment at 80 degrees C almost completely inactivated AAO in broccoli. AAO inactivation followed 1st order kinetics in the temperature range of 55 to 65 degrees C. Based on this study, a thermal treatment above 70 degrees C is recommended for crushed vegetable products to prevent oxidation of l-AA to DHAA, the onset of vitamin C degradation. The results reported in this study are applicable for both domestic and industrial processing of vegetables into products such as juices, soups, and purees. In this report, we have demonstrated that processing crushed broccoli in a temperature range of 30 to 60 degrees C could result in the conversion of l-ascorbic acid to dehydroascorbic (DHAA), a very important reaction in regard to vitamin C degradation because DHAA could be easily converted to other compounds that do not have the biological activity of vitamin C.

Influence of Tridax procumbens on lysyl oxidase activity and wound healing.

PubMed

Udupa, S L; Udupa, A L; Kulkarni, D R

1991-08-01

The effects of an indigenous drug, Tridax procumbens L. (Compositae), on developing granulation tissue in rats were studied. Subcutaneously harvested granuloma tissue formed on dead space wound was removed at 4 day intervals up to 32 days of wounding. Lysyl oxidase activity, protein content, specific activity, and breaking strength were all increased in drug-treated animals as compared to controls. A fall in the lysyl oxidase activity was observed in drug-treated animals after day 8. The drug may be having a dual role: one a stimulatory (direct) effect in the initial phase of wound healing and the other a depressant (indirect) effect in the later stage.

Targeting NADPH oxidases in vascular pharmacology

PubMed Central

Schramm, Agata; Matusik, Paweł; Osmenda, Grzegorz; Guzik, Tomasz J

2012-01-01

Oxidative stress is a molecular dysregulation in reactive oxygen species (ROS) metabolism, which plays a key role in the pathogenesis of atherosclerosis, vascular inflammation and endothelial dysfunction. It is characterized by a loss of nitric oxide (NO) bioavailability. Large clinical trials such as HOPE and HPS have not shown a clinical benefit of antioxidant vitamin C or vitamin E treatment, putting into question the role of oxidative stress in cardiovascular disease. A change in the understanding of the molecular nature of oxidative stress has been driven by the results of these trials. Oxidative stress is no longer perceived as a simple imbalance between the production and scavenging of ROS, but as a dysfunction of enzymes involved in ROS production. NADPH oxidases are at the center of these events, underlying the dysfunction of other oxidases including eNOS uncoupling, xanthine oxidase and mitochondrial dysfunction. Thus NADPH oxidases are important therapeutic targets. Indeed, HMG-CoA reductase inhibitors (statins) as well as drugs interfering with the renin-angiotensin-aldosterone system inhibit NADPH oxidase activation and expression. Angiotensin-converting enzyme (ACE) inhibitors, AT1 receptor antagonists (sartans) and aliskiren, as well as spironolactone or eplerenone, have been discussed. Molecular aspects of NADPH oxidase regulation must be considered, while thinking about novel pharmacological targeting of this family of enzymes consisting of several homologs Nox1, Nox2, Nox3, Nox4 and Nox5 in humans. In order to properly design trials of antioxidant therapies, we must develop reliable techniques for the assessment of local and systemic oxidative stress. Classical antioxidants could be combined with novel oxidase inhibitors. In this review, we discuss NADPH oxidase inhibitors such as VAS2870, VAS3947, GK-136901, S17834 or plumbagin. Therefore, our efforts must focus on generating small molecular weight inhibitors of NADPH oxidases, allowing the

A new methodology for the determination of enzyme activity based on carbon nanotubes and glucose oxidase.

PubMed

Yeşiller, Gülden; Sezgintürk, Mustafa Kemal

2015-11-10

In this research, a novel enzyme activity analysis methodology is introduced as a new perspective for this area. The activity of elastase enzyme, which is a digestive enzyme mostly of found in the digestive system of vertebrates, was determined by an electrochemical device composed of carbon nanotubes and a second enzyme, glucose oxidase, which was used as a signal generator enzyme. In this novel methodology, a complex bioactive layer was constructed by using carbon nanotubes, glucose oxidase and a supporting protein, gelatin on a solid, conductive substrate. The activity of elastase was determined by monitoring the hydrolysis rate of elastase enzyme in the bioactive layer. As a result of this hydrolysis of elastase, glucose oxidase was dissociated from the bioactive layer, and following this the electrochemical signal due to glucose oxidase was decreased. The progressive elastase-catalyzed digestion of the bioactive layer containing glucose oxidase decreased the layer's enzymatic efficiency, resulting in a decrease of the glucose oxidation current as a function of the enzyme activity. The ratio of the decrease was correlated to elastase activity level. In this study, optimization experiments of bioactive components and characterization of the resulting new electrochemical device were carried out. A linear calibration range from 0.0303U/mL to 0.0729U/mL of elastase was reported. Real sample analyses were also carried out by the new electrochemical device. Copyright © 2015 Elsevier B.V. All rights reserved.

Improvement of exopolysaccharide production in Lactobacillus casei LC2W by overexpression of NADH oxidase gene.

PubMed

Li, Nan; Wang, Yuanlong; Zhu, Ping; Liu, Zhenmin; Guo, Benheng; Ren, Jing

2015-02-01

Lactobacillus casei LC2W is an exopolysaccharide (EPS)-producing strain with probiotic effects. To investigate the regulation mechanism of EPS biosynthesis and to improve EPS production through cofactor engineering, a H₂O-forming NADH oxidase gene was cloned from Streptococcus mutans and overexpressed in L. casei LC2W under the control of constitutive promoter P₂₃. The recombinant strain LC-nox exhibited 0.854 U/mL of NADH oxidase activity, which was elevated by almost 20-fold in comparison with that of wild-type strain. As a result, overexpression of NADH oxidase resulted in a reduction in growth rate. In addition, lactate production was decreased by 22% in recombinant strain. It was proposed that more carbon source was saved and used for the biosynthesis of EPS, the production of which was reached at 219.4 mg/L, increased by 46% compared to that of wild-type strain. This work provided a novel and convenient genetic approach to manipulate metabolic flux and to increase EPS production. To the best of our knowledge, this is the first report which correlates cofactor engineering with EPS production. Copyright © 2015 Elsevier GmbH. All rights reserved.

Direct electrochemistry and intramolecular electron transfer of ascorbate oxidase confined on L-cysteine self-assembled gold electrode.

PubMed

Patil, Bhushan; Kobayashi, Yoshiki; Fujikawa, Shigenori; Okajima, Takeyoshi; Mao, Lanqun; Ohsaka, Takeo

2014-02-01

A direct electrochemistry and intramolecular electron transfer of multicopper oxidases are of a great importance for the fabrication of these enzyme-based bioelectrochemical-devices. Ascorbate oxidase from Acremonium sp. (ASOM) has been successfully immobilized via a chemisorptive interaction on the l-cysteine self-assembled monolayer modified gold electrode (cys-SAM/AuE). Thermodynamics and kinetics of adsorption of ASOM on the cys-SAM/AuE were studied using cyclic voltammetry. A well-defined redox wave centered at 166±3mV (vs. Ag│AgCl│KCl(sat.)) was observed in 5.0mM phosphate buffer solution (pH7.0) at the fabricated ASOM electrode, abbreviated as ASOM/cys-SAM/AuE, confirming a direct electrochemistry, i.e., a direct electron transfer (DET) between ASOM and cys-SAM/AuE. The direct electrochemistry of ASOM was further confirmed by taking into account the chemical oxidation of ascorbic acid (AA) by O2 via an intramolecular electron transfer in the ASOM as well as the electrocatalytic oxidation of AA at the ASOM/cys-SAM/AuE. Thermodynamics and kinetics of the adsorption of ASOM on the cys-SAM/AuE have been elaborated along with its direct electron transfer at the modified electrodes on the basis of its intramolecular electron transfer and electrocatalytic activity towards ascorbic acid oxidation and O2 reduction. ASOM saturated surface area was obtained as 2.41×10(-11)molcm(-2) with the apparent adsorption coefficient of 1.63×10(6)Lmol(-1). The ASOM confined on the cys-SAM/AuE possesses its essential enzymatic function. © 2013.

Isolated sulfite oxidase deficiency.

PubMed

Rupar, C A; Gillett, J; Gordon, B A; Ramsay, D A; Johnson, J L; Garrett, R M; Rajagopalan, K V; Jung, J H; Bacheyie, G S; Sellers, A R

1996-12-01

Isolated sulfite oxidase (SO) deficiency is an autosomal recessively inherited inborn error of sulfur metabolism. In this report of a ninth patient the clinical history, laboratory results, neuropathological findings and a mutation in the sulfite oxidase gene are described. The data from this patient and previously published patients with isolated sulfite oxidase deficiency and molybdenum cofactor deficiency are summarized to characterize this rare disorder. The patient presented neonatally with intractable seizures and did not progress developmentally beyond the neonatal stage. Dislocated lenses were apparent at 2 months. There was increased urine excretion of sulfite and S-sulfocysteine and a decreased concentration of plasma cystine. A lactic acidemia was present for 6 months. Liver sulfite oxidase activity was not detectable but xanthine dehydrogenase activity was normal. The boy died of respiratory failure at 32 months. Neuropathological findings of cortical necrosis and extensive cavitating leukoencephalopathy were reminiscent of those seen in severe perinatal asphyxia suggesting an etiology of energy deficiency. A point mutation that resulted in a truncated protein missing the molybdenum-binding site has been identified.

NADPH Oxidases in Vascular Pathology

PubMed Central

Konior, Anna; Schramm, Agata; Czesnikiewicz-Guzik, Marta

2014-01-01

Abstract Significance: Reactive oxygen species (ROS) play a critical role in vascular disease. While there are many possible sources of ROS, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases play a central role. They are a source of “kindling radicals,” which affect other enzymes, such as nitric oxide synthase endothelial nitric oxide synthase or xanthine oxidase. This is important, as risk factors for atherosclerosis (hypertension, diabetes, hypercholesterolemia, and smoking) regulate the expression and activity of NADPH oxidases in the vessel wall. Recent Advances: There are seven isoforms in mammals: Nox1, Nox2, Nox3, Nox4, Nox5, Duox1 and Duox2. Nox1, Nox2, Nox4, and Nox5 are expressed in endothelium, vascular smooth muscle cells, fibroblasts, or perivascular adipocytes. Other homologues have not been found or are expressed at very low levels; their roles have not been established. Nox1/Nox2 promote the development of endothelial dysfunction, hypertension, and inflammation. Nox4 may have a role in protecting the vasculature during stress; however, when its activity is increased, it may be detrimental. Calcium-dependent Nox5 has been implicated in oxidative damage in human atherosclerosis. Critical Issues: NADPH oxidase-derived ROS play a role in vascular pathology as well as in the maintenance of normal physiological vascular function. We also discuss recently elucidated mechanisms such as the role of NADPH oxidases in vascular protection, vascular inflammation, pulmonary hypertension, tumor angiogenesis, and central nervous system regulation of vascular function and hypertension. Future Directions: Understanding the role of individual oxidases and interactions between homologues in vascular disease is critical for efficient pharmacological regulation of vascular NADPH oxidases in both the laboratory and clinical practice. Antioxid. Redox Signal. 20, 2794–2814. PMID:24180474

Levels and interactions of plasma xanthine oxidase, catalase and liver function parameters in Nigerian children with Plasmodium falciparum infection.

PubMed

Iwalokun, B A; Bamiro, S B; Ogunledun, A

2006-12-01

Elevated plasma levels of xanthine oxidase and liver function parameters have been associated with inflammatory events in several human diseases. While xanthine oxidase provides in vitro protection against malaria, its pathophysiological functions in vivo and interactions with liver function parameters remain unclear. This study examined the interactions and plasma levels of xanthine oxidase (XO) and uric acid (UA), catalase (CAT) and liver function parameters GOT, GPT and bilirubin in asymptomatic (n=20), uncomplicated (n=32), and severe (n=18) falciparum malaria children aged 3-13 years. Compared to age-matched control (n=16), significant (p<0.05) elevation in xanthine oxidase by 100-550%, uric acid by 15.4-153.8%, GOT and GPT by 22.1-102.2%, and total bilirubin by 2.3-86% according to parasitaemia (geometric mean parasite density (GMPD)=850-87100 parasites/microL) was observed in the malarial children. Further comparison with control revealed higher CAT level (16.2+/-0.5 vs 14.6+/-0.4 U/L; p<0.05) lacking significant (p>0.05) correlation with XO, but lower CAT level (13.4-5.4 U/L) with improved correlations (r=-0.53 to -0.91; p<0.05) with XO among the asymptomatic and symptomatic malaria children studied. 75% of control, 45% of asymptomatic, 21.9% of uncomplicated, and none of severe malaria children had Hb level>11.0 g/dL. Multivariate analyses further revealed significant (p<0.05) correlations between liver function parameters and xanthine oxidase (r=0.57-0.64) only in the severe malaria group. We conclude that elevated levels of XO and liver enzymes are biochemical features of Plasmodium falciparum parasitaemia in Nigerian children, with both parameters interacting differently to modulate the catalase response in asymptomatic and symptomatic falciparum malaria.

Impacts on the metabolome of down-regulating polyphenol oxidase in transgenic potato tubers

USDA-ARS?s Scientific Manuscript database

Tubers of potato (Solanum tuberosum L. cv. Estima) genetically modified (GM) to reduce polyphenol oxidase (PPO) activity and enzymatic discolouration were assessed for changes in the metabolome using Liquid Chromatography-Mass Spectrometry (LC-MS) and Gas Chromatography (GC)-MS. Metabolome changes ...

Permethrin Induces Overexpression of Cytochrome c Oxidase Subunit 3 in Aedes aegypti

USDA-ARS?s Scientific Manuscript database

Using quantitative PCR (QPCR), the relative transcriptional levels of cytochrome c oxidase subunit 3 (CO3) were studied in Aedes aegypti (L.) in response to treatments with acetone, permethrin, or fipronil. The transcriptional levels of CO3 were significantly (p <0.05) higher in acetone-treated Ae. ...

SPERMINE OXIDASE: AN AMINE OXIDASE WITH SPECIFICITY FOR SPERMINE AND SPERMIDINE

PubMed Central

Hirsch, James G.

1953-01-01

Sheep serum and bovine serum contain an enzyme which brings about a rapid oxidative deamination of certain biological amines. This enzyme differs from previously described amine oxidases in several regards and especially in its substrate specificity. Studies thus far indicate that only spermine and the closely related compound spermidine serve as substrates for the enzyme in sheep serum. For this reason, the enzyme has been named spermine oxidase. Spermine oxidase is active in a variety of fluids of various ionic strength and buffer composition. The reaction takes place between pH 6.0 and pH 8.0 with an optimal rate in the vicinity of neutrality. Under certain conditions, the rate of oxygen consumption during the initial phase of the reaction is independent of the concentration of substrate. The diminution in rate observed during the latter phase of the enzymatic attack appears to be due to an alteration in the kinetics at low concentrations of substrate, or to competitive inhibition by a product of the reaction. Carbonyl reagents almost completely block the action of spermine oxidase, while certain amines and the cyanide ion bring about partial inhibition. Thiol reagents and sequestering compounds do not alter the course of the oxidative process. In the presence of low concentrations of mercuric chloride, the sheep serum-spermine system consumes approximately twice as much oxygen as controls containing no mercuric ion. The mechanism by which the mercuric ion stimulates additional oxygen uptake is obscure. PMID:13052805

RNA interference of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO1 and ACO2) genes expression prolongs the shelf life of Eksotika (Carica papaya L.) papaya fruit.

PubMed

Sekeli, Rogayah; Abdullah, Janna Ong; Namasivayam, Parameswari; Muda, Pauziah; Abu Bakar, Umi Kalsom; Yeong, Wee Chien; Pillai, Vilasini

2014-06-19

The purpose of this study was to evaluate the effectiveness of using RNA interference in down regulating the expression of 1-aminocyclopropane-1-carboxylic acid oxidase gene in Eksotika papaya. One-month old embryogenic calli were separately transformed with Agrobacterium strain LBA 4404 harbouring the three different RNAi pOpOff2 constructs bearing the 1-aminocyclopropane-1-carboxylic acid oxidase gene. A total of 176 putative transformed lines were produced from 15,000 calli transformed, selected, then regenerated on medium supplemented with kanamycin. Integration and expression of the targeted gene in putatively transformed lines were verified by PCR and real-time RT-PCR. Confined field evaluation of a total of 31 putative transgenic lines planted showed a knockdown expression of the targeted ACO1 and ACO2 genes in 13 lines, which required more than 8 days to achieve the full yellow colour (Index 6). Fruits harvested from lines pRNAiACO2 L2-9 and pRNAiACO1 L2 exhibited about 20 and 14 days extended post-harvest shelf life to reach Index 6, respectively. The total soluble solids contents of the fruits ranged from 11 to 14° Brix, a range similar to fruits from non-transformed, wild type seed-derived plants.

Various applications of immobilized glucose oxidase and polyphenol oxidase in a conducting polymer matrix.

PubMed

Cil, M; Böyükbayram, A E; Kiralp, S; Toppare, L; Yağci, Y

2007-06-01

In this study, glucose oxidase and polyphenol oxidase were immobilized in conducting polymer matrices; polypyrrole and poly(N-(4-(3-thienyl methylene)-oxycarbonyl phenyl) maleimide-co-pyrrole) via electrochemical method. Fourier transform infrared and scanning electron microscope were employed to characterize the copolymer of (N-(4-(3-thienyl methylene)-oxycarbonyl phenyl) maleimide) with pyrrole. Kinetic parameters, maximum reaction rate and Michealis-Menten constant, were determined. Effects of temperature and pH were examined for immobilized enzymes. Also, storage and operational stabilities of enzyme electrodes were investigated. Glucose and polyphenol oxidase enzyme electrodes were used for determination of the glucose amount in orange juices and human serum and phenolic amount in red wines, respectively.

Leptin Induces Oxidative Stress Through Activation of NADPH Oxidase in Renal Tubular Cells: Antioxidant Effect of L-Carnitine.

PubMed

Blanca, Antonio J; Ruiz-Armenta, María V; Zambrano, Sonia; Salsoso, Rocío; Miguel-Carrasco, José L; Fortuño, Ana; Revilla, Elisa; Mate, Alfonso; Vázquez, Carmen M

2016-10-01

Leptin is a protein involved in the regulation of food intake and in the immune and inflammatory responses, among other functions. Evidences demonstrate that obesity is directly associated with high levels of leptin, suggesting that leptin may directly link obesity with the elevated cardiovascular and renal risk associated with increased body weight. Adverse effects of leptin include oxidative stress mediated by activation of NADPH oxidase. The aim of this study was to evaluate the effect of L-carnitine (LC) in rat renal epithelial cells (NRK-52E) exposed to leptin in order to generate a state of oxidative stress characteristic of obesity. Leptin increased superoxide anion (O2 (•) -) generation from NADPH oxidase (via PI3 K/Akt pathway), NOX2 expression and nitrotyrosine levels. On the other hand, NOX4 expression and hydrogen peroxide (H2 O2 ) levels diminished after leptin treatment. Furthermore, the expression of antioxidant enzymes, catalase, and superoxide dismutase, was altered by leptin, and an increase in the mRNA expression of pro-inflammatory factors was also found in leptin-treated cells. LC restored all changes induced by leptin to those levels found in untreated cells. In conclusion, stimulation of NRK-52E cells with leptin induced a state of oxidative stress and inflammation that could be reversed by preincubation with LC. Interestingly, LC induced an upregulation of NOX4 and restored the release of its product, hydrogen peroxide, which suggests a protective role of NOX4 against leptin-induced renal damage. J. Cell. Biochem. 117: 2281-2288, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Stepwise Hydrogen Atom and Proton Transfers in Dioxygen Reduction by Aryl-Alcohol Oxidase.

PubMed

Carro, Juan; Ferreira, Patricia; Martínez, Angel T; Gadda, Giovanni

2018-03-20

The mechanism of dioxygen reduction by the flavoenzyme aryl-alcohol oxidase was investigated with kinetic isotope, viscosity, and pL (pH/pD) effects in rapid kinetics experiments by stopped-flow spectrophotometry of the oxidative half-reaction of the enzyme. Double mixing of the enzyme in a stopped-flow spectrophotometer with [α- 2 H 2 ]- p-methoxybenzyl alcohol and oxygen at varying aging times established a slow rate constant of 0.0023 s -1 for the wash-out of the D atom from the N5 atom of the reduced flavin. Thus, the deuterated substrate could be used to probe the cleavage of the N-H bond of the reduced flavin in the oxidative half-reaction. A significant and pH-independent substrate kinetic isotope effect (KIE) of 1.5 between pH 5.0 and 8.0 demonstrated that H transfer is partially limiting the oxidative half-reaction of the enzyme; a negligible solvent KIE of 1.0 between pD 5.0 and 8.0 proved a fast H + transfer reaction that does not contribute to determining the flavin oxidation rates. Thus, a mechanism for dioxygen reduction in which the H atom originating from the reduced flavin and a H + from a solvent exchangeable site are transferred in separate kinetic steps is proposed. The spectroscopic and kinetic data presented also showed a lack of stabilization of transient flavin intermediates. The substantial differences in the mechanistic details of O 2 reduction by aryl-alcohol oxidase with respect to other alcohol oxidases like choline oxidase, pyranose 2-oxidase, and glucose oxidase further demonstrate the high level of versatility of the flavin cofactor in flavoenzymes.

Purification, characterization and amino acid content of cholesterol oxidase produced by Streptomyces aegyptia NEAE 102.

PubMed

El-Naggar, Noura El-Ahmady; Deraz, Sahar F; Soliman, Hoda M; El-Deeb, Nehal M; El-Shweihy, Nancy M

2017-03-29

There is an increasing demand on cholesterol oxidase for its various industrial and clinical applications. The current research was focused on extracellular cholesterol oxidase production under submerged fermentation by a local isolate previously identified as Streptomyces aegyptia NEAE 102. The crude enzyme extract was purified by two purification steps, protein precipitation using ammonium sulfate followed by ion exchange chromatography using DEAE Sepharose CL-6B. The kinetic parameters of purified cholesterol oxidase from Streptomyces aegyptia NEAE 102 were studied. The best conditions for maximum cholesterol oxidase activity were found to be 105 min of incubation time, an initial pH of 7 and temperature of 37 °C. The optimum substrate concentration was found to be 0.4 mM. The higher thermal stability behavior of cholesterol oxidase was at 50 °C. Around 63.86% of the initial activity was retained by the enzyme after 20 min of incubation at 50 °C. The apparent molecular weight of the purified enzyme as sized by sodium dodecyl sulphate-polyacryalamide gel electrophoresis was approximately 46 KDa. On DEAE Sepharose CL-6B column cholesterol oxidase was purified to homogeneity with final specific activity of 16.08 U/mg protein and 3.14-fold enhancement. The amino acid analysis of the purified enzyme produced by Streptomyces aegyptia NEAE 102 illustrated that, cholesterol oxidase is composed of 361 residues with glutamic acid as the most represented amino acid with concentration of 11.49 μg/mL. Taking into account the extracellular production, wide pH tolerance, thermal stability and shelf life, cholesterol oxidase produced by Streptomyces aegyptia NEAE 102 suggested that the enzyme could be industrially useful.

In vitro assessment of anticholinesterase and NADH oxidase inhibitory activities of an edible fern, Diplazium esculentum.

PubMed

Roy, Subhrajyoti; Dutta, Somit; Chaudhuri, Tapas Kumar

2015-07-01

Diplazium esculentum is the most commonly consumed edible fern throughout Asia and Oceania. Several studies have been performed so far to determine different functional properties of this plant, but there have been no reports on the anticholinesterase and nicotinamide adenine dinucleotide (NADH) oxidase inhibitory activities of this plant. Therefore, the present study was conducted to determine the anticholinesterase and NADH oxidase inhibitory activities of 70% methanolic extract of D. esculentum. The D. esculentum extract was investigated for its acetylcholinesterase and NADH oxidase inhibitory activities as well as its free radical scavenging and total antioxidant activities in the linoleic acid system. The free radical scavenging activity of the extract was determined by the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) method. The total antioxidant activity of the extract was evaluated by ferric thiocyanate (FTC) and thiobarbituric acid (TBA) methods. The D. esculentum extract inhibited acetylcholinesterase and NADH oxidase in a dose-dependent manner, with IC50 values of 272.97±19.38 and 265.81±21.20 μg/mL, respectively. The extract also showed a potent DPPH radical scavenging activity with an IC50 value of 402.88±12.70 μg/mL. Moreover, the extract showed 27.41% and 33.22% of total antioxidant activities determined by FTC and TBA methods, respectively. Results indicated that 70% methanolic extract of D. esculentum effectively inhibited the enzymes acetylcholinesterase and NADH oxidase and acted as a potent antioxidant and free radical scavenger. These in vitro assays indicate that this plant extract is a significant source of natural antioxidants, which may be helpful in preventing the progression of various neurodegenerative disorders associated with oxidative stress.

Urate oxidase for the prevention and treatment of tumour lysis syndrome in children with cancer.

PubMed

Cheuk, Daniel Kl; Chiang, Alan Ks; Chan, Godfrey Cf; Ha, Shau Yin

2017-03-08

under the curve of uric acid at four days (MD -201.00 mg/dLhr, 95% CI -258.05 mg/dLhr to -143.95 mg/dLhr; P < 0.00001) were significantly better in the treatment group.One CCT evaluated the primary outcome; no clear evidence of a difference was identified between the treatment and the control groups (RR 0.77, 95% CI 0.44 to 1.33; P = 0.34). Pooled results of three CCTs showed significantly lower mortality due to TLS in the treatment group (RR 0.05, 95% CI 0.00 to 0.89; P = 0.04); no clear evidence of a difference in all-cause mortality was identified between the groups (RR 0.19, 95% CI 0.01 to 3.42; P = 0.26). Pooled results from five CCTs showed significantly lower incidence of renal failure in the treatment group (RR 0.26, 95% CI 0.08 to 0.89; P = 0.03). Results of CCTs also showed significantly lower uric acid in the treatment group at two days (three CCTs: MD -3.80 mg/dL, 95% CI -7.37 mg/dL to -0.24 mg/dL; P = 0.04), three days (two CCTs: MD -3.13 mg/dL, 95% CI -6.12 mg/dL to -0.14 mg/dL; P = 0.04), four days (two CCTs: MD -4.60 mg/dL, 95% CI -6.39 mg/dL to -2.81 mg/dL; P < 0.00001), and seven days (one CCT: MD -1.74 mg/dL, 95% CI -3.01 mg/dL to -0.47 mg/dL; P = 0.007) after therapy, but not one day (three CCTs: MD -3.00 mg/dL, 95% CI -7.61 mg/dL to 1.60 mg/dL; P = 0.2), five days (one CCT: MD -1.02 mg/dL, 95% CI -2.24 mg/dL to 0.20 mg/dL; P = 0.1), and 12 days (one CCT: MD -0.80 mg/dL, 95% CI -2.51 mg/dL to 0.91 mg/dL; P = 0.36) after therapy. Pooled results from three CCTs showed higher frequency of adverse effects in participants who received urate oxidase (RR 9.10, 95% CI 1.29 to 64.00; P = 0.03).Another included RCT, with 30 participants, compared different doses of rasburicase (0.2 mg/kg versus 0.15 mg/kg). The primary outcome was not evaluated. No clear evidence of a difference in mortality (all-cause mortality (Fisher's exact test P = 1.0) and mortality due to TLS (no deaths in both groups)) and renal failure (no renal failure in both groups) was identified

Amperometric L-lactate biosensor based on screen-printed carbon electrode containing cobalt phthalocyanine, coated with lactate oxidase-mesoporous silica conjugate layer.

PubMed

Shimomura, Takeshi; Sumiya, Touru; Ono, Masatoshi; Ito, Tetsuji; Hanaoka, Taka-aki

2012-02-10

A novel amperometric biosensor for the measurement of L-lactate has been developed. The device comprises a screen-printed carbon electrode containing cobalt phthalocyanine (CoPC-SPCE), coated with lactate oxidase (LOD) that is immobilized in mesoporous silica (FSM8.0) using a polymer matrix of denatured polyvinyl alcohol; a Nafion layer on the electrode surface acts as a barrier to interferents. The sampling unit attached to the SPCE requires only a small sample volume of 100 μL for each measurement. The measurement of l-lactate is based on the signal produced by hydrogen peroxide, the product of the enzymatic reaction. The behavior of the biosensor, LOD-FSM8.0/Naf/CoPC-SPCE, was examined in terms of pH, applied potential, sensitivity and operational range, selectivity, and storage stability. The sensor showed an optimum response at a pH of 7.4 and an applied potential of +450 mV. The determination range and the response time for L-lactate were 18.3 μM to 1.5 mM and approximately 90s, respectively. In addition, the sensor exhibited high selectivity for L-lactate and was quite stable in storage, showing no noticeable change in its initial response after being stored for over 9 months. These results indicate that our method provides a simple, cost-effective, high-performance biosensor for l-lactate. Copyright © 2011 Elsevier B.V. All rights reserved.

The neurochemical and behavioral effects of the novel cholinesterase–monoamine oxidase inhibitor, ladostigil, in response to L-dopa and L-tryptophan, in rats

PubMed Central

Sagi, Yotam; Driguès, Noam; Youdim, Moussa B H

2005-01-01

The novel drugs, ladostigil (TV3326) and TV3279, are R and S isomers, respectively, derived from a combination of the carbamate cholinesterase (ChE) inhibitor, rivastigmine, and the pharmacophore of the monoamine oxidase (MAO) B inhibitor, rasagiline. They were developed for the treatment of comorbidity of dementia with Parkinsonism. In the present study, we determined the effects of these drugs on both aminergic neurotransmitter levels and motor behavioral activity in naïve and in L-dopa- or L-tryptophan-induced rats. Chronic treatment of rats with ladostigil (52 mg kg−1 for 21 days) inhibited hippocampal and striatal MAO A and B activities by >90%, increased striatal levels of dopamine and serotonin, and inhibited striatal ChE activity by ∼50%. Chronic TV3279 (26 mg kg−1 for 21 days) similarly inhibited ∼50% of striatal ChE activity, but did not affect MAO activity or amine levels. In sharp contrast to the inductive effect of the MAO A/B inhibitor, tranylcypromine (TCP), on stereotyped hyperactivity in response to L-dopa (50 mg kg−1) or L-tryptophan (100 mg kg−1), ladostigil completely inhibited these behavioral hyperactivity syndromes. Accordingly, acute rivastigmine (2 mg kg−1) and chronic TV3279 abolished the ability of TCP to initiate L-dopa-induced hyperactivity, while scopolamine (0.5 mg kg−1) reversed the inhibitory effect of chronic ladostigil on L-dopa-induced hyperactivity, suggesting that ladostigil may attenuate successive locomotion by activating central cholinergic muscarinic receptors. Finally, while chronic ladostigil administration to naïve rats resulted in preserved spontaneous motor behavior, acute treatment with ladostigil decreased motor performance, compared to control animals. In contrast, chronic as well as acute treatments with TV3279 reduced spontaneous motor activity. Thus, the aminergic potentiation by ladostigil may counteract its cholinergic inhibitory effect on spontaneous motor behavior

Glycosylation site-targeted PEGylation of glucose oxidase retains native enzymatic activity.

PubMed

Ritter, Dustin W; Roberts, Jason R; McShane, Michael J

2013-04-10

Targeted PEGylation of glucose oxidase at its glycosylation sites was investigated to determine the effect on enzymatic activity, as well as the bioconjugate's potential in an optical biosensing assay. Methoxy-poly(ethylene glycol)-hydrazide (4.5kDa) was covalently coupled to periodate-oxidized glycosylation sites of glucose oxidase from Aspergillus niger. The bioconjugate was characterized using gel electrophoresis, liquid chromatography, mass spectrometry, and dynamic light scattering. Gel electrophoresis data showed that the PEGylation protocol resulted in a drastic increase (ca. 100kDa) in the apparent molecular mass of the protein subunit, with complete conversion to the bioconjugate; liquid chromatography data corroborated this large increase in molecular size. Mass spectrometry data proved that the extent of PEGylation was six poly(ethylene glycol) chains per glucose oxidase dimer. Dynamic light scattering data indicated the absence of higher-order oligomers in the PEGylated GOx sample. To assess stability, enzymatic activity assays were performed in triplicate at multiple time points over the course of 29 days in the absence of glucose, as well as before and after exposure to 5% w/v glucose for 24h. At a confidence level of 95%, the bioconjugate's performance was statistically equivalent to native glucose oxidase in terms of activity retention over the 29 day time period, as well as following the 24h glucose exposure. Finally, the bioconjugate was entrapped within a poly(2-hydroxyethyl methacrylate) hydrogel containing an oxygen-sensitive phosphor, and the construct was shown to respond approximately linearly with a 220±73% signal change (n=4, 95% confidence interval) over the physiologically-relevant glucose range (i.e., 0-400mg/dL); to our knowledge, this represents the first demonstration of PEGylated glucose oxidase incorporated into an optical biosensing assay. Copyright © 2013 Elsevier Inc. All rights reserved.

Utilization of quercetin and quercetin glycosides from onion (Allium cepa L.) solid waste as an antioxidant, urease and xanthine oxidase inhibitors.

PubMed

Nile, Shivraj Hariram; Nile, Arti Shivraj; Keum, Young Soo; Sharma, Kavita

2017-11-15

This study aimed to determine the flavonol glycosides from onion solid waste (OSW) using HPLC analysis, with antioxidant and enzyme inhibitory activities. We found considerable amount of quercetin-4'-O-monoglucoside (QMG: 254.85), quercetin-3,4'-O-diglucoside (QDG: 162.34), quercetin (Q: 60.44), and isorhamnetin-3-glucoside (IMG: 23.92) (mg/100g) dry weight (DW) of OSW. For OSW, the methanol and ethanol showed the strongest antioxidant activities, followed by ethyl acetate, chloroform, and n-hexane extracts. Among the flavonols, Q and QDG possessed higher antioxidant activities. OSW and flavonol glycosides displayed significant enzyme inhibitory activity, with IC 50 values ranging from 12.5±0.11 to 32.5±0.28 for OSW, 8.2±0.07 to 16.8±0.02 for flavonol glycosides, and 4.2±0.05μg/mL for thiourea (positive control) towards urease; while 15.2±0.8 to 35.8±0.2 (μg/mL) for OSW, 10.5±0.06 to 20.8±0.05 (μg/mL) for flavonol glycosides, and 6.5±0.05μg/mL for allopurinol (positive control) towards xanthine oxidase, respectively. The OSW and flavonol glycosides may thus be considered as potential antioxidant and antigout agents. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cellular mechanism of resistance of human colorectal adenocarcinoma cells against apoptosis-induction by Russell's Viper venom L-amino acid oxidase (Rusvinoxidase).

PubMed

Mukherjee, Ashis K; Saviola, Anthony J; Mackessy, Stephen P

2018-04-24

The present study highlights the cellular mechanism of resistance in human adenocarcinoma (Colo-205) cells against apoptosis induction by Rusvinoxidase, an L-amino acid oxidase purified from Russell's Viper venom (RVV). The significantly lower cytotoxicity as well as apoptotic activity of Rusvinoxidase towards Colo-205 cells (compared to MCF-7 breast cancer cells) is correlated with lower depletion of cellular glutathione content and increased down-regulation of catalase activity of Colo-205 cells following Rusvinoxidase treatment. Exposure to Rusvinoxidase subsequently diminished reactive oxygen species (ROS) production and failed to impair mitochondrial membrane potential, resulting in apoptosis induction resistance in Colo-205 cells. Further, higher expression levels of caspase 8, compared to caspase 9, indicate that Rusvinoxidase preferentially triggers the extrinsic pathway of apoptosis in Colo-205 cells. A time-dependent lower ratio of the relative expression of Bax and Bcl-xL (pro- and anti-apoptotic proteins) in Colo-205 cells, compared to our previous study on MCF-7 cells, unambiguously supports a higher cellular resistance mechanism in Colo-205 cells against Rusvinoxidase-induced apoptosis. Copyright © 2018. Published by Elsevier B.V.

The GA5 locus of Arabidopsis thaliana encodes a multifunctional gibberellin 20-oxidase: molecular cloning and functional expression.

PubMed

Xu, Y L; Li, L; Wu, K; Peeters, A J; Gage, D A; Zeevaart, J A

1995-07-03

The biosynthesis of gibberellins (GAs) after GA12-aldehyde involves a series of oxidative steps that lead to the formation of bioactive GAs. Previously, a cDNA clone encoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing), EC 1.14.11.-] was isolated by immunoscreening a cDNA library from liquid endosperm of pumpkin (Cucurbita maxima L.) with antibodies against partially purified GA 20-oxidase. Here, we report isolation of a genomic clone for GA 20-oxidase from a genomic library of the long-day species Arabidopsis thaliana Heynh., strain Columbia, by using the pumpkin cDNA clone as a heterologous probe. This genomic clone contains a GA 20-oxidase gene that consists of three exons and two introns. The three exons are 1131-bp long and encode 377 amino acid residues. A cDNA clone corresponding to the putative GA 20-oxidase genomic sequence was constructed with the reverse transcription-PCR method, and the identity of the cDNA clone was confirmed by analyzing the capability of the fusion protein expressed in Escherichia coli to convert GA53 to GA44 and GA19 to GA20. The Arabidopsis GA 20-oxidase shares 55% identity and > 80% similarity with the pumpkin GA 20-oxidase at the derived amino acid level. Both GA 20-oxidases share high homology with other 2-oxoglutarate-dependent dioxygenases (2-ODDs), but the highest homology was found between the two GA 20-oxidases. Mapping results indicated tight linkage between the cloned GA 20-oxidase and the GA5 locus of Arabidopsis. The ga5 semidwarf mutant contains a G-->A point mutation that inserts a translational stop codon in the protein-coding sequence, thus confirming that the GA5 locus encodes GA 20-oxidase. Expression of the GA5 gene in Ara-bidopsis leaves was enhanced after plants were transferred from short to long days; it was reduced by GA4 treatment, suggesting end-product repression in the GA biosynthetic pathway.

Expression and Characterization of Glucose Oxidase from Aspergillus niger in Yarrowia lipolytica.

PubMed

Khadivi Derakshan, Fatemeh; Darvishi, Farshad; Dezfulian, Mehrouz; Madzak, Catherine

2017-08-01

Glucose oxidase (GOX) is currently used in clinical, pharmaceutical, food and chemical industries. The aim of this study was expression and characterization of Aspergillus niger glucose oxidase gene in the yeast Yarrowia lipolytica. For the first time, the GOX gene of A. niger was successfully expressed in Y. lipolytica using a mono-integrative vector containing strong hybrid promoter and secretion signal. The highest total glucose oxidase activity was 370 U/L after 7 days of cultivation. An innovative method was used to cell wall disruption in current study, and it could be recommended to use for efficiently cell wall disruption of Y. lipolytica. Optimum pH and temperature for recombinant GOX activity were 5.5 and 37 °C, respectively. A single band with a molecular weight of 80 kDa similar to the native and pure form of A. niger GOX was observed for the recombinant GOX in SDS-PAGE analysis. Y. lipolytica is a suitable and efficient eukaryotic expression system to production of recombinant GOX in compered with other yeast expression systems and could be used to production of pure form of GOX for industrial applications.

Antibacterial action of a heat-stable form of L-amino acid oxidase isolated from king cobra (Ophiophagus hannah) venom.

PubMed

Lee, Mui Li; Tan, Nget Hong; Fung, Shin Yee; Sekaran, Shamala Devi

2011-03-01

The major l-amino acid oxidase (LAAO, EC 1.4.3.2) of king cobra (Ophiophagus hannah) venom is known to be an unusual form of snake venom LAAO as it possesses unique structural features and unusual thermal stability. The antibacterial effects of king cobra venom LAAO were tested against several strains of clinical isolates including Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli using broth microdilution assay. For comparison, the antibacterial effects of several antibiotics (cefotaxime, kanamycin, tetracycline, vancomycin and penicillin) were also examined using the same conditions. King cobra venom LAAO was very effective in inhibiting the two Gram-positive bacteria (S. aureus and S. epidermidis) tested, with minimum inhibitory concentration (MIC) of 0.78μg/mL (0.006μM) and 1.56μg/mL (0.012μM) against S. aureus and S. epidermidis, respectively. The MICs are comparable to the MICs of the antibiotics tested, on a weight basis. However, the LAAO was only moderately effective against three Gram-negative bacteria tested (P. aeruginosa, K. pneumoniae and E. coli), with MIC ranges from 25 to 50μg/mL (0.2-0.4μM). Catalase at the concentration of 1mg/mL abolished the antibacterial effect of LAAO, indicating that the antibacterial effect of the enzyme involves generation of hydrogen peroxide. Binding studies indicated that king cobra venom LAAO binds strongly to the Gram-positive S. aureus and S. epidermidis, but less strongly to the Gram-negative E. coli and P. aeruginosa, indicating that specific binding to bacteria is important for the potent antibacterial activity of the enzyme. Copyright © 2010 Elsevier Inc. All rights reserved.

Rosuvastatin prevents angiotensin II-induced vascular changes by inhibition of NAD(P)H oxidase and COX-1

PubMed Central

Colucci, Rocchina; Fornai, Matteo; Duranti, Emiliano; Antonioli, Luca; Rugani, Ilaria; Aydinoglu, Fatma; Ippolito, Chiara; Segnani, Cristina; Bernardini, Nunzia; Taddei, Stefano; Blandizzi, Corrado; Virdis, Agostino

2013-01-01

Background and Purpose NAD(P)H oxidase and COX-1 participate in vascular damage induced by angiotensin II. We investigated the effect of rosuvastatin on endothelial dysfunction, vascular remodelling, changes in extracellular matrix components and mechanical properties of small mesenteric arteries from angiotensin II-infused rats. Experimental Approach Male rats received angiotensin II (120 ng·kg−1·min−1, subcutaneously) for 14 days with or without rosuvastatin (10 mg·kg−1·day−1, oral gavage) or vehicle. Vascular functions and morphological parameters were assessed by pressurized myography. Key Results In angiotensin II-infused rats, ACh-induced relaxation was attenuated compared with controls, less sensitive to L-NAME, enhanced by SC-560 (COX-1 inhibitor) or SQ-29548 (prostanoid TP receptor antagonist), and normalized by the antioxidant ascorbic acid or NAD(P)H oxidase inhibitors. After rosuvastatin, relaxations to ACh were normalized, fully sensitive to L-NAME, and no longer affected by SC-560, SQ-29548 or NAD(P)H oxidase inhibitors. Angiotensin II enhanced intravascular superoxide generation, eutrophic remodelling, collagen and fibronectin depositions, and decreased elastin content, resulting in increased vessel stiffness. All these changes were prevented by rosuvastatin. Angiotensin II increased phosphorylation of NAD(P)H oxidase subunit p47phox and its binding to subunit p67phox, effects inhibited by rosuvastatin. Rosuvastatin down-regulated vascular Nox4/NAD(P)H isoform and COX-1 expression, attenuated the vascular release of 6-keto-PGF1α, and enhanced copper/zinc-superoxide dismutase expression. Conclusion and Implications Rosuvastatin prevents angiotensin II-induced alterations in resistance arteries in terms of function, structure, mechanics and composition. These effects depend on restoration of NO availability, prevention of NAD(P)H oxidase-derived oxidant excess, reversal of COX-1 induction and its prostanoid production, and stimulation of

Cytotoxic, Anti-Proliferative and Apoptosis Activity of l-Amino Acid Oxidase from Malaysian Cryptelytrops purpureomaculatus (CP-LAAO) Venom on Human Colon Cancer Cells.

PubMed

Zainal Abidin, Syafiq Asnawi; Rajadurai, Pathmanathan; Hoque Chowdhury, Md Ezharul; Othman, Iekhsan; Naidu, Rakesh

2018-06-08

The aim of this study is to investigate the potential anti-cancer activity of l-amino acid oxidase (CP-LAAO) purified from the venom of Cryptelytrops purpureomaculatus on SW480 and SW620 human colon cancer cells. Mass spectrometry guided purification was able to identify and purify CP-LAAO. Amino acid variations identified from the partial protein sequence of CP-LAAO may suggest novel variants of these proteins. The activity of the purified CP-LAAO was confirmed with o-phenyldiamine (OPD)-based spectrophotometric assay. CP-LAAO demonstrated time- and dose-dependent cytotoxic activity and the EC 50 value was determined at 13 µg/mL for both SW480 and SW620 cells. Significant increase of caspase-3 activity, reduction of Bcl-2 levels, as well as morphological changes consistent with apoptosis were demonstrated by CP-LAAO. Overall, these data provide evidence on the potential anti-cancer activity of CP-LAAO from the venom of Malaysian C. purpureomaculatus for therapeutic intervention of human colon cancer.

Correlation Between Monoamine Oxidase Inhibitors and Anticonvulsants

PubMed Central

Dwivedi, Chandradhar; Misra, Radhey S.; Chaudhari, Anshumali; Parmar, Surendra S.

1980-01-01

Monoamine oxidase inhibitory and anticonvulsant properties of 2-substituted styryl-6-bromo-3-(4-ethylbenzoate/4 benzhydrazide)-4-quinazoles are studied. All styryl quinazolone esters except compound number 9 exhibited monoamine oxidase inhibitory properties during oxidative deamination of kynuramine. Corresponding hydrazides were found to have relatively higher activity. All these quinazolones were able to protect against pentylenetetrazol induced seizures. These observations in general do not prove that monoamine oxidase inhibitory properties represent the biochemical basis for the anticonvulsant activity of these compounds. PMID:7420438

Involvement of abscisic acid in regulating antioxidative defense systems and IAA-oxidase activity and improving adventitious rooting in mung bean [Vigna radiata (L.) Wilczek] seedlings under cadmium stress.

PubMed

Li, Shi-Weng; Leng, Yan; Feng, Lin; Zeng, Xiao-Ying

2014-01-01

In vitro experiments were conducted to investigate the effects of abscisic acid (ABA) and Cd on antioxidative defense systems and indole-3-acetic acid (IAA) oxidase during adventitious rooting in mung bean [Vigna radiata (L.) Wilczek] seedlings. The exogenous ABA significantly enhanced the number and fresh weight of the adventitious roots. CdCl2 strongly inhibited adventitious rooting. Pretreatment with 10 μM ABA clearly alleviated the inhibitory effect of Cd on rooting. ABA significantly reduced superoxide dismutase (SOD), ascorbate peroxidase (APX), peroxidase (POD), and catalase (CAT) activities, as well as the levels of glutathione (GSH) and ascorbic acid (ASA) during adventitious rooting. ABA strongly increased IAA-oxidase activity during the induction (0-12 h) and expression (after 48 h) phases and increased the phenols levels. Cd treatment significantly reduced the activities of SOD, APX, POD, and IAA oxidase, as well as GSH level. Cd strongly increased ASA levels. ABA pretreatment counteracted Cd-induced alterations of certain antioxidants and antioxidative enzymes, e.g., remarkably rescued APX and POD activities, reduced the elevated SOD and CAT activities and ASA levels, and recovered the reduced GSH levels, caused by Cd stress. Thus, the physiological effects of the combination of ABA and Cd treatments were opposite of those obtained with Cd treatment alone, suggesting that ABA involved in the regulation of antioxidative defense systems and the alleviation of wounding- and Cd-induced oxidative stress.

Vascular oxidative stress and endothelial dysfunction in patients with chronic heart failure: role of xanthine-oxidase and extracellular superoxide dismutase.

PubMed

Landmesser, Ulf; Spiekermann, Stephan; Dikalov, Sergey; Tatge, Helma; Wilke, Ragna; Kohler, Christoph; Harrison, David G; Hornig, Burkhard; Drexler, Helmut

2002-12-10

Impaired flow-dependent, endothelium-mediated vasodilation (FDD) in patients with chronic heart failure (CHF) results, at least in part, from accelerated degradation of nitric oxide by oxygen radicals. The mechanisms leading to increased vascular radical formation, however, remain unclear. Therefore, we determined endothelium-bound activities of extracellular superoxide dismutase (ecSOD), a major vascular antioxidant enzyme, and xanthine-oxidase, a potent radical producing enzyme, and their relation to FDD in patients with CHF. ecSOD and xanthine-oxidase activities, released from endothelium into plasma by heparin bolus injection, were determined in 14 patients with CHF and 10 control subjects. FDD of the radial artery was measured using high-resolution ultrasound and was assessed before and after administration of the antioxidant vitamin C (25 mg/min; IA). In patients with CHF, endothelium-bound ecSOD activity was substantially reduced (5.0+/-0.7 versus 14.4+/-2.6 U x mL(-1) x min(-1); P<0.01) and closely related to FDD (r=0.61). Endothelium-bound xanthine-oxidase activity was increased by >200% (38+/-10 versus 12+/-4 nmol O2*- x microL(-1); P<0.05) and inversely related to FDD (r=-0.35) in patients with CHF. In patients with low ecSOD and high xanthine-oxidase activity, a greater benefit of vitamin C on FDD was observed, ie, the portion of FDD inhibited by radicals correlated negatively with ecSOD (r=-0.71) but positively with xanthine-oxidase (r=0.75). These results demonstrate that both increased xanthine-oxidase and reduced ecSOD activity are closely associated with increased vascular oxidative stress in patients with CHF. This loss of vascular oxidative balance likely represents a novel mechanism contributing to endothelial dysfunction in CHF.

Glucose biosensor based on immobilization of glucose oxidase on a carbon paste electrode modified with microsphere-attached l-glycine.

PubMed

Donmez, Soner; Arslan, Fatma; Sarı, Nurşen; Hasanoğlu Özkan, Elvan; Arslan, Halit

2017-09-01

In the present study, a novel biosensor that is sensitive to glucose was prepared using the microspheres modified with (4-formyl-3-methoxyphenoxymethyl)polystyrene (FMPS) with l-glycine. Polymeric microspheres having Schiff bases were prepared from FMPS using the glycine condensation method. Glucose oxidase enzyme was immobilized onto modified carbon paste electrode by cross-linking with glutaraldehyde. Oxidation of enzymatically produced H 2 O 2 (+0.5 V vs. Ag/AgCl) was used for determination of glucose. Optimal temperature and pH were found as 50 °C and 8.0, respectively. The glucose biosensor showed a linear working range from 5.0 × 10 -4 to 1.0 × 10 -2 M, R 2 = 0.999. Storage and operational stability of the biosensor were also investigated. The biosensor gave perfect reproducible results after 20 measurements with 3.3% relative standard deviation. It also had good storage stability. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

Regulation of tyramine oxidase synthesis in Klebsiella aerogenes.

PubMed Central

Okamura, H; Murooka, Y; Harada, T

1976-01-01

Tyramine oxidase in Klebsiella aerogenes is highly specific for tyramine, dopamine, octopamine, and norepinephrine, and its synthesis is induced specifically by these compounds. The enzyme is present in a membrane-bound form. The Km value for tyramine is 9 X 10(-4) M. Tyramine oxidase synthesis was subjected to catabolite repression by glucose in the presence of ammonium salts. Addition of cyclic adenosine 3',5'-monophosphate (cAMP) overcame the catabolite repression. A mutant strain, K711, which can produce a high level of beta-galactosidase in the presence of glucose and ammonium chloride, can also synthesize tyramine oxidase and histidase in the presence of inducer in glucose ammonium medium. Catabolite repression of tyramine oxidase synthesis was relieved when the cells were grown under conditions of nitrogen limitation, whereas beta-galactosidase was strongly repressed under these conditions. A cAMP-requiring mutant, MK54, synthesized tyramine oxidase rapidly when tyramine was used as the sole source of nitrogen in the absence of cAMP. However, a glutamine synthetase-constitutive mutant, MK94, failed to synthesize tyramine oxidase in the presence of glucose and ammonium chloride, although it synthesized histidase rapidly under these conditions. These results suggest that catabolite repression of tyramine oxidase synthesis in K. aerogenes is regulated by the intracellular level of cAMP and an unknown cytoplasmic factor that acts independently of cAMP and is formed under conditions of nitrogen limitation. PMID:179974

Mechanism of Flavoprotein l-6-Hydroxynicotine Oxidase: pH and Solvent Isotope Effects and Identification of Key Active Site Residues.

PubMed

Fitzpatrick, Paul F; Chadegani, Fatemeh; Zhang, Shengnan; Dougherty, Vi

2017-02-14

The flavoenzyme l-6-hydroxynicotine oxidase is a member of the monoamine oxidase family that catalyzes the oxidation of (S)-6-hydroxynicotine to 6-hydroxypseudooxynicotine during microbial catabolism of nicotine. While the enzyme has long been understood to catalyze oxidation of the carbon-carbon bond, it has recently been shown to catalyze oxidation of a carbon-nitrogen bond [Fitzpatrick, P. F., et al. (2016) Biochemistry 55, 697-703]. The effects of pH and mutagenesis of active site residues have now been utilized to study the mechanism and roles of active site residues. Asn166 and Tyr311 bind the substrate, while Lys287 forms a water-mediated hydrogen bond with flavin N5. The N166A and Y311F mutations result in ∼30- and ∼4-fold decreases in k cat /K m and k red for (S)-6-hydroxynicotine, respectively, with larger effects on the k cat /K m value for (S)-6-hydroxynornicotine. The K287M mutation results in ∼10-fold decreases in these parameters and a 6000-fold decrease in the k cat /K m value for oxygen. The shapes of the pH profiles are not altered by the N166A and Y311F mutations. There is no solvent isotope effect on the k cat /K m value for amines. The results are consistent with a model in which both the charged and neutral forms of the amine can bind, with the former rapidly losing a proton to a hydrogen bond network of water and amino acids in the active site prior to the transfer of hydride to the flavin.

Contribution of the NADH-oxidase (Nox) to the aerobic life of Lactobacillus sanfranciscensis DSM20451T.

PubMed

Jänsch, André; Freiding, Simone; Behr, Jürgen; Vogel, Rudi F

2011-02-01

Lactobacillus sanfranciscensis is the key bacterium in traditional sourdough fermentation. The molecular background of its oxygen tolerance was investigated by comparison of wild type and NADH-oxidase (Nox) knock out mutants. The nox gene of L. sanfranciscensis DSM20451(T) coding for a NADH-oxidase (Nox) was inactivated by single crossover integration to yield strain L. sanfranciscensis DSM20451Δnox. By inactivation of the native NADH-oxidase gene, it was ensured that besides fructose, O(2) can react as an electron acceptor. In aerated cultures the mutant strain was only able to grow in MRS media supplemented with fructose as electron acceptor, whereas the wild type strain showed a fructose independent growth response. The use of oxygen as an external electron acceptor enables L. sanfranciscensis to shift from acetyl-phosphate into the acetate branch and gain an additionally ATP, while the reduced cofactors were regenerated by Nox-activity. In aerated cultures the wild type strain formed a fermentation ratio of lactate to acetate of 1.09 in MRS supplemented with fructose after 24 h of fermentation, while the mutant strain formed a fermentation ratio of 3.05. Additionally, L. sanfranciscensis showed manganese-dependent growth response in aerated cultures, the final OD and growth velocity was increased in media supplemented with manganese. The expression of two predicted Mn(2+)/Fe(2+) transporters MntH1 and MntH2 in L. sanfranciscensis DSM20451(T) was verified by amplification of a 318 bp fragment of MntH1 and a 239 bp fragment of MntH2 from cDNA library obtained from aerobically, exponentially growing cells of L. sanfranciscensis DSM20451(T) in MRS. Moreover, the mutant strain DSM20451Δnox was more sensitive to the superoxide generating agent paraquat and showed inhibition of growth on diamide-treated MRS-plates without fructose supplementation. Copyright © 2010 Elsevier Ltd. All rights reserved.

NADPH oxidases of the brain: distribution, regulation, and function.

PubMed

Infanger, David W; Sharma, Ram V; Davisson, Robin L

2006-01-01

The NADPH oxidase is a multi-subunit enzyme that catalyzes the reduction of molecular oxygen to form superoxide (O(2)(-)). While classically linked to the respiratory burst in neutrophils, recent evidence now shows that O(2)(-) (and associated reactive oxygen species, ROS) generated by NADPH oxidase in nonphagocytic cells serves myriad functions in health and disease. An entire new family of NADPH Oxidase (Nox) homologues has emerged, which vary widely in cell and tissue distribution, as well as in function and regulation. A major concept in redox signaling is that while NADPH oxidase-derived ROS are necessary for normal cellular function, excessive oxidative stress can contribute to pathological disease. This certainly is true in the central nervous system (CNS), where normal NADPH oxidase function appears to be required for processes such as neuronal signaling, memory, and central cardiovascular homeostasis, but overproduction of ROS contributes to neurotoxicity, neurodegeneration, and cardiovascular diseases. Despite implications of NADPH oxidase in normal and pathological CNS processes, still relatively little is known about the mechanisms involved. This paper summarizes the evidence for NADPH oxidase distribution, regulation, and function in the CNS, emphasizing the diversity of Nox isoforms and their new and emerging role in neuro-cardiovascular function. In addition, perspectives for future research and novel therapeutic targets are offered.

Importance of NADPH oxidase-mediated redox signaling in the detrimental effect of CRP on pancreatic insulin secretion.

PubMed

Chan, Pei-Chi; Wang, Ya-Chin; Chen, Yi-Ling; Hsu, Wan-Ning; Tian, Yu-Feng; Hsieh, Po-Shiuan

2017-11-01

Elevations in C-reactive protein (CRP) levels are positively correlated with the progress of type 2 diabetes mellitus. However, the effect of CRP on pancreatic insulin secretion is unknown. Here, we showed that purified human CRP impaired insulin secretion in isolated mouse islets and NIT-1 insulin-secreting cells in dose- and time-dependent manners. CRP increased NADPH oxidase-mediated ROS (reactive oxygen species) production, which simultaneously promoted the production of nitrotyrosine (an indicator of RNS, reactive nitrogen species) and TNFα, to diminish cell viability, insulin secretion in islets and insulin-secreting cells. These CRP-mediated detrimental effects on cell viability and insulin secretion were significantly reversed by adding NAC (a potent antioxidant), apocynin (a selective NADPH oxidase inhibitor), L-NAME (a non-selective nitric oxide synthase (NOS) inhibitor), aminoguanidine (a selective iNOS inhibitor), PDTC (a selective NFκB inhibitor) or Enbrel (an anti-TNFα fusion protein). However, CRP-induced ROS production failed to change after adding L-NAME, aminoguanidine or PDTC. In isolated islets and NIT-1 cells, the elevated nitrotyrosine contents by CRP pretreatment were significantly suppressed by adding L-NAME but not PDTC. Conversely, CRP-induced increases in TNF-α production were significantly reversed by administration of PDTC but not L-NAME. In addition, wild-type mice treated with purified human CRP showed significant decreases in the insulin secretion index (HOMA-β cells) and the insulin stimulation index in isolated islets that were reversed by the addition of L-NAME, aminoguanidine or NAC. It is suggested that CRP-activated NADPH-oxidase redox signaling triggers iNOS-mediated RNS and NFκB-mediated proinflammatory cytokine production to cause β cell damage in state of inflammation. Copyright © 2017 Elsevier Inc. All rights reserved.

Gravity Responsive NADH Oxidase of the Plasma Membrane

NASA Technical Reports Server (NTRS)

Morre, D. James (Inventor)

2002-01-01

A method and apparatus for sensing gravity using an NADH oxidase of the plasma membrane which has been found to respond to unit gravity and low centrifugal g forces. The oxidation rate of NADH supplied to the NADH oxidase is measured and translated to represent the relative gravitational force exerted on the protein. The NADH oxidase of the plasma membrane may be obtained from plant or animal sources or may be produced recombinantly.

Reductive trapping of substrate to bovine plasma amine oxidase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hartmann, C.; Klinman, J.P.

1987-01-25

Plasma amine oxidases catalyze the oxidative deamination of amines to aldehydes, followed by a 2e- reduction of O/sub 2/ to H/sub 2/O/sub 2/. Pyrroloquinoline quinone (PQQ), previously believed to be restricted to prokaryotes, has recently been proposed to be the cofactor undergoing reduction in the first half-reaction of bovine plasma amine oxidase (Ameyama, M., Hayashi, U., Matsushita, K., Shinagawa, E., and Adachi, O. (1984) Agric. Biol. Chem. 48, 561-565; Lobenstein-Verbeek, C. L., Jongejan, J. A., Frank, J., and Duine, J. A. (1984) FEBS Lett. 170, 305-309). This result is unexpected, since model studies with PQQ implicate Schiff's base formation betweenmore » a reactive carbonyl and substrates, whereas experiments with bovine plasma amine oxidase have failed to provide evidence for a carbonyl cofactor. We have, therefore, re-examined putative adducts between substrate and enzyme-bound cofactor, employing a combination of (/sup 14/C)benzylamine and (/sup 3/H)NaCNBH/sub 3/. The use of the relatively weak reductant, NaCNBH/sub 3/, affords Schiff's base specificity and permits the study of enzyme below pH 7.0. As we show, enzyme can only be inactivated by NaCNBH/sub 3/ in the presence of substrate, leading to the incorporation of 1 mol of (/sup 14/C)benzylamine/mol of enzyme subunit at complete inactivation. By contrast, we are unable to detect any labeling with (/sup 3/H)NaCNBH/sub 3/, analogous to an earlier study with (/sup 3/H)NaCNBH/sub 4/ (Suva, R. H., and Abeles, R. H. (1978) Biochemistry 17, 3538-3545). We conclude, first, that our inability to obtain adducts containing both carbon 14 and tritium rules out the reductive trapping either of amine substrate with pyridoxal phosphate or of aldehyde product with a lysyl side chain and, second, that the observed pattern of labeling is fully consistent with the presence of PQQ at the active site of bovine plasma amine oxidase.« less

Increased Production of Hydrogen Peroxide by Lactobacillus delbrueckii subsp. bulgaricus upon Aeration: Involvement of an NADH Oxidase in Oxidative Stress

PubMed Central

Marty-Teysset, C.; de la Torre, F.; Garel, J.-R.

2000-01-01

The growth of Lactobacillus delbrueckii subsp. bulgaricus (L. delbrueckii subsp. bulgaricus) on lactose was altered upon aerating the cultures by agitation. Aeration caused the bacteria to enter early into stationary phase, thus reducing markedly the biomass production but without modifying the maximum growth rate. The early entry into stationary phase of aerated cultures was probably related to the accumulation of hydrogen peroxide in the medium. Indeed, the concentration of hydrogen peroxide in aerated cultures was two to three times higher than in unaerated ones. Also, a similar shift from exponential to stationary phase could be induced in unaerated cultures by adding increasing concentrations of hydrogen peroxide. A significant fraction of the hydrogen peroxide produced by L. delbrueckii subsp. bulgaricus originated from the reduction of molecular oxygen by NADH catalyzed by an NADH:H2O2 oxidase. The specific activity of this NADH oxidase was the same in aerated and unaerated cultures, suggesting that the amount of this enzyme was not directly regulated by oxygen. Aeration did not change the homolactic character of lactose fermentation by L. delbrueckii subsp. bulgaricus and most of the NADH was reoxidized by lactate dehydrogenase with pyruvate. This indicated that NADH oxidase had no (or a very small) energetic role and could be involved in eliminating oxygen. PMID:10618234

Immobilization of Pichia pastoris cells containing alcohol oxidase activity

PubMed Central

Maleknia, S; Ahmadi, H; Norouzian, D

2011-01-01

Background and Objectives The attempts were made to describe the development of a whole cell immobilization of P. pastoris by entrapping the cells in polyacrylamide gel beads. The alcohol oxidase activity of the whole cell Pichia pastoris was evaluated in comparison with yeast biomass production. Materials and Methods Methylotrophic yeast P. pastoris was obtained from Collection of Standard Microorganisms, Department of Bacterial Vaccines, Pasteur Institute of Iran (CSMPI). Stock culture was maintained on YPD agar plates. Alcohol oxidase was strongly induced by addition of 0.5% methanol as the carbon source. The cells were harvested by centrifugation then permeabilized. Finally the cells were immobilized in polyacrylamide gel beads. The activity of alcohol oxidase was determined by method of Tane et al. Results At the end of the logarithmic phase of cell culture, the alcohol oxidase activity of the whole cell P. Pastoris reached the highest level. In comparison, the alcohol oxidase activity was measured in an immobilized P. pastoris when entrapped in polyacrylamide gel beads. The alcohol oxidase activity of cells was induced by addition of 0.5% methanol as the carbon source. The cells were permeabilized by cetyltrimethylammonium bromide (CTAB) and immobilized. CTAB was also found to increase the gel permeability. Alcohol oxidase activity of immobilized cells was then quantitated by ABTS/POD spectrophotometric method at OD 420. There was a 14% increase in alcohol oxidase activity in immobilized cells as compared with free cells. By addition of 2-butanol as a substrate, the relative activity of alcohol oxidase was significantly higher as compared with other substrates added to the reaction media. Conclusion Immobilization of cells could eliminate lengthy and expensive procedures of enzyme separation and purification, protect and stabilize enzyme activity, and perform easy separation of the enzyme from the reaction media. PMID:22530090

Secreted fungal sulfhydryl oxidases: sequence analysis and characterisation of a representative flavin-dependent enzyme from Aspergillus oryzae.

PubMed

Faccio, Greta; Kruus, Kristiina; Buchert, Johanna; Saloheimo, Markku

2010-08-20

Sulfhydryl oxidases are flavin-dependent enzymes that catalyse the formation of de novo disulfide bonds from free thiol groups, with the reduction of molecular oxygen to hydrogen peroxide. Sulfhydryl oxidases have been investigated in the food industry to remove the burnt flavour of ultraheat-treated milk and are currently studied as potential crosslinking enzymes, aiming at strengthening wheat dough and improving the overall bread quality. In the present study, potential sulfhydryl oxidases were identified in the publicly available fungal genome sequences and their sequence characteristics were studied. A representative sulfhydryl oxidase from Aspergillus oryzae, AoSOX1, was expressed in the fungus Trichoderma reesei. AoSOX1 was produced in relatively good yields and was purified and biochemically characterised. The enzyme catalysed the oxidation of thiol-containing compounds like glutathione, D/L-cysteine, beta-mercaptoethanol and DTT. The enzyme had a melting temperature of 57°C, a pH optimum of 7.5 and its enzymatic activity was completely inhibited in the presence of 1 mM ZnSO4. Eighteen potentially secreted sulfhydryl oxidases were detected in the publicly available fungal genomes analysed and a novel proline-tryptophan dipeptide in the characteristic motif CXXC, where X is any amino acid, was found. A representative protein, AoSOX1 from A. oryzae, was produced in T. reesei in an active form and had the characteristics of sulfhydryl oxidases. Further testing of the activity on thiol groups within larger peptides and on protein level will be needed to assess the application potential of this enzyme.

Crystal Structure of Alcohol Oxidase from Pichia pastoris

PubMed Central

Valerius, Oliver; Feussner, Ivo; Ficner, Ralf

2016-01-01

FAD-dependent alcohol oxidases (AOX) are key enzymes of methylotrophic organisms that can utilize lower primary alcohols as sole source of carbon and energy. Here we report the crystal structure analysis of the methanol oxidase AOX1 from Pichia pastoris. The crystallographic phase problem was solved by means of Molecular Replacement in combination with initial structure rebuilding using Rosetta model completion and relaxation against an averaged electron density map. The subunit arrangement of the homo-octameric AOX1 differs from that of octameric vanillyl alcohol oxidase and other dimeric or tetrameric alcohol oxidases, due to the insertion of two large protruding loop regions and an additional C-terminal extension in AOX1. In comparison to other alcohol oxidases, the active site cavity of AOX1 is significantly reduced in size, which could explain the observed preference for methanol as substrate. All AOX1 subunits of the structure reported here harbor a modified flavin adenine dinucleotide, which contains an arabityl chain instead of a ribityl chain attached to the isoalloxazine ring. PMID:26905908

Calcium transport in vesicles energized by cytochrome oxidase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rosier, Randy N.

1979-01-01

Experiments on the reconstitution of cytochrome oxidase into phospholipid vesicles were carried out using techniques of selectivity energizing the suspensions with ascorbate and cytochrome c or ascorbate, PMS, and internally trapped cytochrome c. It was found that the K + selective ionophore valinomycin stimulated the rate of respiration of cytochrome oxidase vesicles regardless of the direction of the K + flux across the vesicle membranes. The stimulation occurred in the presence of protonophoric uncouplers and in the complete absence of potassium or in detergent-lysed suspensions. Gramicidin had similar effects and it was determined that the ionophores acted by specific interactionmore » with cytochrome oxidase rather than by the previously assumed collapse of membrane potentials. When hydrophobic proteins and appropriate coupling factors were incorporated into the cytochrome oxidase, vesicles phosphorylation of ADP could be coupled to the oxidation reaction of cytochrome oxidase. Relatively low P:O, representing poor coupling of the system, were problematical and precluded measurements of protonmotive force. However the system was used to study ion translocation.« less

Genetic mapping of new seed-expressed polyphenol oxidase genes in wheat (Triticum aestivum L.).

PubMed

Beecher, Brian S; Carter, Arron H; See, Deven R

2012-05-01

Polyphenol oxidase (PPO) enzymatic activity is a major cause in time-dependent discoloration in wheat dough products. The PPO-A1 and PPO-D1 genes have been shown to contribute to wheat kernel PPO activity. Recently a novel PPO gene family consisting of the PPO-A2, PPO-B2, and PPO-D2 genes has been identified and shown to be expressed in wheat kernels. In this study, the sequences of these five kernel PPO genes were determined for the spring wheat cultivars Louise and Penawawa. The two cultivars were found to be polymorphic at each of the PPO loci. Three novel alleles were isolated from Louise. The Louise X Penawawa mapping population was used to genetically map all five PPO genes. All map to the long arm of homeologous group 2 chromosomes. PPO-A2 was found to be located 8.9 cM proximal to PPO-A1 on the long arm of chromosome 2A. Similarly, PPO-D1 and PPO-D2 were separated by 10.7 cM on the long arm of chromosome 2D. PPO-B2 mapped to the long arm of chromosome 2B and was the site of a novel QTL for polyphenol oxidase activity. Five other PPO QTL were identified in this study. One QTL corresponds to the previously described PPO-D1 locus, one QTL corresponds to the PPO-D2 locus, whereas the remaining three are located on chromosome 2B.

Pacific oyster polyamine oxidase: a protein missing link in invertebrate evolution.

PubMed

Cervelli, Manuela; Polticelli, Fabio; Angelucci, Emanuela; Di Muzio, Elena; Stano, Pasquale; Mariottini, Paolo

2015-05-01

Polyamine oxidases catalyse the oxidation of polyamines and acetylpolyamines and are responsible for the polyamine interconversion metabolism in animal cells. Polyamine oxidases from yeast can oxidize spermine, N(1)-acetylspermine, and N(1)-acetylspermidine, while in vertebrates two different enzymes, namely spermine oxidase and acetylpolyamine oxidase, specifically catalyse the oxidation of spermine, and N(1)-acetylspermine/N(1)-acetylspermidine, respectively. In this work we proved that the specialized vertebrate spermine and acetylpolyamine oxidases have arisen from an ancestor invertebrate polyamine oxidase with lower specificity for polyamine substrates, as demonstrated by the enzymatic activity of the mollusc polyamine oxidase characterized here. This is the first report of an invertebrate polyamine oxidase, the Pacific oyster Crassostrea gigas (CgiPAO), overexpressed as a recombinant protein. This enzyme was biochemically characterized and demonstrated to be able to oxidase both N(1)-acetylspermine and spermine, albeit with different efficiency. Circular dichroism analysis gave an estimation of the secondary structure content and modelling of the three-dimensional structure of this protein and docking studies highlighted active site features. The availability of this pluripotent enzyme can have applications in crystallographic studies and pharmaceutical biotechnologies, including anticancer therapy as a source of hydrogen peroxide able to induce cancer cell death.

The GA5 locus of Arabidopsis thaliana encodes a multifunctional gibberellin 20-oxidase: Molecular cloning and functional expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Yun-Ling; Li, Li; Wu, Keqiang

1995-07-03

The biosynthesis of gibberellins (GAs) after GA{sub 12}-aldehyde involves a series of oxidative steps that lead to the formation of bioactive GAs. Previously, a cDNA clone encoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing), EC 1.14.11-] was isolated by immunoscreening a cDNA library from liquid endosperm of pumpkin (Cucurbita maxima L.) with antibodies against partially purified GA 20-oxidase. Here, we report isolation of a genomic clone for GA 20-oxidase from a genomic library of the long-day species Arabidopsis thaliana Heynh., strain Columbia, by using the pumpkin cDNA clone as a heterologous probe. This genomic clone contains a GA 20-oxidasemore » gene that consists of three exons and two introns. The three exons are 1131-bp long and encode 377 amino acid residues. A cDNA clone corresponding to the putative GA 20-oxidase genomic sequence was constructed with the reverse transcription-PCR method, and the identity of the cDNA clone was confirmed by analyzing the capability of the fusion protein expressed in Escherichia coli to convert GA{sub 53} to GA{sub 44} and GA{sub 19} to GA{sub 20}. The Arabidopsis GA 20-oxidase shares 55% identity and >80% similarity with the pumpkin GA 20-oxidase at the derived amino acid level. Both GA 20-oxidases share high homology with other 2-oxoglutarate-dependent dioxygenases (2-ODDs), but the highest homology was found between the two GA 20-oxidases. Mapping results indicated tight linkage between the cloned GA 20-oxidase and the GA locus of Arabidopsis. The ga5 semidwarf mutant contains a G {yields} A point mutation that inserts a translational stop codon in the protein-coding sequence, thus confirming that the GA5 locus encodes GA 20-oxidase. Expression of the GA5 gene in Arabidopsis leaves was enhanced after plants were transferred from short to long days; it was reduced by GA{sub 4} treatment, suggesting end-product repression in the GA biosynthetic pathway. 28 refs., 6

Evolution of cytochrome oxidase, an enzyme older than atmospheric oxygen.

PubMed

Castresana, J; Lübben, M; Saraste, M; Higgins, D G

1994-06-01

Cytochrome oxidase is a key enzyme in aerobic metabolism. All the recorded eubacterial (domain Bacteria) and archaebacterial (Archaea) sequences of subunits 1 and 2 of this protein complex have been used for a comprehensive evolutionary analysis. The phylogenetic trees reveal several processes of gene duplication. Some of these are ancient, having occurred in the common ancestor of Bacteria and Archaea, whereas others have occurred in specific lines of Bacteria. We show that eubacterial quinol oxidase was derived from cytochrome c oxidase in Gram-positive bacteria and that archaebacterial quinol oxidase has an independent origin. A considerable amount of evidence suggests that Proteobacteria (Purple bacteria) acquired quinol oxidase through a lateral gene transfer from Gram-positive bacteria. The prevalent hypothesis that aerobic metabolism arose several times in evolution after oxygenic photosynthesis, is not sustained by two aspects of the molecular data. First, cytochrome oxidase was present in the common ancestor of Archaea and Bacteria whereas oxygenic photosynthesis appeared in Bacteria. Second, an extant cytochrome oxidase in nitrogen-fixing bacteria shows that aerobic metabolism is possible in an environment with a very low level of oxygen, such as the root nodules of leguminous plants. Therefore, we propose that aerobic metabolism in organisms with cytochrome oxidase has a monophyletic and ancient origin, prior to the appearance of eubacterial oxygenic photosynthetic organisms.

Cloning and characterization of the gene for L-amino acid oxidase in hybrid tilapia.

PubMed

Shen, Yubang; Fu, Gui Hong; Liu, Feng; Yue, Gen Hua

2015-12-01

Tilapia is the common name for a group of cichlid fishes. Identification of DNA markers significantly associated with important traits in candidate genes may speed up genetic improvement. L-Amino acid oxidase (LAO) plays a crucial role in the innate immune defences of animals. Previously, whether LAO variants were associated with economic traits had not been studied in fish. We characterized the cDNA sequence of the LAO gene of hybrid tilapia (Oreochromis spp.). Its ORF was 1536 bp, encoding a flavoenzyme of 511 amino acids. This gene consisted of seven exons and six introns. Its expression was detected in the intestine, blood, kidney, skin, liver. It was highly expressed in the intestine. After a challenge with a bacterial pathogen, Streptococcus agalactiae, its expression was up-regulated significantly in the liver, intestine and spleen (P < 0.05). We identified one SNP in the genomic sequence of the gene and found that this SNP was associated significantly with body length (P < 0.05), but not with resistance to S. agalactiae. The results of this study suggest that the LAO gene plays an important role in innate immune responses to the bacterial pathogen in tilapia. The investigation of relationship between polymorphism of LAO gene and disease resistance and growth in tilapia showed that one SNP was associated significantly with body length. Further experiments on whether SNPs in the LAO gene are associated with growth in tilapia and other populations could be useful in understanding more functions of the LAO gene.

Monoamine Oxidase and Dopamine β-Hydroxylase Inhibitors from the Fruits of Gardenia jasminoides

PubMed Central

Kim, Ji Ho; Kim, Gun Hee; Hwang, Keum Hee

2012-01-01

This research was designed to determine what components of Gardenia jasminoides play a major role in inhibiting the enzymes related antidepressant activity of this plant. In our previous research, the ethyl acetate fraction of G. jasminosides fruits inhibited the activities of both monoamine oxidase-A (MAO-A) and monoamine oxidase-B (MAO-B), and oral administration of the ethanolic extract slightly increased serotonin concentrations in the brain tissues of rats and decreased MAO-B activity. In addition, we found through in vitro screening test that the ethyl acetate fraction showed modest inhibitory activity on dopamine-β hydroxylase (DBH). The bioassay-guided fractionation led to the isolation of five bio-active compounds, protocatechuic acid (1), geniposide (2), 6'-O-trans-p-coumaroylgeniposide (3), 3,5-d-ihydroxy-1,7-bis (4-hydroxyphenyl) heptanes (4), and ursolic acid (5), from the ethyl acetate fraction of G. jasminoides fruits. The isolated compounds showed different inhibitory potentials against MAO-A, -B, and DBH. Protocatechuic acid showed potent inhibition against MAO-B (IC50 300 μmol/L) and DBH (334 μmol/L), exhibiting weak MAO-A inhibition (2.41 mmol/L). Two iridoid glycosides, geniposide (223 μmol/L) and 6'-O-trans-p-coumaroylgeniposide (127μmol/L), were selective MAO-B inhibitor. Especially, 6'-O-trans-p-coumaroylgeniposide exhibited more selective MAO-B inhibition than deprenyl, well-known MAO-B inhibitor for the treatment of early-stage Parkinson’s disease. The inhibitory activity of 3,5-di-hydroxy-1,7-bis (4-hydroxyphenyl) heptane was strong for MAO-B (196 μmol/L), modest for MAO-A (400 μmol/L), and weak for DBH (941 μmol/L). Ursolic acid exhibited significant inhibition of DBH (214 μmol/L), weak inhibition of MAO-B (780 μmol/L), and no inhibition against MAO-A. Consequently, G. jasminoides fruits are considerable for development of biofunctional food materials for the combination treatment of depression and neurodegenerative disorders

Inhibition of Human Vascular NADPH Oxidase by Apocynin Derived Oligophenols

PubMed Central

Mora-Pale, Mauricio; Weïwer, Michel; Yu, Jingjing; Linhardt, Robert J.; Dordick, Jonathan S.

2009-01-01

Enzymatic oxidation of apocynin, which may mimic in vivo metabolism, affords a large number of oligomers (apocynin oxidation products, AOP) that inhibit vascular NADPH oxidase. In vitro studies of NADPH oxidase activity were performed to identify active inhibitors, resulting in a trimer hydroxylated quinone (IIIHyQ) that inhibited NADPH oxidase with an IC50 = 31 nM. Apocynin itself possessed minimal inhibitory activity. NADPH oxidase is believed to be inhibited through prevention of the interaction between two NADPH oxidase subunits, p47phox and p22phox. To that end, while apocynin was unable to block the interaction of his-tagged p47phox with a surface immobilized biotinalyted p22phox peptide, the IIIHyQ product strongly interfered with this interaction (apparent IC50 = 1.6 μM). These results provide evidence that peroxidase-catalyzed AOP, which consist of oligomeric phenols and quinones, inhibit critical interactions that are involved in the assembly and activation of human vascular NADPH oxidase. PMID:19523836

Current status of NADPH oxidase research in cardiovascular pharmacology.

PubMed

Rodiño-Janeiro, Bruno K; Paradela-Dobarro, Beatriz; Castiñeiras-Landeira, María Isabel; Raposeiras-Roubín, Sergio; González-Juanatey, José R; Alvarez, Ezequiel

2013-01-01

The implications of reactive oxygen species in cardiovascular disease have been known for some decades. Rationally, therapeutic antioxidant strategies combating oxidative stress have been developed, but the results of clinical trials have not been as good as expected. Therefore, to move forward in the design of new therapeutic strategies for cardiovascular disease based on prevention of production of reactive oxygen species, steps must be taken on two fronts, ie, comprehension of reduction-oxidation signaling pathways and the pathophysiologic roles of reactive oxygen species, and development of new, less toxic, and more selective nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors, to clarify both the role of each NADPH oxidase isoform and their utility in clinical practice. In this review, we analyze the value of NADPH oxidase as a therapeutic target for cardiovascular disease and the old and new pharmacologic agents or strategies to prevent NADPH oxidase activity. Some inhibitors and different direct or indirect approaches are available. Regarding direct NADPH oxidase inhibition, the specificity of NADPH oxidase is the focus of current investigations, whereas the chemical structure-activity relationship studies of known inhibitors have provided pharmacophore models with which to search for new molecules. From a general point of view, small-molecule inhibitors are preferred because of their hydrosolubility and oral bioavailability. However, other possibilities are not closed, with peptide inhibitors or monoclonal antibodies against NADPH oxidase isoforms continuing to be under investigation as well as the ongoing search for naturally occurring compounds. Likewise, some different approaches include inhibition of assembly of the NADPH oxidase complex, subcellular translocation, post-transductional modifications, calcium entry/release, electron transfer, and genetic expression. High-throughput screens for any of these activities could provide new

Current status of NADPH oxidase research in cardiovascular pharmacology

PubMed Central

Rodiño-Janeiro, Bruno K; Paradela-Dobarro, Beatriz; Castiñeiras-Landeira, María Isabel; Raposeiras-Roubín, Sergio; González-Juanatey, José R; Álvarez, Ezequiel

2013-01-01

The implications of reactive oxygen species in cardiovascular disease have been known for some decades. Rationally, therapeutic antioxidant strategies combating oxidative stress have been developed, but the results of clinical trials have not been as good as expected. Therefore, to move forward in the design of new therapeutic strategies for cardiovascular disease based on prevention of production of reactive oxygen species, steps must be taken on two fronts, ie, comprehension of reduction-oxidation signaling pathways and the pathophysiologic roles of reactive oxygen species, and development of new, less toxic, and more selective nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors, to clarify both the role of each NADPH oxidase isoform and their utility in clinical practice. In this review, we analyze the value of NADPH oxidase as a therapeutic target for cardiovascular disease and the old and new pharmacologic agents or strategies to prevent NADPH oxidase activity. Some inhibitors and different direct or indirect approaches are available. Regarding direct NADPH oxidase inhibition, the specificity of NADPH oxidase is the focus of current investigations, whereas the chemical structure-activity relationship studies of known inhibitors have provided pharmacophore models with which to search for new molecules. From a general point of view, small-molecule inhibitors are preferred because of their hydrosolubility and oral bioavailability. However, other possibilities are not closed, with peptide inhibitors or monoclonal antibodies against NADPH oxidase isoforms continuing to be under investigation as well as the ongoing search for naturally occurring compounds. Likewise, some different approaches include inhibition of assembly of the NADPH oxidase complex, subcellular translocation, post-transductional modifications, calcium entry/release, electron transfer, and genetic expression. High-throughput screens for any of these activities could provide new

Oxygen activation in flavoprotein oxidases: the importance of being positive.

PubMed

Gadda, Giovanni

2012-04-03

The oxidation of flavin hydroquinones by O(2) in solution is slow, with second-order rate constants of ~250 M(-1) s(-1). This is due to the obligatory, single-electron transfer that initiates the reaction being thermodynamically unfavored and poorly catalyzed. Notwithstanding considerations of O(2) accessibility to the reaction site, its desolvation and geometry and other factors that can also contribute to further rate acceleration, flavoprotein oxidases must activate O(2) for reaction with flavin hydroquinones to be able to achieve the 100-1000-fold rate enhancements typically observed. Protein positive charges have been identified in glucose oxidase, monomeric sarcosine oxidase, N-methyltryptophan oxidase and fructosamine oxidase that electrostatically stabilize the transition state for the initial single electron transfer that generates the O(2)(-•)/flavin semiquinone radical pair. In choline oxidase despite the presence of three histidines in the active site, the trimethylammonium group of the reaction product provides such an electrostatic stabilization. A nonpolar site proximal to the flavin C(4a) atom in choline oxidase has also been identified, which contributes to the geometry and desolvation of the O(2) reaction site. The relevance of O(2) activation by product charges to other flavoprotein oxidases, such as for example those catalyzing amine oxidations, is discussed in this review. A nonpolar site close to the flavin C(4a) atom and a positive charge is identified through structural analysis in several flavoprotein oxidases. Mutagenesis has disclosed nonpolar sites in O(2)-reducing enzymes that utilize copper/TPQ or iron. It is predicted that classes of O(2)-reducing enzymes utilizing other cofactors also contain a similar catalytic motif.

Genetic Mapping of a new family of Seed-Expressed Polyphenol Oxidase genes in Wheat (Triticum aestivum L.)

USDA-ARS?s Scientific Manuscript database

Polyphenol oxidase (PPO) enzymatic activity is a major cause in time-dependent discoloration in wheat dough products. The PPO-A1 and PPO-D1 genes have been shown to contribute to wheat kernel PPO activity. However it has been shown that wheat contains multiple PPO genes. Recently a novel PPO gene...

The First Mammalian Aldehyde Oxidase Crystal Structure

PubMed Central

Coelho, Catarina; Mahro, Martin; Trincão, José; Carvalho, Alexandra T. P.; Ramos, Maria João; Terao, Mineko; Garattini, Enrico; Leimkühler, Silke; Romão, Maria João

2012-01-01

Aldehyde oxidases (AOXs) are homodimeric proteins belonging to the xanthine oxidase family of molybdenum-containing enzymes. Each 150-kDa monomer contains a FAD redox cofactor, two spectroscopically distinct [2Fe-2S] clusters, and a molybdenum cofactor located within the protein active site. AOXs are characterized by broad range substrate specificity, oxidizing different aldehydes and aromatic N-heterocycles. Despite increasing recognition of its role in the metabolism of drugs and xenobiotics, the physiological function of the protein is still largely unknown. We have crystallized and solved the crystal structure of mouse liver aldehyde oxidase 3 to 2.9 Å. This is the first mammalian AOX whose structure has been solved. The structure provides important insights into the protein active center and further evidence on the catalytic differences characterizing AOX and xanthine oxidoreductase. The mouse liver aldehyde oxidase 3 three-dimensional structure combined with kinetic, mutagenesis data, molecular docking, and molecular dynamics studies make a decisive contribution to understand the molecular basis of its rather broad substrate specificity. PMID:23019336

Purification and Characterization of Pyranose Oxidase from the White Rot Fungus Trametes multicolor

PubMed Central

Leitner, Christian; Volc, Jindrich; Haltrich, Dietmar

2001-01-01

We purified an intracellular pyranose oxidase from mycelial extracts of the white rot fungus Trametes multicolor by using ammonium sulfate fractionation, hydrophobic interaction, ion-exchange chromatography, and gel filtration. The native enzyme has a molecular mass of 270 kDa as determined by equilibrium ultracentrifugation and is composed of four identical 68-kDa subunits as determined by matrix-assisted laser desorption ionization mass spectrometry. Each subunit contains one covalently bound flavin adenine dinucleotide as its prosthetic group. The enzyme oxidizes several aldopyranoses specifically at position C-2, and its preferred electron donor substrates are d-glucose, d-xylose, and l-sorbose. During this oxidation reaction electrons are transferred to oxygen, yielding hydrogen peroxide. In addition, the enzyme catalyzes the two-electron reduction of 1,4-benzoquinone, several substituted benzoquinones, and 2,6-dichloroindophenol, as well as the one-electron reduction of the ABTS [2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid)] cation radical. As judged by the catalytic efficiencies (kcat/Km), some of these quinone electron acceptors are much better substrates for pyranose oxidase than oxygen. The optimum pH of the pyranose oxidase-catalyzed reaction depends strongly on the electron acceptor employed and varies from 4 to 8. It has been proposed that the main metabolic function of pyranose oxidase is as a constituent of the ligninolytic system of white rot fungi that provides peroxidases with H2O2. An additional function could be reduction of quinones, key intermediates that are formed during mineralization of lignin. PMID:11472941

CotA, a Multicopper Oxidase from Bacillus pumilus WH4, Exhibits Manganese-Oxidase Activity

PubMed Central

Su, Jianmei; Bao, Peng; Bai, Tenglong; Deng, Lin; Wu, Hui; Liu, Fan; He, Jin

2013-01-01

Multicopper oxidases (MCOs) are a family of enzymes that use copper ions as cofactors to oxidize various substrates. Previous research has demonstrated that several MCOs such as MnxG, MofA and MoxA can act as putative Mn(II) oxidases. Meanwhile, the endospore coat protein CotA from Bacillus species has been confirmed as a typical MCO. To study the relationship between CotA and the Mn(II) oxidation, the cotA gene from a highly active Mn(II)-oxidizing strain Bacillus pumilus WH4 was cloned and overexpressed in Escherichia coli strain M15. The purified CotA contained approximately four copper atoms per molecule and showed spectroscopic properties typical of blue copper oxidases. Importantly, apart from the laccase activities, the CotA also displayed substantial Mn(II)-oxidase activities both in liquid culture system and native polyacrylamide gel electrophoresis. The optimum Mn(II) oxidase activity was obtained at 53°C in HEPES buffer (pH 8.0) supplemented with 0.8 mM CuCl2. Besides, the addition of o-phenanthroline and EDTA both led to a complete suppression of Mn(II)-oxidizing activity. The specific activity of purified CotA towards Mn(II) was 0.27 U/mg. The Km, Vmax and kcat values towards Mn(II) were 14.85±1.17 mM, 3.01×10−6±0.21 M·min−1 and 0.32±0.02 s−1, respectively. Moreover, the Mn(II)-oxidizing activity of the recombinant E. coli strain M15-pQE-cotA was significantly increased when cultured both in Mn-containing K liquid medium and on agar plates. After 7-day liquid cultivation, M15-pQE-cotA resulted in 18.2% removal of Mn(II) from the medium. Furthermore, the biogenic Mn oxides were clearly observed on the cell surfaces of M15-pQE-cotA by scanning electron microscopy. To our knowledge, this is the first report that provides the direct observation of Mn(II) oxidation with the heterologously expressed protein CotA, Therefore, this novel finding not only establishes the foundation for in-depth study of Mn(II) oxidation mechanisms, but also offers a

Generating disulfides with the quiescin sulfhydryl oxidases

PubMed Central

Heckler, Erin J.; Rancy, Pumtiwitt C.; Kodali, Vamsi K.; Thorpe, Colin

2008-01-01

The Quiescin-sulfhydryl oxidase (QSOX) family of flavoenzymes catalyzes the direct and facile insertion of disulfide bonds into unfolded reduced proteins with concomitant reduction of oxygen to hydrogen peroxide. This review discusses the chemical mechanism of these enzymes and the involvement of thioredoxin and flavin-binding domains in catalysis. The variability of CxxC motifs in the QSOX family is highlighted and attention is drawn to the steric factors that may promote efficient thiol/disulfide exchange during oxidative protein folding. The varied cellular location of these multi-domain sulfhydryl oxidases is reviewed and potential intracellular and extracellular roles are summarized. Finally, this review identifies important unresolved questions concerning this ancient family of sulfhydryl oxidases. PMID:17980160

Role of Angiotensin II type 1 receptor on renal NAD(P)H oxidase, oxidative stress and inflammation in nitric oxide inhibition induced-hypertension.

PubMed

Rincón, J; Correia, D; Arcaya, J L; Finol, E; Fernández, A; Pérez, M; Yaguas, K; Talavera, E; Chávez, M; Summer, R; Romero, F

2015-03-01

Activation of the renin-angiotensin system (RAS), renal oxidative stress and inflammation are constantly present in experimental hypertension. Nitric oxide (NO) inhibition with N(w)-nitro-L-arginine methyl ester (L-NAME) has previously been reported to produce hypertension, increased expression of Angiotensin II (Ang II) and renal dysfunction. The use of Losartan, an Ang II type 1 receptor (AT1R) antagonist has proven to be effective reducing hypertension and renal damage; however, the mechanism by which AT1R blockade reduced kidney injury and normalizes blood pressure in this experimental model is still complete unknown. The current study was designed to test the hypothesis that AT1R activation promotes renal NAD(P)H oxidase up-regulation, oxidative stress and cytokine production during L-NAME induced-hypertension. Male Sprague-Dawley rats were distributed in three groups: L-NAME, receiving 70 mg/100ml of L-NAME, L-NAME+Los, receiving 70 mg/100ml of L-NAME and 40 mg/kg/day of Losartan; and Controls, receiving water instead of L-NAME or L-NAME and Losartan. After two weeks, L-NAME induced high blood pressure, renal overexpression of AT1R, NAD(P)H oxidase sub-units gp91, p22 and p47, increased levels of oxidative stress, interleukin-6 (IL-6) and interleukin-17 (IL-17). Also, we found increased renal accumulation of lymphocytes and macrophages. Losartan treatment abolished the renal expression of gp91, p22, p47, oxidative stress and reduced NF-κB activation and IL-6 expression. These findings indicate that NO induced-hypertension is associated with up-regulation of NADPH oxidase, oxidative stress production and overexpression of key inflammatory mediators. These events are associated with up-regulation of AT1R, as evidenced by their reversal with AT1R blocker treatment. Copyright © 2015 Elsevier Inc. All rights reserved.

Arginase Inhibition Suppresses Native Low-Density Lipoprotein-Stimulated Vascular Smooth Muscle Cell Proliferation by NADPH Oxidase Inactivation.

PubMed

Koo, Bon Hyeock; Yi, Bong Gu; Wang, Wi Kwang; Ko, In Young; Hoe, Kwang Lae; Kwon, Young Guen; Won, Moo Ho; Kim, Young Myeong; Lim, Hyun Kyo; Ryoo, Sungwoo

2018-05-01

Vascular smooth muscle cell (VSMC) proliferation induced by native low-density lipoprotein (nLDL) stimulation is dependent on superoxide production from activated NADPH oxidase. The present study aimed to investigate whether the novel arginase inhibitor limonin could suppress nLDL-induced VSMC proliferation and to examine related mechanisms. Isolated VSMCs from rat aortas were treated with nLDL, and cell proliferation was measured by WST-1 and BrdU assays. NADPH oxidase activation was evaluated by lucigenin-induced chemiluminescence, and phosphorylation of protein kinase C (PKC) βII and extracellular signal-regulated kinase (ERK) 1/2 was determined by western blot analysis. Mitochondrial reactive oxygen species (ROS) generation was assessed using MitoSOX-red, and intracellular L-arginine concentrations were determined by high-performance liquid chromatography (HPLC) in the presence or absence of limonin. Limonin inhibited arginase I and II activity in the uncompetitive mode, and prevented nLDL-induced VSMC proliferation in a p21Waf1/Cip1-dependent manner without affecting arginase protein levels. Limonin blocked PKCβII phosphorylation, but not ERK1/2 phosphorylation, and translocation of p47phox to the membrane was decreased, as was superoxide production in nLDL-stimulated VSMCs. Moreover, mitochondrial ROS generation was increased by nLDL stimulation and blocked by preincubation with limonin. Mitochondrial ROS production was responsible for the phosphorylation of PKCβII. HPLC analysis showed that arginase inhibition with limonin increases intracellular L-arginine concentrations, but decreases polyamine concentrations. L-Arginine treatment prevented PKCβII phosphorylation without affecting ERK1/2 phosphorylation. Increased L-arginine levels following limonin-dependent arginase inhibition prohibited NADPH oxidase activation in a PKCβII-dependent manner, and blocked nLDL-stimulated VSMC proliferation. © Copyright: Yonsei University College of Medicine 2018.

Arginase Inhibition Suppresses Native Low-Density Lipoprotein-Stimulated Vascular Smooth Muscle Cell Proliferation by NADPH Oxidase Inactivation

PubMed Central

Wang, Wi-Kwang; Ko, In-Young; Hoe, Kwang-Lae; Kwon, Young-Guen; Won, Moo-Ho; Kim, Young-Myeong

2018-01-01

Purpose Vascular smooth muscle cell (VSMC) proliferation induced by native low-density lipoprotein (nLDL) stimulation is dependent on superoxide production from activated NADPH oxidase. The present study aimed to investigate whether the novel arginase inhibitor limonin could suppress nLDL-induced VSMC proliferation and to examine related mechanisms. Materials and Methods Isolated VSMCs from rat aortas were treated with nLDL, and cell proliferation was measured by WST-1 and BrdU assays. NADPH oxidase activation was evaluated by lucigenin-induced chemiluminescence, and phosphorylation of protein kinase C (PKC) βII and extracellular signal-regulated kinase (ERK) 1/2 was determined by western blot analysis. Mitochondrial reactive oxygen species (ROS) generation was assessed using MitoSOX-red, and intracellular L-arginine concentrations were determined by high-performance liquid chromatography (HPLC) in the presence or absence of limonin. Results Limonin inhibited arginase I and II activity in the uncompetitive mode, and prevented nLDL-induced VSMC proliferation in a p21Waf1/Cip1-dependent manner without affecting arginase protein levels. Limonin blocked PKCβII phosphorylation, but not ERK1/2 phosphorylation, and translocation of p47phox to the membrane was decreased, as was superoxide production in nLDL-stimulated VSMCs. Moreover, mitochondrial ROS generation was increased by nLDL stimulation and blocked by preincubation with limonin. Mitochondrial ROS production was responsible for the phosphorylation of PKCβII. HPLC analysis showed that arginase inhibition with limonin increases intracellular L-arginine concentrations, but decreases polyamine concentrations. L-Arginine treatment prevented PKCβII phosphorylation without affecting ERK1/2 phosphorylation. Conclusion Increased L-arginine levels following limonin-dependent arginase inhibition prohibited NADPH oxidase activation in a PKCβII-dependent manner, and blocked nLDL-stimulated VSMC proliferation. PMID

THE PREPARATION AND PROPERTIES OF HIGHLY PURIFIED ASCORBIC ACID OXIDASE

PubMed Central

Powers, Wendell H.; Lewis, Stanley; Dawson, Charles R.

1944-01-01

1. A method is described for the preparation of a highly purified ascorbic acid oxidase containing 0.24 per cent copper. 2. Using comparable activity measurements, this oxidase is about one and a half times as active on a dry weight basis as the hitherto most highly purified preparation described by Lovett-Janison and Nelson. The latter contained 0.15 per cent copper. 3. The oxidase activity is proportional to the copper content and the proportionality factor is the same as that reported by Lovett-Janison and Nelson. 4. When dialyzed free of salt, the blue concentrated oxidase solutions precipitate a dark green-blue protein which carries the activity. This may be prevented by keeping the concentrated solutions about 0.1 M in Na2HPO4. 5. When highly diluted for activity measurements the oxidase rapidly loses activity (irreversibly) previous to the measurement, unless the dilution is made with a dilute inert protein (gelatin) solution. Therefore activity values obtained using such gelatin-stabilized dilute solutions of the oxidase run considerably higher than values obtained by the Lovett-Janison and Nelson technique. 6. The effect of pH and substrate concentration on the activity of the purified oxidase in the presence and absence of inert protein was studied. PMID:19873382

Expression cloning of a gibberellin 20-oxidase, a multifunctional enzyme involved in gibberellin biosynthesis.

PubMed Central

Lange, T; Hedden, P; Graebe, J E

1994-01-01

In the biosynthetic pathway to the gibberellins (GAs), carbon-20 is removed by oxidation to give the C19-GAs, which include the biologically active plant hormones. We report the isolation of a cDNA clone encoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing) EC 1.14.11.-] by screening a cDNA library from developing cotyledons of pumpkin (Cucurbita maxima L.) for expression of this enzyme. When mRNA from either the cotyledons or the endosperm was translated in vitro using rabbit reticulocyte lysates, the products contained GA12 20-oxidase activity. A polyclonal antiserum was raised against the amino acid sequence of a peptide released by tryptic digestion of purified GA 20-oxidase from the endosperm. A cDNA expression library in lambda gt11 was prepared from cotyledon mRNA and screened with the antiserum. The identity of positive clones was confirmed by the demonstration of GA12 20-oxidase activity in single bacteriophage plaques. Recombinant protein from a selected clone catalyzed the three-step conversions of GA12 to GA25 and of GA53 to GA17, as well as the formation of the C19-GAs, GA1, GA9, and GA20, from their respective aldehyde precursors, GA23, GA24, and GA19. The nucleotide sequence of the cDNA insert contains an open reading frame of 1158 nt encoding a protein of 386 amino acid residues. The predicted M(r) (43,321) and pI (5.3) are similar to those determined experimentally for the native GA 20-oxidase. Furthermore, the derived amino acid sequence includes sequences obtained from the N terminus and two tryptic peptides from the native enzyme. It also contains regions that are highly conserved in a group of non-heme Fe-containing dioxygenases. Images PMID:8078921

Amine oxidases as important agents of pathological processes of rhabdomyolysis in rats.

PubMed

Gudkova, O O; Latyshko, N V; Shandrenko, S G

2016-01-01

In this study we have tested an idea on the important role of amine oxidases (semicarbazide-sensitive amine oxidase, diamine oxidase, polyamine oxidase) as an additional source of oxidative/carbonyl stress under glycerol-induced rhabdomyolysis, since the enhanced formation of reactive oxygen species and reactive carbonyl species in a variety of tissues is linked to various diseases. In our experiments we used the sensitive fluorescent method devised for estimation of amine oxidases activity in the rat kidney and thymus as targeted organs under rhabdomyolysis. We have found in vivo the multiple rises in activity of semicarbazide-sensitive amine oxidase, diamine oxidase, polyamine oxidase (2-4.5 times) in the corresponding cell fractions, whole cells or their lysates at the 3-6th day after glycerol injection. Aberrant antioxidant activities depended on rhabdomyolysis stage and had organ specificity. Additional treatment of animals with metal chelator ‘Unithiol’ adjusted only the activity of antioxidant enzymes but not amine oxidases in both organs. Furthermore the in vitro experiment showed that Fenton reaction (hydrogen peroxide in the presence of iron) products alone had no effect on semicarbazide-sensitive amine oxidase activity in rat liver cell fraction whereas supplementation with methylglyoxal resulted in its significant 2.5-fold enhancement. Combined action of the both agents had additive effect on semicarbazide-sensitive amine oxidase activity. We can assume that biogenic amine and polyamine catabolism by amine oxidases is upregulated by oxidative and carbonyl stress factors directly under rhabdomyolysis progression, and the increase in catabolic products concentration contributes to tissue damage in glycerol-induced acute renal failure and apoptosis stimulation in thymus.

Dexamethasone but not indomethacin inhibits human phagocyte nicotinamide adenine dinucleotide phosphate oxidase activity by down-regulating expression of genes encoding oxidase components.

PubMed

Condino-Neto, A; Whitney, C; Newburger, P E

1998-11-01

We investigated the effects of dexamethasone or indomethacin on the NADPH oxidase activity, cytochrome b558 content, and expression of genes encoding the components gp91-phox and p47-phox of the NADPH oxidase system in the human monocytic THP-1 cell line, differentiated with IFN-gamma and TNF-alpha, alone or in combination, for up to 7 days. IFN-gamma and TNF-alpha, alone or in combination, caused a significant up-regulation of the NADPH oxidase system as reflected by an enhancement of the PMA-stimulated superoxide release, cytochrome b558 content, and expression of gp91-phox and p47-phox genes on both days 2 and 7 of cell culture. Noteworthy was the tremendous synergism between IFN-gamma and TNF-alpha for all studied parameters. Dexamethasone down-regulated the NADPH oxidase system of cytokine-differentiated THP-1 cells as assessed by an inhibition on the PMA-stimulated superoxide release, cytochrome b558 content, and expression of the gp91-phox and p47-phox genes. The nuclear run-on assays indicated that dexamethasone down-regulated the NADPH oxidase system at least in part by inhibiting the transcription of gp91-phox and p47-phox genes. Indomethacin inhibited only the PMA-stimulated superoxide release of THP-1 cells differentiated with IFN-gamma and TNF-alpha during 7 days. None of the other parameters was affected by indomethacin. We conclude that dexamethasone down-regulates the NADPH oxidase system at least in part by inhibiting the expression of genes encoding the gp91-phox and p47-phox components of the NADPH oxidase system.

Heterologous expression and characterization of mouse spermine oxidase.

PubMed

Cervelli, Manuela; Polticelli, Fabio; Federico, Rodolfo; Mariottini, Paolo

2003-02-14

Polyamine oxidases are key enzymes responsible of the polyamine interconversion metabolism in animal cells. Recently, a novel enzyme belonging to this class of enzymes has been characterized for its capability to oxidize preferentially spermine and designated as spermine oxidase. This is a flavin adenine dinucleotide-containing enzyme, and it has been expressed both in vitro and in vivo systems. The primary structure of mouse spermine oxidase (mSMO) was deduced from a cDNA clone (Image Clone 264769) recovered by a data base search utilizing the human counterpart of polyamine oxidases, PAOh1. The open reading frame predicts a 555-amino acid protein with a calculated M(r) of 61,852.30, which shows a 95.1% identity with PAOh1. To understand the biochemical properties of mSMO and its structure/function relationship, the mSMO cDNA has been subcloned and expressed in secreted and secreted-tagged forms into Escherichia coli BL21 DE3 cells. The recombinant enzyme shows an optimal pH value of 8.0 and is able to oxidize rapidly spermine to spermidine and 3-aminopropanal and fails to act upon spermidine and N(1)-acetylpolyamines. The purified recombinant-tagged form enzyme (M(r) approximately 68,000) has K(m) and k(cat) values of 90 microm and 4.5 s(-1), respectively, using spermine as substrate at pH 8.0. Molecular modeling of mSMO protein based on maize polyamine oxidase three-dimensional structure suggests that the general features of maize polyamine oxidase active site are conserved in mSMO.

Putting together a plasma membrane NADH oxidase: a tale of three laboratories.

PubMed

Löw, Hans; Crane, Frederick L; Morré, D James

2012-11-01

The observation that high cellular concentrations of NADH were associated with low adenylate cyclase activity led to a search for the mechanism of the effect. Since cyclase is in the plasma membrane, we considered the membrane might have a site for NADH action, and that NADH might be oxidized at that site. A test for NADH oxidase showed very low activity, which could be increased by adding growth factors. The plasma membrane oxidase was not inhibited by inhibitors of mitochondrial NADH oxidase such as cyanide, rotenone or antimycin. Stimulation of the plasma membrane oxidase by iso-proterenol or triiodothyronine was different from lack of stimulation in endoplasmic reticulum. After 25 years of research, three components of a trans membrane NADH oxidase have been discovered. Flavoprotein NADH coenzyme Q reductases (NADH cytochrome b reductase) on the inside, coenzyme Q in the middle, and a coenzyme Q oxidase on the outside as a terminal oxidase. The external oxidase segment is a copper protein with unique properties in timekeeping, protein disulfide isomerase and endogenous NADH oxidase activity, which affords a mechanism for control of cell growth by the overall NADH oxidase and the remarkable inhibition of oxidase activity and growth of cancer cells by a wide range of anti-tumor drugs. A second trans plasma membrane electron transport system has been found in voltage dependent anion channel (VDAC), which has NADH ferricyanide reductase activity. This activity must be considered in relation to ferricyanide stimulation of growth and increased VDAC antibodies in patients with autism. Copyright © 2012 Elsevier Ltd. All rights reserved.

Cyanobacterial Lactate Oxidases Serve as Essential Partners in N2 Fixation and Evolved into Photorespiratory Glycolate Oxidases in Plants[w

PubMed Central

Hackenberg, Claudia; Kern, Ramona; Hüge, Jan; Stal, Lucas J.; Tsuji, Yoshinori; Kopka, Joachim; Shiraiwa, Yoshihiro; Bauwe, Hermann; Hagemann, Martin

2011-01-01

Glycolate oxidase (GOX) is an essential enzyme involved in photorespiratory metabolism in plants. In cyanobacteria and green algae, the corresponding reaction is catalyzed by glycolate dehydrogenases (GlcD). The genomes of N2-fixing cyanobacteria, such as Nostoc PCC 7120 and green algae, appear to harbor genes for both GlcD and GOX proteins. The GOX-like proteins from Nostoc (No-LOX) and from Chlamydomonas reinhardtii showed high l-lactate oxidase (LOX) and low GOX activities, whereas glycolate was the preferred substrate of the phylogenetically related At-GOX2 from Arabidopsis thaliana. Changing the active site of No-LOX to that of At-GOX2 by site-specific mutagenesis reversed the LOX/GOX activity ratio of No-LOX. Despite its low GOX activity, No-LOX overexpression decreased the accumulation of toxic glycolate in a cyanobacterial photorespiratory mutant and restored its ability to grow in air. A LOX-deficient Nostoc mutant grew normally in nitrate-containing medium but died under N2-fixing conditions. Cultivation under low oxygen rescued this lethal phenotype, indicating that N2 fixation was more sensitive to O2 in the Δlox Nostoc mutant than in the wild type. We propose that LOX primarily serves as an O2-scavenging enzyme to protect nitrogenase in extant N2-fixing cyanobacteria, whereas in plants it has evolved into GOX, responsible for glycolate oxidation during photorespiration. PMID:21828292

R1, a novel repressor of the human monoamine oxidase A.

PubMed

Chen, Kevin; Ou, Xiao-Ming; Chen, Gao; Choi, Si Ho; Shih, Jean C

2005-03-25

Monoamine oxidase catalyzes the oxidative deamination of a number of neurotransmitters. A deficiency in monoamine oxidase A results in aggressive behavior in both humans and mice. Studies on the regulation of monoamine oxidase A gene expression have shown that the Sp1 family is important for monoamine oxidase A expression. To search for novel transcription factors, the sequences of three Sp1 sites in the monoamine oxidase A core promoter were used in the yeast one-hybrid system to screen a human cDNA library. A novel repressor, R1 (RAM2), has been cloned. The R1 cDNA encodes a protein with 454 amino acids and an open reading frame at the 5'-end. The transfection of R1 in a human neuroblastoma cell line, SK-N-BE (2)-C, inhibited the monoamine oxidase A promoter and enzymatic activity. The degree of inhibition of monoamine oxidase A by R1 correlated with the level of R1 protein expression. R1 was also found to repress monoamine oxidase A promoter activity within a natural chromatin environment. A gel-shift assay indicated that the endogenous R1 protein in SK-N-BE (2)-C cells interacted with the R1 binding sequence. R1 also bound directly to the natural monoamine oxidase A promoter in vivo as shown by chromatin immunoprecipitation assay. Immunocytochemical analysis showed that R1 was expressed in both cytosol and nucleus, which suggested a role for R1 in transcriptional regulation. Northern blot analysis revealed the presence of endogenous R1 mRNA in human brain and peripheral tissues. Taken together, this study shows that R1 is a novel repressor that inhibits monoamine oxidase A gene expression.

A novel proteolytic processing of prolysyl oxidase

PubMed Central

Atsawasuwan, Phimon; Mochida, Yoshiyuki; Katafuchi, Michitsuna; Tokutomi, Kentaro; Mocanu, Viorel; Parker, Carol E.; Yamauchi, Mitsuo

2012-01-01

Lysyl oxidase (LOX) is an amine oxidase that is critical for the stability of connective tissues. The secreted proLOX is enzymatically quiescent and is activated through proteolytic cleavage between residue Gly162 and Asp163 (residue numbers according to the mouse LOX) by bone morphogenetic protein (BMP)-1 gene products. Here we report a novel processing of proLOX identified in vitro and in vivo. Two forms of mature LOX were identified and characterized by their immunoreactivity to specific antibodies, amine oxidase activity and mass spectrometry. One form was identified as a well characterized BMP-1 processed LOX protein. Another was found to be a truncated form of LOX (tLOX) resulting from the cleavage at the carboxy terminus of Arg192. The tLOX still appeared to retain amine oxidase activity. The results from the proLOX gene deletion and mutation experiments indicated that the processing occurs independent of the cleavage of proLOX by BMP-1 gene products and likely requires the presence of LOX propeptide. These results indicate that proLOX could be processed by two different mechanisms producing two forms of active LOX. PMID:21591931

A novel proteolytic processing of prolysyl oxidase.

PubMed

Atsawasuwan, Phimon; Mochida, Yoshiyuki; Katafuchi, Michitsuna; Tokutomi, Kentaro; Mocanu, Viorel; Parker, Carol E; Yamauchi, Mitsuo

2011-01-01

Lysyl oxidase (LOX) is an amine oxidase that is critical for the stability of connective tissues. The secreted proLOX is enzymatically quiescent and is activated through proteolytic cleavage between residues Gly(162) and Asp(163) (residue numbers according to the mouse LOX) by bone morphogenetic protein (BMP)-1 gene products. Here we report a novel processing of proLOX identified in vitro and in vivo. Two forms of mature LOX were identified and characterized by their immunoreactivity to specific antibodies, amine oxidase activity, and mass spectrometry. One form was identified as a well-characterized BMP-1 processed LOX protein. Another was found to be a truncated form of LOX resulting from the cleavage at the carboxy terminus of Arg(192). The truncated form of LOX still appeared to retain amine oxidase activity. The results from the proLOX gene deletion and mutation experiments indicated that the processing occurs independent of the cleavage of proLOX by BMP-1 gene products and likely requires the presence of LOX propeptide. These results indicate that proLOX could be processed by two different mechanisms producing two forms of active LOX.

Exploiting algal NADPH oxidase for biophotovoltaic energy

DOE PAGES

Anderson, Alexander; Laohavisit, Anuphon; Blaby, Ian K.; ...

2015-01-29

Photosynthetic microbes exhibit light-dependent electron export across the cell membrane, which can generate electricity in biological photovoltaic (BPV) devices. How electrons are exported remains to be determined; the identification of mechanisms would help selection or generation of photosynthetic microbes capable of enhanced electrical output. We show that plasma membrane NADPH oxidase activity is a significant component of light-dependent generation of electricity by the unicellular green alga Chlamydomonas reinhardtii. NADPH oxidases export electrons across the plasma membrane to form superoxide anion from oxygen. The C. reinhardtii mutant lacking the NADPH oxidase encoded by RBO1 is impaired in both extracellular superoxide anionmore » production and current generation in a BPV device. Complementation with the wild-type gene restores both capacities, demonstrating the role of the enzyme in electron export. Monitoring light-dependent extracellular superoxide production with a colorimetric assay is shown to be an effective way of screening for electrogenic potential of candidate algal strains. Furthermore, the results show that algal NADPH oxidases are important for superoxide anion production and open avenues for optimizing the biological component of these devices.« less

NADPH oxidases: novel therapeutic targets for neurodegenerative diseases.

PubMed

Gao, Hui-Ming; Zhou, Hui; Hong, Jau-Shyong

2012-06-01

Oxidative stress is a key pathologic factor in neurodegenerative diseases such as Alzheimer and Parkinson diseases (AD, PD). The failure of free-radical-scavenging antioxidants in clinical trials pinpoints an urgent need to identify and to block major sources of oxidative stress in neurodegenerative diseases. As a major superoxide-producing enzyme complex in activated phagocytes, phagocyte NADPH oxidase (PHOX) is essential for host defense. However, recent preclinical evidence has underscored a pivotal role of overactivated PHOX in chronic neuroinflammation and progressive neurodegeneration. Deficiency in PHOX subunits mitigates neuronal damage induced by diverse insults/stresses relevant to neurodegenerative diseases. More importantly, suppression of PHOX activity correlates with reduced neuronal impairment in models of neurodegenerative diseases. The discovery of PHOX and non-phagocyte NADPH oxidases in astroglia and neurons further reinforces the crucial role of NADPH oxidases in oxidative stress-mediated chronic neurodegeneration. Thus, proper modulation of NADPH oxidase activity might hold therapeutic potential for currently incurable neurodegenerative diseases. Published by Elsevier Ltd.

Monoamine Oxidase-A Genetic Variants and Childhood Abuse Predict Impulsiveness in Borderline Personality Disorder.

PubMed

Kolla, Nathan J; Meyer, Jeffrey; Sanches, Marcos; Charbonneau, James

2017-11-30

Impulsivity is a core feature of borderline personality disorder (BPD) and antisocial personality disorder (ASPD) that likely arises from combined genetic and environmental influences. The interaction of the low activity variant of the monoamine oxidase-A (MAOA-L) gene and early childhood adversity has been shown to predict aggression in clinical and non-clinical populations. Although impulsivity is a risk factor for aggression in BPD and ASPD, little research has investigated potential gene-environment (G×E) influences impacting its expression in these conditions. Moreover, G×E interactions may differ by diagnosis. Full factorial analysis of variance was employed to investigate the influence of monoamine oxidase-A (MAO-A) genotype, childhood abuse, and diagnosis on Barratt Impulsiveness Scale-11 (BIS-11) scores in 61 individuals: 20 subjects with BPD, 18 subjects with ASPD, and 23 healthy controls. A group×genotype×abuse interaction was present (F(2,49)=4.4, p =0.018), such that the interaction of MAOA-L and childhood abuse predicted greater BIS-11 motor impulsiveness in BPD. Additionally, BPD subjects reported higher BIS-11 attentional impulsiveness versus ASPD participants (t(1,36)=2.3, p =0.025). These preliminary results suggest that MAOA-L may modulate the impact of childhood abuse on impulsivity in BPD. Results additionally indicate that impulsiveness may be expressed differently in BPD and ASPD.

[Involvement of hydrogen peroxide in the regulation of coexpression of alternative oxidase and rotenone-insensitive NADH dehydrogenase in tomato leaves and calluses].

PubMed

Eprintsev, A T; Mal'tseva, E V; Shatskikh, A S; Popov, V N

2011-01-01

The involvement of active oxygen forms in the regulation of the expression of mitochondrial respiratory chain components, which are not related to energy storing, has been in vitro and in vivo studied in Lycopersicum esculentum L. The highest level of transcription of genes encoding alternative oxidase and NADH dehydrogenase has been observed in green tomato leaves. It has been shown that even low H2O2 concentrations activate both aoxlalpha and ndb1 genes, encoding alternative oxidase and external mitochondrial rotenone-insensitive NADH dehydrogenase, respectively. According to our results, in the case of an oxidative stress, alternative oxidase and NADH dehydrogenase are coexpressed in tomato plant tissues, and active oxygen forms serve as the secondary messengers of their coexpression.

Pulse-radiolysis studies on the interaction of one-electron reduced species with blue oxidases. Reduction of type-2-copper-depleted ascorbate oxidase.

PubMed

O'Neill, P; Fielden, E M; Avigliano, L; Marcozzi, G; Ballini, A; Agrò, F

1984-08-15

The interaction of one-electron reduced metronidazole (ArNO2.-) with native and Type-2-copper-depleted ascorbate oxidase were studied in buffered aqueous solution at pH 6.0 and 7.4 by using the technique of pulse radiolysis. With ArNO2.-, reduction of Type 1 copper of the native enzyme and of the Type-2-copper-depleted ascorbate oxidase occurs via a bimolecular step and at the same rate. Whereas the native protein accepts, in the absence of O2, 6-7 reducing equivalents, Type-2-copper-depleted ascorbate oxidase accepts only 3 reducing equivalents with stoichiometric reduction of Type 1 copper. On reaction of O2.- with ascorbate oxidase under conditions of [O2.-] much greater than [ascorbate oxidase], removal of Type 2 copper results in reduction of all the Type 1 copper atoms, in contrast with reduction of the equivalent of only one Type 1 copper atom in the holoprotein. From observations at 610 nm, the rate of reduction of ascorbate oxidase by O2.- is not dependent on the presence of Type 2 copper. For the holoprotein, no significant optical-absorption changes were observed at 330 nm. It is proposed that electrons enter the protein via Type 1 copper in a rate-determining step followed by a fast intramolecular transfer of electrons within the protein. For the Type-2-copper-depleted protein, intramolecular transfer within the protein, however, is slow or does not occur. In the presence of O2, it is also suggested that re-oxidation of the partially reduced holoprotein occurs at steady state, as inferred from the observations at 330 nm and 610 nm. The role of Type 2 copper in ascorbate oxidase is discussed in terms of its involvement in redistribution of electrons within the protein or structural considerations.

Supramolecular organization of cytochrome c oxidase- and alternative oxidase-dependent respiratory chains in the filamentous fungus Podospora anserina.

PubMed

Krause, Frank; Scheckhuber, Christian Q; Werner, Alexandra; Rexroth, Sascha; Reifschneider, Nicole H; Dencher, Norbert A; Osiewacz, Heinz D

2004-06-18

To elucidate the molecular basis of the link between respiration and longevity, we have studied the organization of the respiratory chain of a wild-type strain and of two long-lived mutants of the filamentous fungus Podospora anserina. This established aging model is able to respire by either the standard or the alternative pathway. In the latter pathway, electrons are directly transferred from ubiquinol to the alternative oxidase and thus bypass complexes III and IV. We show that the cytochrome c oxidase pathway is organized according to the mammalian "respirasome" model (Schägger, H., and Pfeiffer, K. (2000) EMBO J. 19, 1777-1783). In contrast, the alternative pathway is composed of distinct supercomplexes of complexes I and III (i.e. I(2) and I(2)III(2)), which have not been described so far. Enzymatic analysis reveals distinct functional properties of complexes I and III belonging to either cytochrome c oxidase- or alternative oxidase-dependent pathways. By a gentle colorless-native PAGE, almost all of the ATP synthases from mitochondria respiring by either pathway were preserved in the dimeric state. Our data are of significance for the understanding of both respiratory pathways as well as lifespan control and aging.

In vitro study of 6-mercaptopurine oxidation catalysed by aldehyde oxidase and xanthine oxidase.

PubMed

Rashidi, Mohammad-Reza; Beedham, Christine; Smith, John S; Davaran, Soodabeh

2007-08-01

In spite of over 40 years of clinical use of 6-mercaptopurine, many aspects of complex pharmacology and metabolism of this drug remain unclear. It is thought that 6-mercaptopurine is oxidized to 6-thiouric acid through 6-thioxanthine or 8-oxo-6-mercaptopurine by one of two molybdenum hydroxylases, xanthine oxidase (XO), however, the role of other molybdenum hydroxylase, aldehyde oxidase (AO), in the oxidation of 6-mercaptopurine and possible interactions of AO substrates and inhibitors has not been investigated in more details. In the present study, the role of AO and XO in the oxidation of 6- mercaptopurine has been investigated. 6-mercaptopurine was incubated with bovine milk xanthine oxidase or partially purified guinea pig liver molybdenum hydroxylase fractions in the absence and presence of XO and AO inhibitor/substrates, and the reactions were monitored by spectrophotometric and HPLC methods. According to the results obtained from the inhibition studies, it is more likely that 6- mercaptopurine is oxidized to 6-thiouric acid via 6-thioxanthine rather than 8-oxo-6-mercaptopurine. The first step which is the rate limiting step is catalyzed solely by XO, whereas both XO and AO are involved in the oxidation of 6-thioxanthine to 6-thiouric acid.

Discovery of a Xylooligosaccharide Oxidase from Myceliophthora thermophila C1.

PubMed

Ferrari, Alessandro R; Rozeboom, Henriëtte J; Dobruchowska, Justyna M; van Leeuwen, Sander S; Vugts, Aniek S C; Koetsier, Martijn J; Visser, Jaap; Fraaije, Marco W

2016-11-04

By inspection of the predicted proteome of the fungus Myceliophthora thermophila C1 for vanillyl-alcohol oxidase (VAO)-type flavoprotein oxidases, a putative oligosaccharide oxidase was identified. By homologous expression and subsequent purification, the respective protein could be obtained. The protein was found to contain a bicovalently bound FAD cofactor. By screening a large number of carbohydrates, several mono- and oligosaccharides could be identified as substrates. The enzyme exhibits a strong substrate preference toward xylooligosaccharides; hence it is named xylooligosaccharide oxidase (XylO). Chemical analyses of the product formed upon oxidation of xylobiose revealed that the oxidation occurs at C1, yielding xylobionate as product. By elucidation of several XylO crystal structures (in complex with a substrate mimic, xylose, and xylobiose), the residues that tune the unique substrate specificity and regioselectivity could be identified. The discovery of this novel oligosaccharide oxidase reveals that the VAO-type flavoprotein family harbors oxidases tuned for specific oligosaccharides. The unique substrate profile of XylO hints at a role in the degradation of xylan-derived oligosaccharides by the fungus M. thermophila C1. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Stability of spermine oxidase to thermal and chemical denaturation: comparison with bovine serum amine oxidase.

PubMed

Cervelli, Manuela; Leonetti, Alessia; Cervoni, Laura; Ohkubo, Shinji; Xhani, Marla; Stano, Pasquale; Federico, Rodolfo; Polticelli, Fabio; Mariottini, Paolo; Agostinelli, Enzo

2016-10-01

Spermine oxidase (SMOX) is a flavin-containing enzyme that specifically oxidizes spermine to produce spermidine, 3-aminopropanaldehyde and hydrogen peroxide. While no crystal structure is available for any mammalian SMOX, X-ray crystallography showed that the yeast Fms1 polyamine oxidase has a dimeric structure. Based on this scenario, we have investigated the quaternary structure of the SMOX protein by native gel electrophoresis, which revealed a composite gel band pattern, suggesting the formation of protein complexes. All high-order protein complexes are sensitive to reducing conditions, showing that disulfide bonds were responsible for protein complexes formation. The major gel band other than the SMOX monomer is the covalent SMOX homodimer, which was disassembled by increasing the reducing conditions, while being resistant to other denaturing conditions. Homodimeric and monomeric SMOXs are catalytically active, as revealed after gel staining for enzymatic activity. An engineered SMOX mutant deprived of all but two cysteine residues was prepared and characterized experimentally, resulting in a monomeric species. High-sensitivity differential scanning calorimetry of SMOX was compared with that of bovine serum amine oxidase, to analyse their thermal stability. Furthermore, enzymatic activity assays and fluorescence spectroscopy were used to gain insight into the unfolding process.

Recombinant expression, purification, and characterization of polyphenol oxidase 2 (VvPPO2) from "Shine Muscat" (Vitis labruscana Bailey × Vitis vinifera L.).

PubMed

Katayama-Ikegami, Ayako; Suehiro, Yuka; Katayama, Takane; Jindo, Kazushi; Itamura, Hiroyuki; Esumi, Tomoya

2017-12-01

Polyphenol oxidases (PPOs) catalyze browning reactions in various plant organs, therefore controlling the reactions is important for the food industry. PPOs have been assumed to be involved in skin browning of white grape cultivars; however, the molecular mechanism underlying PPO-mediated browning process remains elusive. We have recently identified a new PPO gene named VvPPO2 from "Shine Muscat" (Vitis labruscana Bailey × V. vinifera L.), and have shown that the gene is transcribed at a higher level than the previously identified VvPPO1 in browning, physiologically disordered berry skins at the maturation stage. In this study, we expressed VvPPO2 in Escherichia coli and, using the purified preparation, revealed unique physicochemical characteristics of the enzyme. Our study opens up a way to not only understand the berry skin browning process but also to elucidate the enzymatic maturation process of grape PPOs.

POLYAMINE OXIDASE 1 from rice (Oryza sativa) is a functional ortholog of Arabidopsis POLYAMINE OXIDASE 5.

PubMed

Liu, Taibo; Wook Kim, Dong; Niitsu, Masaru; Berberich, Thomas; Kusano, Tomonobu

2014-01-01

POLYAMINE OXIDASE 1 (OsPAO1), from rice (Oryza sativa), and POLYAMINE OXIDASE 5 (AtPAO5), from Arabidopsis (Arabidopsis thaliana), are enzymes sharing high identity at the amino acid level and with similar characteristics, such as polyamine specificity and pH preference; furthermore, both proteins localize to the cytosol. A loss-of-function Arabidopsis mutant, Atpao5-2, was hypersensitive to low doses of exogenous thermospermine but this phenotype could be rescued by introduction of the wild-type AtPAO5 gene. Introduction of OsPAO1, under the control of a constitutive promoter, into Atpao5-2 mutants also restored normal thermospermine sensitivity, allowing growth in the presence of low levels of thermospermine, along with a concomitant decrease in thermospermine content in plants. By contrast, introduction of OsPAO3, which encodes a peroxisome-localized polyamine oxidase, into Atpao5-2 plants could not rescue any of the mutant phenotypes in the presence of thermospermine. These results suggest that OsPAO1 is the functional ortholog of AtPAO5.

Aurone synthase is a catechol oxidase with hydroxylase activity and provides insights into the mechanism of plant polyphenol oxidases

PubMed Central

Molitor, Christian; Mauracher, Stephan Gerhard

2016-01-01

Tyrosinases and catechol oxidases belong to the family of polyphenol oxidases (PPOs). Tyrosinases catalyze the o-hydroxylation and oxidation of phenolic compounds, whereas catechol oxidases were so far defined to lack the hydroxylation activity and catalyze solely the oxidation of o-diphenolic compounds. Aurone synthase from Coreopsis grandiflora (AUS1) is a specialized plant PPO involved in the anabolic pathway of aurones. We present, to our knowledge, the first crystal structures of a latent plant PPO, its mature active and inactive form, caused by a sulfation of a copper binding histidine. Analysis of the latent proenzyme’s interface between the shielding C-terminal domain and the main core provides insights into its activation mechanisms. As AUS1 did not accept common tyrosinase substrates (tyrosine and tyramine), the enzyme is classified as a catechol oxidase. However, AUS1 showed hydroxylase activity toward its natural substrate (isoliquiritigenin), revealing that the hydroxylase activity is not correlated with the acceptance of common tyrosinase substrates. Therefore, we propose that the hydroxylase reaction is a general functionality of PPOs. Molecular dynamics simulations of docked substrate–enzyme complexes were performed, and a key residue was identified that influences the plant PPO’s acceptance or rejection of tyramine. Based on the evidenced hydroxylase activity and the interactions of specific residues with the substrates during the molecular dynamics simulations, a novel catalytic reaction mechanism for plant PPOs is proposed. The presented results strongly suggest that the physiological role of plant catechol oxidases were previously underestimated, as they might hydroxylate their—so far unknown—natural substrates in vivo. PMID:26976571

Synthesis of three novel fluorine-18 labeled analogues of L-deprenyl for positron emission tomography (PET) studies of monoamine oxidase B (MAO-B).

PubMed

Nag, Sangram; Lehmann, Lutz; Heinrich, Tobias; Thiele, Andrea; Kettschau, Georg; Nakao, Ryuji; Gulyás, Balázs; Halldin, Christer

2011-10-27

The aim in this project was to synthesize and to study fluorine-18 labeled analogues of l-deprenyl which bind selectively to the enzyme monoamine oxidase B (MAO-B). Three fluorinated l-deprenyl analogues have been generated in multistep organic syntheses. The most promising fluorine-18 compound N-[(2S)-1-[(18)F]fluoro-3-phenylpropan-2-yl]-N-methylprop-2-yn-1-amine (4c) was synthesized by a one-step fluorine-18 nucleophilic substitution reaction. Autoradiography on human brain tissue sections demonstrated specific binding for compound 4c to brain regions known to have a high content of MAO-B. In addition, the corresponding nonradioactive fluorine-19 compound (13) inhibited recombinant human MAO-B with an IC(50) of 170.5 ± 29 nM but did not inhibit recombinant human MAO-A (IC(50) > 2000 nM), demonstrating its specificity. Biodistribution of 4c in mice showed high initial brain uptake leveling at 5.2 ± 0.04%ID/g after 2 min post injection. In conclusion, compound 4c is a specific inhibitor of MAO-B with high initial brain uptake in mice and is, therefore, a candidate for further investigation in PET.

Activation of Polyphenol Oxidase in Dormant Wild Oat Caryopses by a Seed-Decay Isolate of Fusarium avenaceum

USDA-ARS?s Scientific Manuscript database

Incubation of dormant wild oat (Avena fatua L., isoline M73) caryopses for 1 to 3 days with Fusarium avenaceum seed-decay isolate F.a.1 induced activity of the plant defense enzyme polyphenol oxidase (PPO). Both extracts and leachates obtained from F.a.1-treated caryopses had decreased abundance of ...

Three-dimensional organization of three-domain copper oxidases: A review

NASA Astrophysics Data System (ADS)

Zhukhlistova, N. E.; Zhukova, Yu. N.; Lyashenko, A. V.; Zaĭtsev, V. N.; Mikhaĭlov, A. M.

2008-01-01

“Blue” copper-containing proteins are multidomain proteins that utilize a unique redox property of copper ions. Among other blue multicopper oxidases, three-domain oxidases belong to the group of proteins that exhibit a wide variety of compositions in amino acid sequences, functions, and occurrences in organisms. This paper presents a review of the data obtained from X-ray diffraction investigations of the three-dimensional structures of three-domain multicopper oxidases, such as the ascorbate oxidase catalyzing oxidation of ascorbate to dehydroascorbate and its three derivatives; the multicopper oxidase CueO (the laccase homologue); the laccases isolated from the basidiomycetes Coprinus cinereus, Trametes versicolor, Coriolus zonatus, Cerrena maxima, and Rigidoporus lignosus and the ascomycete Melanocarpus albomyces; and the bacterial laccases CotA from the endospore coats of Bacillus subtilis. A comparison of the molecular structures of the laccases of different origins demonstrates that, structurally, these objects are highly conservative. This obviously indicates that the catalytic activity of the enzymes under consideration is characterized by similar mechanisms.

NADPH OXIDASE: STRUCTURE AND ACTIVATION MECHANISMS (REVIEW). NOTE I.

PubMed

Filip-Ciubotaru, Florina; Manciuc, Carmen; Stoleriu, Gabriela; Foia, Liliana

2016-01-01

NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase), with its generically termed NOX isoforms, is the major source of ROS (reactive oxigen species) in biological systems. ROS are small oxygen-derived molecules with an important role in various biological processes (physiological or pathological). If under physiological conditions some processes are beneficial and necessary for life, under pathophysiological conditions they are noxious, harmful. NADPH oxidases are present in phagocytes and in a wide variety of nonphagocytic cells. The enzyme generates superoxide by transferring electrons from NADPH inside the cell across the membrane and coupling them to molecular oxygen to produce superoxide anion, a reactive free-radical. Structurally, NADPH oxidase is a multicomponent enzyme which includes two integral membrane proteins, glycoprotein gp9 1 Phox and adaptor protein p22(phox), which together form the heterodimeric flavocytochrome b558 that constitutes the core of the enzyme. During the resting state, the multidomain regulatory subunits p40P(phox), p47(phox), p67(Phox) are located in the cytosol organized as a complex. The activation of phagocytic NADPH oxidase occurs through a complex series of protein interactions.

The use of glucose oxidase and catalase for the enzymatic reduction of the potential ethanol content in wine.

PubMed

Röcker, Jessica; Schmitt, Matthias; Pasch, Ludwig; Ebert, Kristin; Grossmann, Manfred

2016-11-01

Due to the increase of sugar levels in wine grapes as one of the impacts of climate change, alcohol reduction in wines becomes a major focus of interest. This study combines the use of glucose oxidase and catalase activities with the aim of rapid conversion of glucose into non-fermentable gluconic acid. The H2O2 hydrolysing activity of purified catalase is necessary in order to stabilize glucose oxidase activity. After establishing the adequate enzyme ratio, the procedure was applied in large-scale trials (16L- and 220L-scale) of which one was conducted in a winery under industrial wine making conditions. Both enzyme activity and wine flavour were clearly influenced by the obligatory aeration in the different trials. With the enzyme treatment an alcohol reduction of 2%vol. was achieved after 30h of aeration. However the enzyme treated wines were significantly more acidic and less typical. Copyright © 2016. Published by Elsevier Ltd.

Batch production of Pyranose 2-oxidase from Trametes versicolor (ATCC 11235) in medium with a lignocellulosic substrate and enzymatic bleaching of cotton fabrics.

PubMed

Pazarlioglu, Nurdan Kasikara; Erden, Emre; Ucar, M Cigdem; Akkaya, Alper; Sariisik, A Merih

2012-04-01

The aim of this work was to determine new, different and low-cost substrates that can be used for enzyme production from the white rot fungus Trametes versicolor (ATCC 11235) by taking advantage of the broad substrate specificity of pyranose 2-oxidase. In this report, we investigated the production of pyranose 2-oxidase from T. versicolor (ATCC 11235) using ten different agricultural residues such as clover straw, almond shells, hazelnut cobs, grass and others. Pyranose 2-oxidase activity was determined as 2.332 U/g at the 9th day in a submerged culture containing clover straw and tap water shaken at 150 rpm and 26°C, and the optimum clover straw concentration was determined to be 12 g/l. The effects of different glucose, nitrogen and phosphate sources on the production of pyranose 2-oxidase were studied in the clover straw medium. Analyses of biomass, protein, reduced sugar and nitrogen concentrations were also monitored in a clover straw medium that did not contain carbon or nitrogen and phosphate sources under the parameters determined. The produced pyranose 2-oxidase was used for improving the properties of cotton fabrics.

Expression and Chloroplast Targeting of Cholesterol Oxidase in Transgenic Tobacco Plants

PubMed Central

Corbin, David R.; Grebenok, Robert J.; Ohnmeiss, Thomas E.; Greenplate, John T.; Purcell, John P.

2001-01-01

Cholesterol oxidase represents a novel type of insecticidal protein with potent activity against the cotton boll weevil (Anthonomus grandis grandis Boheman). We transformed tobacco (Nicotiana tabacum) plants with the cholesterol oxidase choM gene and expressed cytosolic and chloroplast-targeted versions of the ChoM protein. Transgenic leaf tissues expressing cholesterol oxidase exerted insecticidal activity against boll weevil larvae. Our results indicate that cholesterol oxidase can metabolize phytosterols in vivo when produced cytosolically or when targeted to chloroplasts. The transgenic plants exhibiting cytosolic expression accumulated low levels of saturated sterols known as stanols, and displayed severe developmental aberrations. In contrast, the transgenic plants expressing chloroplast-targeted cholesterol oxidase maintained a greater accumulation of stanols, and appeared phenotypically and developmentally normal. These results are discussed within the context of plant sterol distribution and metabolism. PMID:11457962

Comparative Activity-Based Flavin-Dependent Oxidase Profiling.

PubMed

Krysiak, Joanna; Breinbauer, Rolf

2017-01-01

Activity-based protein profiling (ABPP) has become a powerful chemoproteomic technology allowing for the dissection of complex ligand-protein interactions in their native cellular environment. One of the biggest challenges for ABPP is the extension of the proteome coverage. In this chapter a new ABPP strategy dedicated to monoamine oxidases (MAO) is presented. These enzymes are representative examples of flavin-dependent oxidases, playing a crucial role in the regulation of nervous system signaling.

Multidomain flavin-dependent sulfhydryl oxidases.

PubMed

Coppock, Donald L; Thorpe, Colin

2006-01-01

Eukaryotic flavin-dependent sulfhydryl oxidases catalyze oxidative protein folding with the generation of disulfides and the reduction of oxygen to hydrogen peroxide. This review deals principally with the Quiescinsulfhydryl oxidases (QSOX) that are found in multiple forms in multicellular organisms and singly in a number of protozoan parasites. QSOX is an ancient fusion of thioredoxin domains and an FAD-binding module, ERV1/ALR. Interdomain disulfide exchanges transmit reducing equivalents from substrates to the flavin cofactor and thence to molecular oxygen. The in vitro substrate specificity of avian QSOX1 and the likely substrates of QSOXs in vivo are discussed. The location of QSOX immunoreactivity and mRNA expression levels in human cells and tissues is reviewed. Generally, there is a marked association of QSOX1 expression with cell types that have a high secretory load of disulfide-containing peptides and proteins. The abundance of sulfhydryl oxidases in the islets of Langerhans suggests that oxidative protein folding may directly contribute to the oxidative stress believed to be a factor in the progression to type II diabetes. Finally, the structure and mechanism of QSOX proteins is compared to their smaller stand-alone cousins: yeast ERV1p and ERV2p, the mammalian augmenter of liver regeneration (ALR), and the viral ALR homologs.

Cytokinin oxidase from Phaseolus vulgaris callus tissues. Enhanced in vitro activity of the enzyme in the presence of copper-imidazole complexes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chatfield, J.M.; Armstrong, D.J.

1987-07-01

The effects of metal ions on cytokinin oxidase activity extracted from callus tissues of Phaseolus vulgaris L. cv Great Northern have been examined using an assay based on the oxidation of N/sup 6/-(..delta../sup 2/-isopentenyl)-adenine-2,8-/sup 3/H (i/sup 6/ Ade) to adenine (Ade). The addition of cupric ions to reaction mixtures containing imidazole buffer markedly enhanced cytokinin oxidase activity. In the presence of optimal concentrations of copper and imidazole, cytokinin oxidase activity was stimulated more than 20-fold. The effect was enzyme dependent, specific for copper, and observed only in the presence of imidazole. The substrate specificity of the copper-imidazole enhanced reaction, asmore » judged by substrate competition tests, was the same as that observed in the absence of copper and imidazole. Similarly, in tests involving DEAE-cellulose chromatography, elution profiles of cytokinin oxidase activity determined using a copper-imidazole enhanced assay were identical to those obtained using an assay without copper and imidazole. On the basis of these results, the addition of copper and imidazole to reaction mixtures used to assay for cytokinin oxidase activity is judged to provide a reliable and specific assay of greatly enhanced sensitivity for the enzyme. The mechanism by which copper and imidazole enhance cytokinin oxidase activity is not certain, but the reaction catalyzed by the enzyme was not inhibited by anaerobic conditions when these reagents were present. This observation suggests that copper-imidazole complexes are substituting for oxygen in the reaction mechanism by which cytokinin oxidase effects cleavage of the N/sup 6/-side chain of i/sup 6/ Ade.« less

Effect of contraceptive steroids on monoamine oxidase activity

PubMed Central

Southgate, Jennifer; Collins, G. G. S.; Pryse-Davies, J.; Sandler, M.

1969-01-01

Cyclical variations in monoamine oxidase activity during the human menstrual cycle, specific to the endometrium and modified in women undergoing contraceptive steroid treatment, may reflect changes in hormonal environment. Treatment of rats with individual constituents of the contraceptive pill causes analogous changes: oestrogens inhibit and progestogens potentiate uterine monoamine oxidase activity. ImagesFig. 2Fig. 3

Design, synthesis, and molecular docking studies of N-(9,10-anthraquinone-2-carbonyl)amino acid derivatives as xanthine oxidase inhibitors.

PubMed

Zhang, Ting-Jian; Li, Song-Ye; Yuan, Wei-Yan; Zhang, Yi; Meng, Fan-Hao

2018-04-01

A series of N-(9,10-anthraquinone-2-carbonyl)amino acid derivatives (1a-j) was designed and synthesized as novel xanthine oxidase inhibitors. Among them, the L/D-phenylalanine derivatives (1d and 1i) and the L/D-tryptophan derivatives (1e and 1j) were effective with micromolar level potency. In particular, the L-phenylalanine derivative 1d (IC 50  = 3.0 μm) and the D-phenylalanine derivative 1i (IC 50  = 2.9 μm) presented the highest potency and were both more potent than the positive control allopurinol (IC 50  = 8.1 μm). Preliminary SAR analysis pointed that an aromatic amino acid fragment, for example, phenylalanine or tryptophan, was essential for the inhibition; the D-amino acid derivative presented equal or greater potency compared to its L-enantiomer; and the 9,10-anthraquinone moiety was welcome for the inhibition. Molecular simulations provided rational binding models for compounds 1d and 1i in the xanthine oxidase active pocket. As a result, compounds 1d and 1i could be promising lead compounds for further investigation. © 2017 John Wiley & Sons A/S.

Phenolic profiles and polyphenol oxidase (PPO) gene expression of red clover (Trifolium pratense) selected for decreased postharvest browning

USDA-ARS?s Scientific Manuscript database

Red clover (Trifolium pratense L.) is a legume forage abundant in phenolic compounds. It tends to brown when cut for hay, due to oxidation of phenolic compounds catalyzed by polyphenol oxidase (PPO), and subsequent binding to proteins. Selecting for a greener hay may provide information about the re...

Development of a Polyphenol Oxidase Biosensor from Jenipapo Fruit Extract (Genipa americana L.) and Determination of Phenolic Compounds in Textile Industrial Effluents.

PubMed

Antunes, Rafael Souza; Ferraz, Denes; Garcia, Luane Ferreira; Thomaz, Douglas Vieira; Luque, Rafael; Lobón, Germán Sanz; Gil, Eric de Souza; Lopes, Flávio Marques

2018-05-15

In this work, an innovative polyphenol oxidase biosensor was developed from Jenipapo ( Genipa americana L.) fruit and used to assess phenolic compounds in industrial effluent samples obtained from a textile industry located in Jaraguá-GO, Brasil. The biosensor was prepared and optimized according to: the proportion of crude vegetal extract, pH and overall voltammetric parameters for differential pulse voltammetry. The calibration curve presented a linear interval from 10 to 310 µM (r² = 0.9982) and a limit of detection of 7 µM. Biosensor stability was evaluated throughout 15 days, and it exhibited 88.22% of the initial response. The amount of catechol standard recovered post analysis varied between 87.50% and 96.00%. Moreover, the biosensor was able to detect phenolic compounds in a real sample, and the results were in accordance with standard spectrophotometric assays. Therefore, the innovatively-designed biosensor hereby proposed is a promising tool for phenolic compound detection and quantification when environmental contaminants are concerned.

Plasma diamine oxidase levels in pregnancy complicated by threatened abortion.

PubMed Central

Legge, M; Duff, G B

1981-01-01

Plasma diamine oxidase levels were assayed in 66 patients who presented with pregnancy complicated by threatened abortion. Levels within the normal range were associated with continuing pregnancies, whereas levels below the normal range were associated with subsequent abortion. Among those patients in whom gestation was greater than eight weeks, 66.6% of diamine oxidase levels correctly predicted the pregnancy outcome. Assay of the diamine oxidase levels at eight weeks of gestation or less gave little useful information. PMID:6785320

Plasma diamine oxidase levels in pregnancy complicated by threatened abortion.

PubMed

Legge, M; Duff, G B

1981-02-01

Plasma diamine oxidase levels were assayed in 66 patients who presented with pregnancy complicated by threatened abortion. Levels within the normal range were associated with continuing pregnancies, whereas levels below the normal range were associated with subsequent abortion. Among those patients in whom gestation was greater than eight weeks, 66.6% of diamine oxidase levels correctly predicted the pregnancy outcome. Assay of the diamine oxidase levels at eight weeks of gestation or less gave little useful information.

Structure-function relationships in the evolutionary framework of spermine oxidase.

PubMed

Cervelli, Manuela; Salvi, Daniele; Polticelli, Fabio; Amendola, Roberto; Mariottini, Paolo

2013-06-01

Spermine oxidase is a FAD-dependent enzyme that specifically oxidizes spermine, and plays a central role in the highly regulated catabolism of polyamines in vertebrates. The spermine oxidase substrate is specifically spermine, a tetramine that plays mandatory roles in several cell functions, such as DNA synthesis, cellular proliferation, modulation of ion channels function, cellular signalling, nitric oxide synthesis and inhibition of immune responses. The oxidative products of spermine oxidase activity are spermidine, H2O2 and the aldehyde 3-aminopropanal that spontaneously turns into acrolein. In this study the reconstruction of the phylogenetic relationships among spermine oxidase proteins from different vertebrate taxa allowed to infer their molecular evolutionary history, and assisted in elucidating the conservation of structural and functional properties of this enzyme family. The amino acid residues, which have been hypothesized or demonstrated to play a pivotal role in the enzymatic activity, and substrate specificity are here analysed to obtain a comprehensive and updated view of the structure-function relationships in the evolution of spermine oxidase.

Three-dimensional organization of three-domain copper oxidases: A review

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhukhlistova, N. E., E-mail: amm@ns.crys.ras.ru; Zhukova, Yu. N.; Lyashenko, A. V.

2008-01-15

'Blue' copper-containing proteins are multidomain proteins that utilize a unique redox property of copper ions. Among other blue multicopper oxidases, three-domain oxidases belong to the group of proteins that exhibit a wide variety of compositions in amino acid sequences, functions, and occurrences in organisms. This paper presents a review of the data obtained from X-ray diffraction investigations of the three-dimensional structures of three-domain multicopper oxidases, such as the ascorbate oxidase catalyzing oxidation of ascorbate to dehydroascorbate and its three derivatives; the multicopper oxidase CueO (the laccase homologue); the laccases isolated from the basidiomycetes Coprinus cinereus, Trametes versicolor, Coriolus zonatus, Cerrenamore » maxima, and Rigidoporus lignosus and the ascomycete Melanocarpus albomyces; and the bacterial laccases CotA from the endospore coats of Bacillus subtilis. A comparison of the molecular structures of the laccases of different origins demonstrates that, structurally, these objects are highly conservative. This obviously indicates that the catalytic activity of the enzymes under consideration is characterized by similar mechanisms.« less

Catalase deficiency may complicate urate oxidase (rasburicase) therapy.

PubMed

Góth, László; Bigler, N William

2007-09-01

Patients with low (inherited and acquired) catalase activities who are treated with infusion of uric acid oxidase because they are at risk of tumour lysis syndrome may experience very high concentrations of hydrogen peroxide. They may suffer from methemoglobinaemia and haemolytic anaemia which may be attributed either to deficiency of glucose-6-phosphate dehydrogenase or to other unknown circumstances. Data have not been reported from catalase deficient patients who were treated with uric acid oxidase. It may be hypothesized that their decreased blood catalase could lead to the increased concentration of hydrogen peroxide which may cause haemolysis and formation of methemoglobin. Blood catalase activity should be measured for patients at risk of tumour lysis syndrome prior to uric acid oxidase treatment.

Effects of Acute Lymphoblastic Leukemia on Ceruloplasmin Oxidase, Copper and Several Markers of Oxidative Damage, in Children.

PubMed

Mehdi, Wesen Adel; Yusof, Faridah; Mehde, Atheer Awad; Zainulabdeen, Jwan Abdulmohsin; Raus, Raha Ahmed; Abdulbari, Alaa Shawqi

2015-01-01

Acute leukaemia is characterized by fast growth of abnormal clones of haemopoietic precursor cells inside bone marrow leading to undue accumulation in the bone marrow. Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer. The study concerned 50 children diagnosed with ALL (mean age, 8.55±2.54) compared to 40 healthy controls (mean age, 8.00±1.85). The Hb, serum copper, ceruloplasmin oxidase, advanced oxidation protein products (AOPPs), total antioxidant activity (TAA) and protein were measured in all groups. One proteinous component was isolated by gel filtration chromatography from the precipitate produced by polyethylene glycol. Significantly higher levels of AOPP, copper and decrease in total antioxidant activity were noted in the cases. Statistical analysis also showed a significant increase (p<0.01) in the activity of serum ceruloplasmin oxidase in patients with ALL compared to normal subjects. The maximum velocity (Vmax) and Michaelis constant had values of 104.2 U/L and 11.7 mM, respectively. The ΔH* values for ceruloplasmin oxidase in ALL patients were positive, confirming the reaction to be endothermic. The results from this study showed a significant increase in AOPP, ceruloplasmine oxidase and decrease in total antioxidant activity .These parameters may play a role in development of DNA damage in childhood patients with acute lymphoblastic leukemia (ALL). The ΔS* and ΔG* values were negative, these refer that the reaction of ES formation is spontaneous, but needs energy in a so-called endergonic reaction. Also the negative ΔS* value of ceruloplasmin oxidase indicates that the complex [ES*] is further modulated through increasing structure arrangement.

[Respiratory oxidases: the enzymes which use most of the oxygen which living things breathe].

PubMed

Toledo-Cuevas, E M

1997-01-01

The respiratory oxidases are the last enzymes of the aerobic respiratory chain. They catalize the reduction of molecular oxygen to water, with generation of an electrochemical gradient useful for the energy demanding cellular processes. Most of the oxidases belong to the heme-copper superfamily. They possess a heme-copper center, constituted of a high spin heme and a CuB center, where the reduction of oxygen takes place and probably where the link to proton pumping is located. The superfamily is divided in two classes: the quinol- and the cytochrome c-oxidases. The latter are divided in the aa3 and the cbb3-type cytochrome c oxidases. The main difference between quinol- and the aa3-type cytochrome c-oxidases is the CuA center, which is absent in the quinol oxidases. The cbb3-type cytochrome oxidases have the binuclear center, but lack the CuA center. They also does not have the classical subunits II and III. These differences seem not to affect the oxygen reduction or the proton pumping. Probably the oxidases have evolved from some denitrification enzymes and prior the photosynthetic process. Also is possible that the cbb3-type cytochrome oxidases or others very similar have been the first oxidases to appear.

Identification of the alternative terminal oxidase of higher plant mitochondria

PubMed Central

Elthon, Thomas E.; McIntosh, Lee

1987-01-01

In addition to cytochrome oxidase, plant mitochondria have a second terminal oxidase called the alternative oxidase. The alternative oxidase is of great interest in that energy is not conserved when electrons flow through it. The potential energy of the system is thus lost as heat, and, in plants with high levels of the alternative oxidase, this results in thermogenesis. We have purified the alternative oxidase from mitochondria of the thermogenic spadix of Sauromatum guttatum and have identified its polypeptide constituents by using polyclonal antibodies. A 166-fold purification was achieved through a combination of cation-exchange (carboxymethyl-Sepharose) and hydrophobic-interaction (phenyl-Sepharose) chromatography. Polyclonal antibodies raised to the CM-Sepharose fractions readily immunoprecipitated alternative oxidase activity and immunoprecipitated four of the proteins that copurify with the activity. These proteins have apparent molecular masses of 37, 36, 35.5, and 35 kDa. Polyclonal antibodies raised individually to the 37-, 36-, and 35.5- plus 35-kDa proteins cross-reacted with all of these proteins, indicating the presence of common antigenic sites. The 37-kDa protein appears to be constitutive in Sauromatum, whereas expression of the 36- and 35-kDa proteins was correlated with presence of alternative pathway activity. The 35.5-kDa protein appears with loss of alternative pathway activity during senescence, indicating that this protein may be a degradation product of the 36-kDa protein. Binding of anti-36-kDa protein antibodies to total mitochondrial protein blots of five plant species indicated that similar proteins were always present when alternative pathway activity was observed. Images PMID:16593898

Molecular and Biochemical Characterization of a Cytokinin Oxidase from Maize1

PubMed Central

Bilyeu, Kristin D.; Cole, Jean L.; Laskey, James G.; Riekhof, Wayne R.; Esparza, Thomas J.; Kramer, Michelle D.; Morris, Roy O.

2001-01-01

It is generally accepted that cytokinin oxidases, which oxidatively remove cytokinin side chains to produce adenine and the corresponding isopentenyl aldehyde, play a major role in regulating cytokinin levels in planta. Partially purified fractions of cytokinin oxidase from various species have been studied for many years, but have yet to clearly reveal the properties of the enzyme or to define its biological significance. Details of the genomic organization of the recently isolated maize (Zea mays) cytokinin oxidase gene (ckx1) and some of its Arabidopsis homologs are now presented. Expression of an intronless ckx1 in Pichia pastoris allowed production of large amounts of recombinant cytokinin oxidase and facilitated detailed kinetic and cofactor analysis and comparison with the native enzyme. The enzyme is a flavoprotein containing covalently bound flavin adenine dinucleotide, but no detectable heavy metals. Expression of the oxidase in maize tissues is described. PMID:11154345

21 CFR 866.2420 - Oxidase screening test for gonorrhea.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Oxidase screening test for gonorrhea. 866.2420 Section 866.2420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices § 866.2420 Oxidase...

21 CFR 866.2420 - Oxidase screening test for gonorrhea.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Oxidase screening test for gonorrhea. 866.2420 Section 866.2420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices § 866.2420 Oxidase...

21 CFR 866.2420 - Oxidase screening test for gonorrhea.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Oxidase screening test for gonorrhea. 866.2420 Section 866.2420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices § 866.2420 Oxidase...

21 CFR 866.2420 - Oxidase screening test for gonorrhea.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Oxidase screening test for gonorrhea. 866.2420 Section 866.2420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices § 866.2420 Oxidase...

21 CFR 866.2420 - Oxidase screening test for gonorrhea.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Oxidase screening test for gonorrhea. 866.2420 Section 866.2420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices § 866.2420 Oxidase...

Identification of a Third Mn(II) Oxidase Enzyme in Pseudomonas putida GB-1

PubMed Central

Smesrud, Logan; Tebo, Bradley M.

2016-01-01

ABSTRACT The oxidation of soluble Mn(II) to insoluble Mn(IV) is a widespread bacterial activity found in a diverse array of microbes. In the Mn(II)-oxidizing bacterium Pseudomonas putida GB-1, two Mn(II) oxidase genes, named mnxG and mcoA, were previously identified; each encodes a multicopper oxidase (MCO)-type enzyme. Expression of these two genes is positively regulated by the response regulator MnxR. Preliminary investigation into putative additional regulatory pathways suggested that the flagellar regulators FleN and FleQ also regulate Mn(II) oxidase activity; however, it also revealed the presence of a third, previously uncharacterized Mn(II) oxidase activity in P. putida GB-1. A strain from which both of the Mn(II) oxidase genes and fleQ were deleted exhibited low levels of Mn(II) oxidase activity. The enzyme responsible was genetically and biochemically identified as an animal heme peroxidase (AHP) with domain and sequence similarity to the previously identified Mn(II) oxidase MopA. In the ΔfleQ strain, P. putida GB-1 MopA is overexpressed and secreted from the cell, where it actively oxidizes Mn. Thus, deletion of fleQ unmasked a third Mn(II) oxidase activity in this strain. These results provide an example of an Mn(II)-oxidizing bacterium utilizing both MCO and AHP enzymes. IMPORTANCE The identity of the Mn(II) oxidase enzyme in Pseudomonas putida GB-1 has been a long-standing question in the field of bacterial Mn(II) oxidation. In the current work, we demonstrate that P. putida GB-1 employs both the multicopper oxidase- and animal heme peroxidase-mediated pathways for the oxidation of Mn(II), rendering this model organism relevant to the study of both types of Mn(II) oxidase enzymes. The presence of three oxidase enzymes in P. putida GB-1 deepens the mystery of why microorganisms oxidize Mn(II) while providing the field with the tools necessary to address this question. The initial identification of MopA as a Mn(II) oxidase in this strain required the

The first mammalian aldehyde oxidase crystal structure: insights into substrate specificity.

PubMed

Coelho, Catarina; Mahro, Martin; Trincão, José; Carvalho, Alexandra T P; Ramos, Maria João; Terao, Mineko; Garattini, Enrico; Leimkühler, Silke; Romão, Maria João

2012-11-23

Aldehyde oxidases have pharmacological relevance, and AOX3 is the major drug-metabolizing enzyme in rodents. The crystal structure of mouse AOX3 with kinetics and molecular docking studies provides insights into its enzymatic characteristics. Differences in substrate and inhibitor specificities can be rationalized by comparing the AOX3 and xanthine oxidase structures. The first aldehyde oxidase structure represents a major advance for drug design and mechanistic studies. Aldehyde oxidases (AOXs) are homodimeric proteins belonging to the xanthine oxidase family of molybdenum-containing enzymes. Each 150-kDa monomer contains a FAD redox cofactor, two spectroscopically distinct [2Fe-2S] clusters, and a molybdenum cofactor located within the protein active site. AOXs are characterized by broad range substrate specificity, oxidizing different aldehydes and aromatic N-heterocycles. Despite increasing recognition of its role in the metabolism of drugs and xenobiotics, the physiological function of the protein is still largely unknown. We have crystallized and solved the crystal structure of mouse liver aldehyde oxidase 3 to 2.9 Å. This is the first mammalian AOX whose structure has been solved. The structure provides important insights into the protein active center and further evidence on the catalytic differences characterizing AOX and xanthine oxidoreductase. The mouse liver aldehyde oxidase 3 three-dimensional structure combined with kinetic, mutagenesis data, molecular docking, and molecular dynamics studies make a decisive contribution to understand the molecular basis of its rather broad substrate specificity.

[Oxygen and the superoxide anion. Modulation of NADPH oxidase?].

PubMed

Delbosc, S; Cristol, J P; Descomps, B; Chénard, J; Sirois, P

2001-01-01

Oxidative stress which results from an imbalance between oxidant production and antioxidant defense mechanisms can promote modifications of lipids, proteins and nucleic acids. This review focuses on the different pathways leading to Reactive Oxygen Species (ROS) production in particular on NADPH oxidase activation. This enzyme is localized in numerous cells including phagocytes and vascular cells and composed of membrane and cytosolic sub-units. The activation of the NADPH oxidase is largely involved in inflammation associated diseases such as asthma, Systemic Inflammatory Response Syndrome and aging associated diseases such as atherosclerosis and neurodeneratives diseases. The modulation of NADPH oxidase could be a way to limit or prevent the development of these diseases.

Aiding and abetting roles of NOX oxidases in cellular transformation

PubMed Central

Block, Karen; Gorin, Yves

2013-01-01

NADPH oxidases of the NADPH oxidase (NOX) family are dedicated reactive oxygen species-generating enzymes that broadly and specifically regulate redox-sensitive signalling pathways that are involved in cancer development and progression. They act at specific cellular membranes and microdomains through the activation of oncogenes and the inactivation of tumour suppressor proteins. In this Review, we discuss primary targets and redox-linked signalling systems that are influenced by NOX-derived ROS, and the biological role of NOX oxidases in the aetiology of cancer. PMID:22918415

Monoamine oxidase B layer-by-layer film fabrication and characterization toward dopamine detection.

PubMed

Miyazaki, Celina Massumi; Pereira, Tamyris Paschoal; Mascagni, Daniela Branco Tavares; de Moraes, Marli Leite; Ferreira, Marystela

2016-01-01

In this work nanostructured film composites of the monoamine oxidase B (MAO-B) enzyme, free or encapsulated in liposomes, were fabricated by the layer-by-layer (LbL) self-assembly technique, employing polyethylene imine (PEI) as polycation. Initially, the MAO-B enzyme was incorporated into liposomes in order to preserve its enzymatic structure ensuring their activity and catalytic stability. The LbL film growth was monitored by surface plasmon resonance (SPR) by gold resonance angle shift analysis after each bilayer deposition. Subsequently, the films were applied as amperometric biosensors for dopamine detection using Prussian Blue (PB) as the electron mediator. The biosensor fabricated by MAO-B incorporated into liposomes composed of DPPG:POPG in the ratio (1:4) (w/w) showed the best performance with a sensitivity of 0.86 (μA cm(-2))/(mmol L(-1)) and a detection limit of 0.33 mmol L(-1).

In vitro effects of acetylcholinesterase reactivators on monoamine oxidase activity.

PubMed

Fišar, Zdeněk; Hroudová, Jana; Korábečný, Jan; Musílek, Kamil; Kuča, Kamil

2011-03-05

Administration of acetylcholinesterase (AChE) reactivators (oximes) is usually used in order to counteract the poisoning effects of nerve agents. The possibility was suggested that oximes may show some therapeutic and/or adverse effects through their action in central nervous system. There are no sufficient data about interaction of oximes with monoaminergic neurotransmitter's systems in the brain. Oxime-type AChE reactivators pralidoxime, obidoxime, trimedoxime, methoxime and HI-6 were tested for their potential to affect the activity of monoamine oxidase of type A (MAO-A) and type B (MAO-B) in crude mitochondrial fraction of pig brains. The compounds were found to inhibit fully MAO-A with half maximal inhibitory concentration (IC(50)) of 0.375 mmol/l (pralidoxime), 1.53 mmol/l (HI-6), 2.31 mmol/l (methoxime), 2.42 mmol/l (obidoxime) and 4.98 mmol/l (trimedoxime). Activity of MAO-B was fully inhibited by HI-6 and pralidoxime only with IC(50) 4.81 mmol/l and 11.01 mmol/l, respectively. Methoxime, obidoxime and trimedoxime displayed non-monotonic concentration dependent effect on MAO-B activity. Because oximes concentrations effective for MAO inhibition could not be achieved in vivo at the cerebral level, we suppose that oximes investigated do not interfere with brain MAO at therapeutically relevant concentrations. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

NADPH Oxidase Activation Contributes to Heavy Ion Irradiation–Induced Cell Death

PubMed Central

Wang, Yupei; Liu, Qing; Zhao, Weiping; Zhou, Xin; Miao, Guoying; Sun, Chao

2017-01-01

Increased oxidative stress plays an important role in heavy ion radiation–induced cell death. The mechanism involved in the generation of elevated reactive oxygen species (ROS) is not fully illustrated. Here we show that NADPH oxidase activation is closely related to heavy ion radiation–induced cell death via excessive ROS generation. Cell death and cellular ROS can be greatly reduced in irradiated cancer cells with the preincubation of diphenyleneiodium, an inhibitor of NADPH oxidase. Most of the NADPH oxidase (NOX) family proteins (NOX1, NOX2, NOX3, NOX4, and NOX5) showed increased expression after heavy ion irradiation. Meanwhile, the cytoplasmic subunit p47phox was translocated to the cell membrane and localized with NOX2 to form reactive NADPH oxidase. Our data suggest for the first time that ROS generation, as mediated by NADPH oxidase activation, could be an important contributor to heavy ion irradiation–induced cell death. PMID:28473742

Immobilization of xanthine oxidase on a polyaniline silicone support.

PubMed

Nadruz, W; Marques, E T; Azevedo, W M; Lima-Filho, J L; Carvalho, L B

1996-03-01

A polyaniline silicone support to immobilize xanthine oxidase is proposed as a reactor coil to monitor the action of xanthine oxidase on hypoxanthine, xanthine and 6-mercaptopurine. A purified xanthine oxidase immobilized on this support lost 80% of the initial activity after 12 min of use. Co-immobilization of superoxide dismutase and catalase increased the stability of immobilized xanthine oxidase so that the derivative maintained 79% of its initial activity after 4.6 h of continuous use in which 1.5 mumol purine bases were converted by the immobilized enzyme system. There is no evidence of either polyaniline or protein leaching from the coil during 3 h of continuous use. When solutions (10 ml) of hypoxanthine, xanthine and 6-mercaptopurine were circulated individually through the xanthine oxidase-superoxide dismutase-catalase-polyaniline coil (1 mm internal diameter and 3 m in length, 3 ml internal volume) activities of 8.12, 11.17 and 1.09 nmol min-1 coil-1, respectively, were obtained. The advantages of the reactor configuration and the redox properties of the polymer, particularly with respect to immobilized oxidoreductases, make this methodology attractive for similar enzyme systems. This immobilized enzyme system using polyaniline-silicone as support converted 6-mercaptopurine to 6-thiouric acid with equal efficiency as resins based on polyacrylamide and polyamide 11.

Cytotoxicity of polyamines to Amoeba proteus: role of polyamine oxidase.

PubMed

Schenkel, E; Dubois, J G; Helson-Cambier, M; Hanocq, M

1996-02-01

It has been shown that oxidation of polyamines by polyamine oxidases can produce toxic compounds (H2O2, aldehydes, ammonia) and that the polyamine oxidase-polyamine system is implicated, in vitro, in the death of several parasites. Using Amoeba proteus as an in vitro model, we studied the cytotoxicity to these cells of spermine, spermidine, their acetyl derivatives, and their hypothetical precursors. Spermine and N1-acetylspermine were more toxic than emetine, an amoebicidal reference drug. Spermine presented a short-term toxicity, but a 48-h contact time was necessary for the high toxicity of spermidine. The uptake by Amoeba cells of the different polyamines tested was demonstrated. On the other hand, a high polyamine oxidase activity was identified in Amoeba proteus crude extract. Spermine (theoretical 100%) and N1-acetylspermine (64%) were the best substrates at pH 9.5, while spermidine, its acetyl derivatives, and putrescine were very poorly oxidized by this enzyme (3-20%). Spermine oxidase activity was inhibited by phenylhydrazine (nil) and isoniazid (approximately 50%). Mepacrine did not inhibit the enzyme activity at pH 8. Neither monoamine nor diamine oxidase activity (approximately 10%) was found. It must be emphasized that spermine, the best enzyme substrate, is the most toxic polyamine. This finding suggests that knowledge of polyamine oxidase specificity can be used to modulate the cytotoxicity of polyamine derivatives. Amoeba proteus was revealed as a simple model for investigation of the connection between cytotoxicity and enzyme activity.

Xanthine oxidase inhibiting effects of noni (Morinda citrifolia) fruit juice.

PubMed

Palu, Afa; Deng, Shixin; West, Brett; Jensen, Jarakae

2009-12-01

Morinda citrifolia L. (noni), family Rubiaceae, has been used in Polynesia for over 2000 years for its reputed health benefits, one of which is its therapeutic effects on gout (langa e hokotanga hui). However, its healing mechanism has not been elucidated. This study showed that in an in vitro bioassay that Tahitian Noni Juice (TNJ) inhibited xanthine oxidase (XO) concentration dependently. Concentrations of 1, 5 and 10 mg/mL of TNJ inhibited XO by 11%, 113% and 148%, respectively, with an IC50 of 3.8 mg compared with an IC50 of 2.4 microm for allopurinol. Noni fruit juice concentrate (NFJC) also inhibited XO concentration dependently. Concentrations of 1 and 5 mg/mL NFJC inhibited XO in vitro by 184% and 159%, respectively. A 0.1 mg/mL methanol extract (NFJME) from the fractionation of noni fruit puree inhibited XO by 64%. It was elucidated that the noni fruit juice inhibitory effect on XO enzymes is the mechanism by which noni ameliorates gout and gout-like diseases. Further, the results also support the traditional usage of noni in the treatment of gout. Copyright (c) 2009 John Wiley & Sons, Ltd.

Simple, high-yield purification of xanthine oxidase from bovine milk.

PubMed

Ozer, N; Müftüoglu, M; Ataman, D; Ercan, A; Ogüs, I H

1999-05-13

Xanthine oxidase, a commercially important enzyme with a wide area of application, was extracted from fresh milk, without added preservatives, using toluene and heat. The short purification procedure, with high yield, consisted of extraction, ammonium sulfate fractionation, and DEAE-Sepharose (fast flow) column chromatography. Xanthine oxidase was eluted as a single activity peak from the column using a buffer gradient. The purification fold, specific activity and yield for the purified xanthine oxidase were 328, 10.161 U/mg and 69%, respectively. The enzyme was concentrated by ultrafiltration, although 31% of the activity was lost during concentration, no change in specific activity was observed. Activity and protein gave coincident staining bands on native polyacrylamide gels. The intensity and the number of bands were dependent on the oxidative state(s) of the enzyme; reduction by 2-mercaptoethanol decreased the intensity of the slow-moving bands and increased the intensity of the fastest-moving band. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two major bands (molecular masses of 152 and 131 kDa) were observed, accounting for > or = 95% of xanthine oxidase. Native- and SDS-PAGE showed that the purified xanthine oxidase becomes a heterodimer due to endogenous proteases.

Urate Oxidase Purification by Salting-in Crystallization: Towards an Alternative to Chromatography

PubMed Central

Giffard, Marion; Ferté, Natalie; Ragot, François; El Hajji, Mohamed; Castro, Bertrand; Bonneté, Françoise

2011-01-01

Background Rasburicase (Fasturtec® or Elitek®, Sanofi-Aventis), the recombinant form of urate oxidase from Aspergillus flavus, is a therapeutic enzyme used to prevent or decrease the high levels of uric acid in blood that can occur as a result of chemotherapy. It is produced by Sanofi-Aventis and currently purified via several standard steps of chromatography. This work explores the feasibility of replacing one or more chromatography steps in the downstream process by a crystallization step. It compares the efficacy of two crystallization techniques that have proven successful on pure urate oxidase, testing them on impure urate oxidase solutions. Methodology/Principal Findings Here we investigate the possibility of purifying urate oxidase directly by crystallization from the fermentation broth. Based on attractive interaction potentials which are known to drive urate oxidase crystallization, two crystallization routes are compared: a) by increased polymer concentration, which induces a depletion attraction and b) by decreased salt concentration, which induces attractive interactions via a salting-in effect. We observe that adding polymer, a very efficient way to crystallize pure urate oxidase through the depletion effect, is not an efficient way to grow crystals from impure solution. On the other hand, we show that dialysis, which decreases salt concentration through its strong salting-in effect, makes purification of urate oxidase from the fermentation broth possible. Conclusions The aim of this study is to compare purification efficacy of two crystallization methods. Our findings show that crystallization of urate oxidase from the fermentation broth provides purity comparable to what can be achieved with one chromatography step. This suggests that, in the case of urate oxidase, crystallization could be implemented not only for polishing or concentration during the last steps of purification, but also as an initial capture step, with minimal changes to the

Construction of Mutant Glucose Oxidases with Increased Dye-Mediated Dehydrogenase Activity

PubMed Central

Horaguchi, Yohei; Saito, Shoko; Kojima, Katsuhiro; Tsugawa, Wakako; Ferri, Stefano; Sode, Koji

2012-01-01

Mutagenesis studies on glucose oxidases (GOxs) were conducted to construct GOxs with reduced oxidase activity and increased dehydrogenase activity. We focused on two representative GOxs, of which crystal structures have already been reported—Penicillium amagasakiense GOx (PDB ID; 1gpe) and Aspergillus niger GOx (PDB ID; 1cf3). We constructed oxygen-interacting structural models for GOxs, and predicted the residues responsible for oxidative half reaction with oxygen on the basis of the crystal structure of cholesterol oxidase as well as on the fact that both enzymes are members of the glucose/methanol/choline (GMC) oxidoreductase family. Rational amino acid substitution resulted in the construction of an engineered GOx with drastically decreased oxidase activity and increased dehydrogenase activity, which was higher than that of the wild-type enzyme. As a result, the dehydrogenase/oxidase ratio of the engineered enzyme was more than 11-fold greater than that of the wild-type enzyme. These results indicate that alteration of the dehydrogenase/oxidase activity ratio of GOxs is possible by introducing a mutation into the putative functional residues responsible for oxidative half reaction with oxygen of these enzymes, resulting in a further increased dehydrogenase activity. This is the first study reporting the alteration of GOx electron acceptor preference from oxygen to an artificial electron acceptor. PMID:23203056

Molecular cloning and photoperiod-regulated expression of gibberellin 20-oxidase from the long-day plant spinach.

PubMed

Wu, K; Li, L; Gage, D A; Zeevaart, J A

1996-02-01

Spinach (Spinacia oleracea L.) is a long-day (LD) rosette plant in which stem growth under LD conditions is mediated by gibberellins (GAs). Major control points in spinach are the later steps of sequential oxidation and elimination of C-20 of C20-GAs. Degenerate oligonucleotide primers were used to obtain a polymerase chain reaction product from spinach genomic DNA that has a high homology with GA 20-oxidase cDNAs from Cucurbita maxima L. and Arabidopsis thaliana Heynh. This polymerase chain reaction product was used as a probe to isolate a full-length cDNA clone with an open reading frame encoding a putative 43-kD protein of 374 amino acid residues. When this cDNA clone was expressed in Escherichia coli, the fusion protein catalyzed the biosynthetic sequence GA53-->GA44-->GA19-->GA20 and GA19-->GA17. This establishes that in spinach a single protein catalyzes the oxidation and elimination of C-20. Transfer of spinach plants from short day (SD) to LD conditions caused an increase in the level of all GAs of the early-13-hydroxylation pathway, except GA53, with GA20, GA1, and GA8 showing the largest increases. Northern blot analysis indicated that the level of GA 20-oxidase mRNA was higher in plants in LD than in SD conditions, with highest level of expression in the shoot tips and elongating stems. This expression pattern of GA 20-oxidase is consistent with the different levels of GA20, GA1, and GA8 found in spinach plants grown in SD and LD conditions.

Development of 2-(Substituted Benzylamino)-4-Methyl-1, 3-Thiazole-5-Carboxylic Acid Derivatives as Xanthine Oxidase Inhibitors and Free Radical Scavengers.

PubMed

Ali, Md Rahmat; Kumar, Suresh; Afzal, Obaid; Shalmali, Nishtha; Sharma, Manju; Bawa, Sandhya

2016-04-01

A series of 2-(substituted benzylamino)-4-methylthiazole-5-carboxylic acid was designed and synthesized as structural analogue of febuxostat. A methylene amine spacer was incorporated between the phenyl ring and thiazole ring in contrast to febuxostat in which the phenyl ring was directly linked with the thiazole moiety. The purpose of incorporating methylene amine was to provide a heteroatom which is expected to favour hydrogen bonding within the active site residues of the enzyme xanthine oxidase. The structure of all the compounds was established by the combined use of FT-IR, NMR and MS spectral data. All the compounds were screened in vitro for their ability to inhibit the enzyme xanthine oxidase as per the reported procedure along with DPPH free radical scavenging assay. Compounds 5j, 5k and 5l demonstrated satisfactory potent xanthine oxidase inhibitory activities with IC50 values, 3.6, 8.1 and 9.9 μm, respectively, whereas compounds 5k, 5n and 5p demonstrated moderate antioxidant activities having IC50 15.3, 17.6 and 19.6 μm, respectively, along with xanthine oxidase inhibitory activity. Compound 5k showed moderate xanthine oxidase inhibitory activity as compared with febuxostat along with antioxidant activity. All the compounds were also studied for their binding affinity in active site of enzyme (PDB ID-1N5X). © 2015 John Wiley & Sons A/S.

Alternative oxidase (AOX) and phenolic metabolism in methyl jasmonate-treated hairy root cultures of Daucus carota L.

PubMed

Sircar, Debabrata; Cardoso, Hélia G; Mukherjee, Chiranjit; Mitra, Adinpunya; Arnholdt-Schmitt, Birgit

2012-05-01

Methyl-jasmonate (MJ)-treated hairy roots of Daucus carota L. were used to study the influence of alternative oxidase (AOX) in phenylpropanoid metabolism. Phenolic acid accumulation, as well as total flavonoids and lignin content of the MJ-treated hairy roots were decreased by treatment with salicylhydroxamic acid (SHAM), a known inhibitor of AOX. The inhibitory effect of SHAM was concentration dependent. Treatment with propyl gallate (PG), another inhibitor of AOX, also had a similar inhibitory effect on accumulation of phenolic acid, total flavonoids and lignin. The transcript levels of two DcAOX genes (DcAOX2a and DcAOX1a) were monitored at selected post-elicitation time points. A notable rise in the transcript levels of both DcAOX genes was observed preceding the MJ-induced enhanced accumulation of phenolics, flavonoids and lignin. An appreciable increase in phenylalanine ammonia-lyase (PAL) transcript level was also observed prior to enhanced phenolics accumulation. Both DcAOX genes showed differential transcript accumulation patterns after the onset of elicitation. The transcript levels of DcAOX1a and DcAOX2a attained peak at 6hours post elicitation (hpe) and 12hpe, respectively. An increase in the transcript levels of both DcAOX genes preceding the accumulation of phenylpropanoid-derivatives and lignin showed a positive correlation between AOX activity and phenylpropanoid biosynthesis. The results provide important new insight about the influence of AOX in phenylpropanoid biosynthesis. Copyright © 2012 Elsevier GmbH. All rights reserved.

Structure of caa(3) cytochrome c oxidase--a nature-made enzyme-substrate complex.

PubMed

Noor, Mohamed Radzi; Soulimane, Tewfik

2013-05-01

Aerobic respiration, the energetically most favorable metabolic reaction, depends on the action of terminal oxidases that include cytochrome c oxidases. The latter forms a part of the heme-copper oxidase superfamily and consists of three different families (A, B, and C types). The crystal structures of all families have now been determined, allowing a detailed structural comparison from evolutionary and functional perspectives. The A2-type oxidase, exemplified by the Thermus thermophilus caa(3) oxidase, contains the substrate cytochrome c covalently bound to the enzyme complex. In this article, we highlight the various features of caa(3) enzyme and provide a discussion of their importance, including the variations in the proton and electron transfer pathways.

The Importance of NADPH Oxidases and Redox Signaling in Angiogenesis

PubMed Central

Prieto-Bermejo, Rodrigo; Hernández-Hernández, Angel

2017-01-01

Eukaryotic cells have to cope with the constant generation of reactive oxygen species (ROS). Although the excessive production of ROS might be deleterious for cell biology, there is a plethora of evidence showing that moderate levels of ROS are important for the control of cell signaling and gene expression. The family of the nicotinamide adenine dinucleotide phosphate oxidases (NADPH oxidases or Nox) has evolved to produce ROS in response to different signals; therefore, they fulfil a central role in the control of redox signaling. The role of NADPH oxidases in vascular physiology has been a field of intense study over the last two decades. In this review we will briefly analyze how ROS can regulate signaling and gene expression. We will address the implication of NADPH oxidases and redox signaling in angiogenesis, and finally, the therapeutic possibilities derived from this knowledge will be discussed. PMID:28505091

Degradation of oxalate in rats implanted with immobilized oxalate oxidase.

PubMed

Raghavan, K G; Tarachand, U

1986-01-20

Accumulation of oxalate leads to hyperoxaluria and calcium oxalate nephrolithiasis in man. Since oxalate is a metabolic end product in mammals, the feasibility of its enzymic degradation has been tested in vivo in rats by administering exogenous oxalate oxidase. Oxalate oxidase, isolated from banana fruit peels, in its native form was found to be non-active at the physiological pH of the recipient animal. However, its functional viability in the recipient animal was ensured by its prior binding with ethylenemaleic anhydride, thus shifting its pH activity curve towards the alkaline range. Rats implanted with dialysis membrane capsules containing such immobilized oxalate oxidase in their peritoneal cavities effectively metabolized intraperitoneally injected [14C]oxalate as well as its precursor [14C]glyoxalate. The implantation of capsules containing coentrapped multienzyme preparations of oxalate oxidase, catalase and peroxidase led to a further degradation of administered [14C]oxalate in rats.

Urate oxidase is imported into peroxisomes recognizing the C-terminal SKL motif of proteins.

PubMed

Miura, S; Oda, T; Funai, T; Ito, M; Okada, Y; Ichiyama, A

1994-07-01

Rat liver urate oxidase synthesized from cDNA through coupled transcription and translation was incubated at 26 degrees C for 60 min with purified peroxisomes from rat liver. Urate oxidase was efficiently imported into the peroxisomes, as determined by resistance to externally added proteinase K. The amount of imported urate oxidase increased with time and the import was temperature dependent. A synthetic peptide composed of the C-terminal 10 amino acid residues of acyl-CoA oxidase (the C-terminal tripeptide is Ser-Lys-Leu) inhibited the import of urate oxidase, whereas other peptides, in which the C-terminal Ser-Lys-Leu (SKL) sequence was deleted or mutated, were not effective. Two mutant urate oxidase proteins in which the C-terminal Ser-Arg-Leu (SRL) sequence was deleted or mutated to Ser-Glu-Leu (SEL) were not imported into peroxisomes. With substitution of a lysine residue for arginine in the SRL tripeptide at the C-terminus the import activity was retained. These results show that urate oxidase is important into peroxisomes via a common pathway with acyl-CoA oxidase, and that the C-terminal SRL sequence functions as a peroxisomal-targeting signal.

Multilayered Polyelectrolyte Microcapsules: Interaction with the Enzyme Cytochrome C Oxidase

PubMed Central

Pastorino, Laura; Dellacasa, Elena; Noor, Mohamed R.; Soulimane, Tewfik; Bianchini, Paolo; D'Autilia, Francesca; Antipov, Alexei; Diaspro, Alberto; Tofail, Syed A. M.; Ruggiero, Carmelina

2014-01-01

Cell-sized polyelectrolyte capsules functionalized with a redox-driven proton pump protein were assembled for the first time. The interaction of polyelectrolyte microcapsules, fabricated by electrostatic layer-by-layer assembly, with cytochrome c oxidase molecules was investigated. We found that the cytochrome c oxidase retained its functionality, that the functionalized microcapsules interacting with cytochrome c oxidase were permeable and that the permeability characteristics of the microcapsule shell depend on the shell components. This work provides a significant input towards the fabrication of an integrated device made of biological components and based on specific biomolecular functions and properties. PMID:25372607

Purification and partial amino-acid sequence of gibberellin 20-oxidase from Cucurbita maxima L. endosperm.

PubMed

Lange, T

1994-01-01

Gibberellin (GA) 20-oxidase was purified to apparent homogeneity from Cucurbita maxima endosperm by fractionated ammonium-sulphate precipitation, gel-filtration chromatography and anion-exchange and hydrophobic-interaction high-performance liquid chromatography (HPLC). Average purification after the last step was 55-fold with 3.9% of the activity recovered. The purest single fraction was enriched 101-fold with 0.2% overall recovery. Apparent relative molecular mass of the enzyme was 45 kDa, as determined by gel-filtration HPLC and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, indicating that GA 20-oxidase is probably a monomeric enzyme. The purified enzyme degraded on two-dimensional gel electrophoresis, giving two protein spots: a major one corresponding to a molecular mass of 30 kDa and a minor one at 45 kDa. The isoelectric point for both was 5.4. The amino-acid sequences of the amino-terminus of the purified enzyme and of two peptides from a tryptic digest were determined. The purified enzyme catalysed the sequential conversion of [14C]GA12 to [14C]GA15, [14C]GA24 and [14C]GA25, showing that carbon atom 20 was oxidised to the corresponding alcohol, aldehyde and carboxylic acid in three consecutive reactions. [14C]Gibberellin A53 was similarly converted to [14C]GA44, [14C]GA19, [14C]GA17 and small amounts of a fourth product, which was preliminarily identified as [14C]GA20, a C19-gibberellin. All GAs except [14C]GA20 were identified by combined gas chromatography-mass spectrometry. The cofactor requirements in the absence of dithiothreitol were essentially as in its presence (Lange et al., Planta 195, 98-107, 1994), except that ascorbate was essential for enzyme activity and the optimal concentration of catalase was lower.

Functional expression of amine oxidase from Aspergillus niger (AO-I) in Saccharomyces cerevisiae.

PubMed

Kolaríková, Katerina; Galuszka, Petr; Sedlárová, Iva; Sebela, Marek; Frébort, Ivo

2009-01-01

The aim of this work was to prepare recombinant amine oxidase from Aspergillus niger after overexpressing in yeast. The yeast expression vector pDR197 that includes a constitutive PMA1 promoter was used for the expression in Saccharomyces cerevisiae. Recombinant amine oxidase was extracted from the growth medium of the yeast, purified to homogeneity and identified by activity assay and MALDI-TOF peptide mass fingerprinting. Similarity search in the newly published A. niger genome identified six genes coding for copper amine oxidase, two of them corresponding to the previously described enzymes AO-I a methylamine oxidase and three other genes coding for FAD amine oxidases. Thus, A. niger possesses an enormous metabolic gear to grow on amine compounds and thus support its saprophytic lifestyle.

Cyanide-insensitive quinol oxidase (CIO) from Gluconobacter oxydans is a unique terminal oxidase subfamily of cytochrome bd.

PubMed

Miura, Hiroshi; Mogi, Tatsushi; Ano, Yoshitaka; Migita, Catharina T; Matsutani, Minenosuke; Yakushi, Toshiharu; Kita, Kiyoshi; Matsushita, Kazunobu

2013-06-01

Cyanide-insensitive terminal quinol oxidase (CIO) is a subfamily of cytochrome bd present in bacterial respiratory chain. We purified CIO from the Gluconobacter oxydans membranes and characterized its properties. The air-oxidized CIO showed some or weak peaks of reduced haemes b and of oxygenated and ferric haeme d, differing from cytochrome bd. CO- and NO-binding difference spectra suggested that haeme d serves as the ligand-binding site of CIO. Notably, the purified CIO showed an extraordinary high ubiquinol-1 oxidase activity with the pH optimum of pH 5-6. The apparent Vmax value of CIO was 17-fold higher than that of G. oxydans cytochrome bo3. In addition, compared with Escherichia coli cytochrome bd, the quinol oxidase activity of CIO was much more resistant to cyanide, but sensitive to azide. The Km value for O2 of CIO was 7- to 10-fold larger than that of G. oxydans cytochrome bo3 or E. coli cytochrome bd. Our results suggest that CIO has unique features attributable to the structure and properties of the O2-binding site, and thus forms a new sub-group distinct from cytochrome bd. Furthermore, CIO of acetic acid bacteria may play some specific role for rapid oxidation of substrates under acidic growth conditions.

Tea polyphenols alleviate high fat and high glucose-induced endothelial hyperpermeability by attenuating ROS production via NADPH oxidase pathway.

PubMed

Zuo, Xuezhi; Tian, Chong; Zhao, Nana; Ren, Weiye; Meng, Yi; Jin, Xin; Zhang, Ying; Ding, Shibin; Ying, Chenjiang; Ye, Xiaolei

2014-03-02

Hyperglycemia-induced endothelial hyperpermeability is crucial to cardiovascular disorders and macro-vascular complications in diabetes mellitus. The objective of this study is to investigate the effects of green tea polyphenols (GTPs) on endothelial hyperpermeability and the role of nicotinamide adenine dinucleotide phosphate (NADPH) pathway. Male Wistar rats fed on a high fat diet (HF) were treated with GTPs (0, 0.8, 1.6, 3.2 g/L in drinking water) for 26 weeks. Bovine aortic endothelial cells (BAECs) were treated with high glucose (HG, 33 mmol/L) and GTPs (0.0, 0.4, or 4 μg/mL) for 24 hours in vitro. The endothelial permeabilities in rat aorta and monolayer BAECs were measured by Evans blue injection method and efflux of fluorescein isothiocyanate (FITC)-dextran, respectively. The reactive oxygen species (ROS) levels in rat aorta and monolayer BAECs were measured by dihydroethidium (DHE) and 2', 7'-dichloro-fluorescein diacetate (DCFH-DA) fluorescent probe, respectively. Protein levels of NADPH oxidase subunits were determined by Western-blot. HF diet-fed increased the endothelial permeability and ROS levels in rat aorta while HG treatments increased the endothelial permeability and ROS levels in cultured BAECs. Co-treatment with GTPs alleviated those changes both in vivo and in vitro. In in vitro studies, GTPs treatments protected against the HG-induced over-expressions of p22phox and p67phox. Diphenylene iodonium chloride (DPI), an inhibitor of NADPH oxidase, alleviated the hyperpermeability induced by HG. GTPs could alleviate endothelial hyperpermeabilities in HF diet-fed rat aorta and in HG treated BAECs. The decrease of ROS production resulting from down-regulation of NADPH oxidase contributed to the alleviation of endothelial hyperpermeability.

Determination of Monoamine Oxidase A and B Activity in Long-Term Treated Patients With Parkinson Disease.

PubMed

Müller, Thomas; Riederer, Peter; Grünblatt, Edna

Biogenic amines and monoamine oxidase inhibitors influence peripheral monoamine oxidase enzyme activity in chronic levodopa/dopa decarboxylase inhibitor-treated patients with Parkinson disease. Rasagiline is an irreversible inhibitor of monoamine oxidase B. Safinamide blocks this isoenzyme in a reversible fashion. The aim of this study was to determine monoamine oxidase A (plasma) and B (platelets) enzyme activity in long-term levodopa-treated patients without and with additional oral intake of 50- or 100-mg safinamide or 1-mg rasagiline or first-time intake of rasagiline. Monoamine oxidase A enzyme activity did not differ between all groups. Patients on rasagiline or safinamide showed lower monoamine oxidase-B enzyme activity compared with patients without monoamine oxidase B inhibitor intake. No impact of the number of previous oral levodopa intakes was found. Rasagiline and safinamide did not essentially differ in terms of inhibition of monoamine oxidase B despite their different pharmacology regarding reversibility of monoamine oxidase B inhibition. In view of the observed, considerable heterogeneity of enzyme activities, we suggest to determine activities of monoamine oxidase A and B to reduce the risk for tyramine-induced hypertension and the serotonergic syndrome during chronic therapy with rasagiline or safinamide.

Platinum Nanoparticles: Efficient and Stable Catechol Oxidase Mimetics.

PubMed

Liu, Yi; Wu, Haohao; Chong, Yu; Wamer, Wayne G; Xia, Qingsu; Cai, Lining; Nie, Zhihong; Fu, Peter P; Yin, Jun-Jie

2015-09-09

Although enzyme-like nanomaterials have been extensively investigated over the past decade, most research has focused on the peroxidase-like, catalase-like, or SOD-like activity of these nanomaterials. Identifying nanomaterials having oxidase-like activities has received less attention. In this study, we demonstrate that platinum nanoparticles (Pt NPs) exhibit catechol oxidase-like activity, oxidizing polyphenols into the corresponding o-quinones. Four unique approaches are employed to demonstrate the catechol oxidase-like activity exerted by Pt NPs. First, UV-vis spectroscopy is used to monitor the oxidation of polyphenols catalyzed by Pt NPs. Second, the oxidized products of polyphenols are identified by ultrahigh-performance liquid chromatography (UHPLC) separation followed by high-resolution mass spectrometry (HRMS) identification. Third, electron spin resonance (ESR) oximetry techniques are used to confirm the O2 consumption during the oxidation reaction. Fourth, the intermediate products of semiquinone radicals formed during the oxidation of polyphenols are determined by ESR using spin stabilization. These results indicate Pt NPs possess catechol oxidase-like activity. Because polyphenols and related bioactive substances have been explored as potent antioxidants that could be useful for the prevention of cancer and cardiovascular diseases, and Pt NPs have been widely used in the chemical industry and medical science, it is essential to understand the potential effects of Pt NPs for altering or influencing the antioxidant activity of polyphenols.

Bienzyme biosensors for glucose, ethanol and putrescine built on oxidase and sweet potato peroxidase.

PubMed

Castillo, Jaime; Gáspár, Szilveszter; Sakharov, Ivan; Csöregi, Elisabeth

2003-05-01

Amperometric biosensors for glucose, ethanol, and biogenic amines (putrescine) were constructed using oxidase/peroxidase bienzyme systems. The H(2)O(2) produced by the oxidase in reaction with its substrate is converted into a measurable signal via a novel peroxidase purified from sweet potato peels. All developed biosensors are based on redox hydrogels formed of oxidases (glucose oxidase, alcohol oxidase, or amine oxidase) and the newly purified sweet potato peroxidase (SPP) cross-linked to a redox polymer. The developed electrodes were characterized (sensitivity, stability, and performances in organic medium) and compared with similarly built ones using the 'classical' horseradish peroxidase (HRP). The SPP-based electrodes displayed higher sensitivity and better detection limit for putrescine than those using HRP and were also shown to retain their activity in organic phase much better than the HPR based ones. The importance of attractive or repulsive electrostatic interactions between the peroxidases and oxidases (determined by their isoelectric points) were found to play an important role in the sensitivity of the obtained sensors.

The increasing role of monoamine oxidase type B inhibitors in Parkinson's disease therapy.

PubMed

Elmer, Lawrence W; Bertoni, John M

2008-11-01

The role of monoamine oxidase type B inhibitors in the treatment of Parkinson's disease has expanded with the new monoamine oxidase B inhibitor rasagiline and a new formulation, selegiline oral disintegrating tablets. As primary therapy in early disease monoamine oxidase B inhibitors reduce motor disability and delay the need for levodopa. In more advanced disease requiring levodopa, adjunctive monoamine oxidase B inhibitors reduce 'off' time and may improve gait and freezing. Rasagiline and selegiline oral disintegrating tablets may reduce the safety risks associated with the amfetamine and methamfetamine metabolites of conventional oral selegiline while retaining or improving therapeutic efficacy. Articles were identified by searches of PubMed and searches on the Internet and reviewed. All articles and other referenced materials were retrieved using the keywords 'Parkinson's disease', 'treatment' and 'monoamine oxidase B inhibitor' and were published between 1960 and 2007, with older references selected for historical significance. Only papers published in English were reviewed. Accumulating data support the use of monoamine oxidase B inhibitors as monotherapy for early and mild Parkinson's disease and as adjunctive therapy for more advanced Parkinson's disease with levodopa-associated motor fluctuations. The recently released monoamine oxidase B inhibitor rasagiline and a new formulation, selegiline oral disintegrating tablets, have potential advantages over conventional oral selegiline.

Photoaffinity labeling of protoporphyrinogen oxidase, the molecular target of diphenylether-type herbicides.

PubMed

Camadro, J M; Matringe, M; Thome, F; Brouillet, N; Mornet, R; Labbe, P

1995-05-01

Diphenylether-type herbicides are extremely potent inhibitors of protoporphyrinogen oxidase, a membrane-bound enzyme involved in the heme and chlorophyll biosynthesis pathways. Tritiated acifluorfen and a diazoketone derivative of tritiated acifluorfen were specifically bound to a single class of high-affinity binding sites on yeast mitochondrial membranes with apparent dissociation constants of 7 nM and 12.5 nM, respectively. The maximum density of specific binding sites, determined by Scatchard analysis, was 3 pmol.mg-1 protein. Protoporphyrinogen oxidase specific activity was estimated to be 2500 nmol protoporphyrinogen oxidized h-1.mol-1 enzyme. The diazoketone derivative of tritiated acifluorfen was used to specifically photolabel yeast protoporphyrinogen oxidase. The specifically labeled polypeptide in wild-type mitochondrial membranes had an apparent molecular mass of 55 kDa, identical to the molecular mass of the purified enzyme. This photolabeled polypeptide was not detected in a protoporphyrinogen-oxidase-deficient yeast strain, but the membranes contained an equivalent amount of inactive immunoreactive protoporphyrinogen oxidase protein.

Amine oxidase from lentil seedlings: energetic domains and effect of temperature on activity.

PubMed

Moosavi-Nejad, S Z; Rezaei-Tavirani, M; Padiglia, A; Floris, G; Moosavi-Movahedi, A A

2001-07-01

Copper/TPQ amine oxidases from mammalian and plant sources have shown many differences in substrate specificity and molecular properties. In this work the activity of lentil seedling amine oxidase was followed at various temperatures in 100 mM potassium phosphate buffer, pH 7, using benzylamine as substrate. The discontinuous Arrhenius plot of lentil amine oxidase showed two distinct phases with a jump between them. Thermal denaturation of the enzyme, using differential scanning calorimetry under the same experimental conditions, showed a transition at the same temperature ranges in the absence of substrate, indicating the occurrence of conformational changes, with an enthalpy change of about 175.9 kJ/mole. The temperature-induced changes of the activity of lentil amine oxidase are compared with those of bovine serum amine oxidase (taken from the literature).

Purification of the Alpha Glycerophosphate Oxidase from African Trypanosomes

DTIC Science & Technology

1987-02-02

oxidase (GPO). This enzyme has not been purified or characterize in detail. Inhibition of this enzyme coupled with inhibition of the anaerobic...more manageable afterwards and remained in the procedure although it only slightly increased the yield. The stability of the solubilized enzyme was...whether the detergent was added during the assay or in the solubilization procedure. However, the successful assay for the enzyme was ubiquinol oxidase

Vascular lysyl oxidase over-expression alters extracellular matrix structure and induces oxidative stress.

PubMed

Varona, Saray; García-Redondo, Ana B; Martínez-González, Jose; Salaices, Mercedes; Briones, Ana M; Rodríguez, Cristina

Lysyl oxidase (LOX) participates in the assembly of collagen and elastin fibres. The impact of vascular LOX over-expression on extracellular matrix (ECM) structure and its contribution to oxidative stress has been analysed. Studies were conducted on mice over-expressing LOX (Tg), specifically in smooth muscle cells (VSMC). Gene expression was assessed by real-time PCR analysis. Sirius Red staining, H 2 O 2 production and NADPH oxidase activity were analysed in different vascular beds. The size and number of fenestra of the internal elastic lamina were determined by confocal microscopy. LOX activity was up-regulated in VSMC of transgenic mice compared with cells from control animals. At the same time, transgenic cells deposited more organised elastin fibres and their supernatants induced a stronger collagen assembly in in vitro assays. Vascular collagen cross-linking was also higher in Tg mice, which showed a decrease in the size of fenestrae and an enhanced expression of Fibulin-5. Interestingly, higher H 2 O 2 production and NADPH oxidase activity was detected in the vascular wall from transgenic mice. The H 2 O 2 scavenger catalase attenuated the stronger deposition of mature elastin fibres induced by LOX transgenesis. LOX over-expression in VSMC was associated with a change in the structure of collagen and elastin fibres. LOX could constitute a novel source of oxidative stress that might participate in elastin changes and contribute to vascular remodelling. Copyright © 2017 Sociedad Española de Arteriosclerosis. Publicado por Elsevier España, S.L.U. All rights reserved.

NADPH oxidase mediates depressive behavior induced by chronic stress in mice.

PubMed

Seo, Ji-Seon; Park, Jin-Young; Choi, Juli; Kim, Tae-Kyung; Shin, Joo-Hyun; Lee, Ja-Kyeong; Han, Pyung-Lim

2012-07-11

Stress is a potent risk factor for depression, yet the underlying mechanism is not clearly understood. In the present study, we explored the mechanism of development and maintenance of depression in a stress-induced animal model. Mice restrained for 2 h daily for 14 d showed distinct depressive behavior, and the altered behavior persisted for >3 months in the absence of intervention. Acute restraint induced a surge of oxidative stress in the brain, and stress-induced oxidative stress progressively increased with repetition of stress. In vitro, the stress hormone glucocorticoid generated superoxide via upregulation of NADPH oxidase. Consistently, repeated restraints increased the expression of the key subunits of NADPH oxidase, p47phox and p67phox, in the brain. Moreover, stressed brains markedly upregulated the expression of p47phox to weak restress evoked in the poststress period, and this molecular response was reminiscent of amplified ROS surge to restress. Pharmacological inhibition of NADPH oxidase by the NADPH oxidase inhibitor apocynin during the stress or poststress period completely blocked depressive behavior. Consistently, heterozygous p47phox knock-out mice (p47phox(+/-)) or molecular inhibition of p47phox with Lenti shRNA-p47phox in the hippocampus suppressed depressive behavior. These results suggest that repeated stress promotes depressive behavior through the upregulation of NADPH oxidase and the resultant metabolic oxidative stress, and that the inhibition of NADPH oxidase provides beneficial antidepression effects.

Direct Identification of a Bacterial Manganese(II) Oxidase, the Multicopper Oxidase MnxG, from Spores of Several Different Marine Bacillus Species▿ †

PubMed Central

Dick, Gregory J.; Torpey, Justin W.; Beveridge, Terry J.; Tebo, Bradley M.

2008-01-01

Microorganisms catalyze the formation of naturally occurring Mn oxides, but little is known about the biochemical mechanisms of this important biogeochemical process. We used tandem mass spectrometry to directly analyze the Mn(II)-oxidizing enzyme from marine Bacillus spores, identified as an Mn oxide band with an in-gel activity assay. Nine distinct peptides recovered from the Mn oxide band of two Bacillus species were unique to the multicopper oxidase MnxG, and one peptide was from the small hydrophobic protein MnxF. No other proteins were detected in the Mn oxide band, indicating that MnxG (or a MnxF/G complex) directly catalyzes biogenic Mn oxide formation. The Mn(II) oxidase was partially purified and found to be resistant to many proteases and active even at high concentrations of sodium dodecyl sulfate. Comparative analysis of the genes involved in Mn(II) oxidation from three diverse Bacillus species revealed a complement of conserved Cu-binding regions not present in well-characterized multicopper oxidases. Our results provide the first direct identification of a bacterial enzyme that catalyzes Mn(II) oxidation and suggest that MnxG catalyzes two sequential one-electron oxidations from Mn(II) to Mn(III) and from Mn(III) to Mn(IV), a novel type of reaction for a multicopper oxidase. PMID:18165363

Highly sensitive luminol electrochemiluminescence immunosensor based on ZnO nanoparticles and glucose oxidase decorated graphene for cancer biomarker detection.

PubMed

Cheng, Yinfeng; Yuan, Ruo; Chai, Yaqin; Niu, Huan; Cao, Yaling; Liu, Huijing; Bai, Lijuan; Yuan, Yali

2012-10-01

In this work, we reported a sandwiched luminol electrochemiluminescence (ECL) immunosensor using ZnO nanoparticles (ZnONPs) and glucose oxidase (GOD) decorated graphene as labels and in situ generated hydrogen peroxide as coreactant. In order to construct the base of the immunosensor, a hybrid architecture of Au nanoparticles and graphene by reduction of HAuCl(4) and graphene oxide (GO) with ascorbic acid was prepared. The resulted hybrid architecture modified electrode provided an excellent platform for immobilization of antibody with good bioactivity and stability. Then, ZnONPs and GOD functionalized graphene labeled secondary antibody was designed for fabricating a novel sandwiched ECL immunosensor. Enhanced sensitivity was obtained by in situ generating hydrogen peroxide with glucose oxidase and the catalysis of ZnONPs to the ECL reaction of luminol-H(2)O(2) system. The as-prepared ECL immunosensor exhibited excellent analytical property for the detection of carcinoembryonic antigen (CEA) in the range from 10 pg mL(-1) to 80 ng mL(-1) and with a detection limit of 3.3 pg mL(-1) (SN(-1)=3). The amplification strategy performed good promise for clinical application of screening of cancer biomarkers. Copyright © 2012 Elsevier B.V. All rights reserved.

The voltage dependence of NADPH oxidase reveals why phagocytes need proton channels

NASA Astrophysics Data System (ADS)

DeCoursey, Thomas E.; Morgan, Deri; Cherny, Vladimir V.

2003-04-01

The enzyme NADPH oxidase in phagocytes is important in the body's defence against microbes: it produces superoxide anions (O2-, precursors to bactericidal reactive oxygen species). Electrons move from intracellular NADPH, across a chain comprising FAD (flavin adenine dinucleotide) and two haems, to reduce extracellular O2 to O2-. NADPH oxidase is electrogenic, generating electron current (Ie) that is measurable under voltage-clamp conditions. Here we report the complete current-voltage relationship of NADPH oxidase, the first such measurement of a plasma membrane electron transporter. We find that Ie is voltage-independent from -100mV to >0mV, but is steeply inhibited by further depolarization, and is abolished at about +190mV. It was proposed that H+ efflux mediated by voltage-gated proton channels compensates Ie, because Zn2+ and Cd2+ inhibit both H+ currents and O2- production. Here we show that COS-7 cells transfected with four NADPH oxidase components, but lacking H+ channels, produce O2- in the presence of Zn2+ concentrations that inhibit O2- production in neutrophils and eosinophils. Zn2+ does not inhibit NADPH oxidase directly, but through effects on H+ channels. H+ channels optimize NADPH oxidase function by preventing membrane depolarization to inhibitory voltages.

Nucleic Acid Homologies Among Oxidase-Negative Moraxella Species

PubMed Central

Johnson, John L.; Anderson, Robert S.; Ordal, Erling J.

1970-01-01

The deoxyribonucleic acid (DNA) base composition and DNA homologies of more than 40 strains of oxidase-negative Moraxella species were determined. These bacteria have also been identified as belonging to the Mima-Herellea-Acinetobacter group and the Bacterium anitratum group, as well as to several other genera including Achromobacter and Alcaligenes. The DNA base content of these strains ranged from 40 to 46% guanine plus cytosine. DNA–DNA competition experiments distinguished five groups whose members were determined by showing 50% or more homology to one of the reference strains: B. anitratum type B5W, Achromobacter haemolyticus var. haemolyticus, Alcaligenes haemolysans, Achromobacter metalcaligenes, and Moraxella lwoffi. A sixth group comprised those strains showing less than 50% homology to any of the reference strains. Negligible homology was found between strains of oxidase-negative and oxidase-positive Moraxella species in DNA–DNA competition experiments. However, evidence of a distant relationship between the two groups was obtained in competition experiments by using ribosomal ribonucleic acid. PMID:5413826

Biochemical, biological and molecular characterization of an L-Amino acid oxidase (LAAO) purified from Bothrops pictus Peruvian snake venom.

PubMed

Lazo, Fanny; Vivas-Ruiz, Dan E; Sandoval, Gustavo A; Rodríguez, Edith F; Kozlova, Edgar E G; Costal-Oliveira, F; Chávez-Olórtegui, Carlos; Severino, Ruperto; Yarlequé, Armando; Sanchez, Eladio F

2017-12-01

An L-amino acid oxidase from Peruvian Bothrops pictus (Bpic-LAAO) snake venom was purified using a combination of size-exclusion and ion-exchange chromatography. Bpic-LAAO is a homodimeric glycosylated flavoprotein with molecular mass of ∼65 kDa under reducing conditions and ∼132 kDa in its native form as analyzed by SDS-PAGE and gel filtration chromatography, respectively. N-terminal amino acid sequencing showed highly conserved residues in a glutamine-rich motif related to binding substrate. The enzyme exhibited optimal activity towards L-Leu at pH 8.5, and like other reported SV-LAAOs, it is stable until 55 °C. Kinetic studies showed that the cations Ca 2+ , Mg 2+ and Mn 2+ did not alter Bpic-LAAO activity; however, Zn 2+ is an inhibitor. Some reagents such as β-mercaptoethanol, glutathione and iodoacetate had inhibitory effect on Bpic-LAAO activity, but PMSF, EDTA and glutamic acid did not affect its activity. Regarding the biological activities of Bpic-LAAO, this enzyme induced edema in mice (MED = 7.8 μg), and inhibited human platelet aggregation induced by ADP in a dose-dependent manner and showed antibacterial activity on Gram (+) and Gram (-) bacteria. Bpic-LAAO cDNA of 1494 bp codified a mature protein with 487 amino acid residues comprising a signal peptide of 11 amino acids. Finally, the phylogenetic tree obtained with other sequences of LAAOs, evidenced its similarity to other homologous enzymes, showing two well-established monophyletic groups in Viperidae and Elapidae families. Bpic-LAAO is evolutively close related to LAAOs from B. jararacussu, B. moojeni and B. atrox, and together with the LAAO from B. pauloensis, form a well-defined cluster of the Bothrops genus. Copyright © 2017 Elsevier Ltd. All rights reserved.

A novel domain of amino-Nogo-A protects HT22 cells exposed to oxygen glucose deprivation by inhibiting NADPH oxidase activity.

PubMed

Guo, Fan; Wang, Huiwen; Li, Liya; Zhou, Heng; Wei, Haidong; Jin, Weilin; Wang, Qiang; Xiong, Lize

2013-04-01

This study aimed to investigate the protective effect of the M9 region (residues 290-562) of amino-Nogo-A fused to the human immunodeficiency virus trans-activator TAT in an in vitro model of ischemia-reperfusion induced by oxygen-glucose deprivation (OGD) in HT22 hippocampal neurons, and to investigate the role of NADPH oxidase in this protection. Transduction of TAT-M9 was analyzed by immunofluorescence staining and western blot. The biologic activity of TAT-M9 was assessed by its effects against OGD-induced HT22 cell damage, compared with a mutant M9 fusion protein or vehicle. Cellular viability and lactate dehydrogenase (LDH) release were assessed. Neuronal apoptosis was evaluated by flow cytometry. The Bax/Bcl-2 ratio was determined by western blotting. Reactive oxygen species (ROS) levels and NADPH oxidase activity were also measured in the presence or absence of an inhibitor or activator of NADPH oxidase. Our results confirmed the delivery of the protein into HT22 cells by immunofluorescence and western blot. Addition of 0.4 μmol/L TAT-M9 to the culture medium effectively improved neuronal cell viability and reduced LDH release induced by OGD. The fusion protein also protected HT22 cells from apoptosis, suppressed overexpression of Bax, and inhibited the reduction in Bcl-2 expression. Furthermore, TAT-M9, as well as apocynin, decreased NADPH oxidase activity and ROS content. The protective effects of the TAT-M9 were reversed by TBCA, an agonist of NADPH oxidase. In conclusion, TAT-M9 could be successfully transduced into HT22 cells, and protected HT22 cells against OGD damage by inhibiting NADPH oxidase-mediated oxidative stress. These findings suggest that the TAT-M9 protein may be an efficient therapeutic agent for neuroprotection.

Oral administration of L-arginine in patients with angina or following myocardial infarction may be protective by increasing plasma superoxide dismutase and total thiols with reduction in serum cholesterol and xanthine oxidase

PubMed Central

Tripathi, Pratima; Chandra, M

2009-01-01

Administration of L-arginine has been shown to control ischemic injury by producing nitric oxide which dilates the vessels and thus maintains proper blood flow to the myocardium. In the present study attempt has been made to determine whether oral administration of L-arginine has any effect on oxidant/antioxidant homeostasis in ischemic myocardial patients [represented by the patients of acute angina (AA) and acute myocardial infarction (MI)]. L-arginine has antioxidant and antiapoptotic properties, decreases endothelin-1 expression and improves endothelial function, thereby controlling oxidative injury caused during myocardial ischemic syndrome. Effect of L-arginine administration on the status of free radical scavenging enzymes, pro-oxidant enzyme and antioxidants viz. total thiols, carbonyl content and plasma ascorbic acid levels in the patients has been evaluated. We have observed that L-arginine administration (three grams per day for 15 days) resulted in increased activity of free radical scavenging enzyme superoxide dismutase (SOD) and increase in the levels of total thiols (T-SH) and ascorbic acid with concomitant decrease in lipid per-oxidation, carbonyl content, serum cholesterol and the activity of proxidant enzyme, xanthine oxidase (XO). These findings suggest that the supplementation of L-arginine along with regular therapy may be beneficial to the patients of ischemic myocardial syndromes. PMID:20716909

[Activation of the alternative oxidase of Yarrowia lipolytica by adenosine 5'-monophosphate].

PubMed

Medentsev, A G; Arinbasarova, A Iu; Smirnova, N M; Akimenko, V K

2004-01-01

The study of the effect of nucleoside phosphates on the activity of cyanide-resistant oxidase in the mitochondria and the submitochondrial particles of Yarrowia lipolytica showed that adenosine monophosphate (5'-AMP, AMP) did not stimulate the respiration of the intact mitochondria. The incubation of the mitochondria at room temperature (25 degrees C) for 3-5 h or their treatment with ultrasound, phospholipase A, and detergent Triton X-100 at a low temperature inactivated the cyanide-resistant alternative oxidase. The inactivated alternative oxidase could be reactivated by AMP. The reactivating effect of AMP was enhanced by azolectin. Some other nucleoside phosphates also showed reactivating ability in the following descending order. AMP = GMP > GDP > GTP > XMP > IMP. The apparent reaction rate constant Km for AMP upon the reactivation of the alternative oxidase of mitochondria treated with Triton X-100 or incubated at 25 degrees C was 12.5 and 20 microM, respectively. The Km for AMP upon the reactivation of the alternative oxidase of submitochondrial particles was 15 microM. During the incubation of yeast cells under conditions promoting the development of alternative oxidase, the content of adenine nucleotides (AMP, ADP, and ATP) in the cells and their respiration tended to decrease. The subsequent addition of cyanide to the cells activated their respiration, diminished the intracellular content of ATP three times, and augmented the content of AMP five times. These data suggest that the stimulation of cell respiration by cyanide may be due to the activation of alternative oxidase by AMP.

Isolation of a polyphenol oxidase (PPO) cDNA from artichoke and expression analysis in wounded artichoke heads.

PubMed

Quarta, Angela; Mita, Giovanni; Durante, Miriana; Arlorio, Marco; De Paolis, Angelo

2013-07-01

The polyphenol oxidase (PPO) enzyme, which can catalyze the oxidation of phenolics to quinones, has been reported to be involved in undesirable browning in many plant foods. This phenomenon is particularly severe in artichoke heads wounded during the manufacturing process. A full-length cDNA encoding for a putative polyphenol oxidase (designated as CsPPO) along with a 1432 bp sequence upstream of the starting ATG codon was characterized for the first time from [Cynara cardunculus var. scolymus (L.) Fiori]. The 1764 bp CsPPO sequence encodes a putative protein of 587 amino acids with a calculated molecular mass of 65,327 Da and an isoelectric point of 5.50. Analysis of the promoter region revealed the presence of cis-acting elements, some of which are putatively involved in the response to light and wounds. Expression analysis of the gene in wounded capitula indicated that CsPPO was significantly induced after 48 h, even though the browning process had started earlier. This suggests that the early browning event observed in artichoke heads was not directly related to de novo mRNA synthesis. Finally, we provide the complete gene sequence encoding for polyphenol oxidase and the upstream regulative region in artichoke. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Systemic Manifestations in Pyridox(am)ine 5'-Phosphate Oxidase Deficiency.

PubMed

Guerriero, Réjean M; Patel, Archana A; Walsh, Brian; Baumer, Fiona M; Shah, Ankoor S; Peters, Jurriaan M; Rodan, Lance H; Agrawal, Pankaj B; Pearl, Phillip L; Takeoka, Masanori

2017-11-01

Pyridoxine is converted to its biologically active form pyridoxal-5-phosphate (P5P) by the enzyme pyridox(am)ine 5'-phosphate oxidase and serves as a cofactor in nearly 200 reactions in the central nervous system. Pyridox(am)ine 5'-phosphate oxidase deficiency leads to P5P dependent epilepsy, typically a neonatal- or infantile-onset epileptic encephalopathy treatable with P5P or in some cases, pyridoxine. Following identification of retinopathy in a patient with pyridox(am)ine 5'-phosphate oxidase deficiency that was reversible with P5P therapy, we describe the systemic manifestations of pyridox(am)ine 5'-phosphate oxidase deficiency. A series of six patients with homozygous mutations of PNPO, the gene coding pyridox(am)ine 5'-phosphate oxidase, were evaluated in our center over the course of two years for phenotyping of neurological and systemic manifestations. Five of six were born prematurely, three had anemia and failure to thrive, and two had elevated alkaline phosphatase. A movement disorder was observed in two children, and a reversible retinopathy was observed in the most severely affected infant. All patients had neonatal-onset epilepsy and were on a continuum of developmental delay to profound encephalopathy. Electroencephalographic features included background slowing and disorganization, absent sleep features, and multifocal and generalized epileptiform discharges. All the affected probands carried a homozygous PNPO mutation (c.674 G>T, c.686 G>A and c.352G>A). In addition to the well-described epileptic encephalopathy, pyridox(am)ine 5'-phosphate oxidase deficiency causes a range of neurological and systemic manifestations. A movement disorder, developmental delay, and encephalopathy, as well as retinopathy, anemia, and failure to thrive add to the broadening clinical spectrum of P5P dependent epilepsy. Copyright © 2017 Elsevier Inc. All rights reserved.

Xanthine Oxidase Induces Foam Cell Formation through LOX-1 and NLRP3 Activation.

PubMed

Dai, Yao; Cao, Yongxiang; Zhang, Zhigao; Vallurupalli, Srikanth; Mehta, Jawahar L

2017-02-01

Xanthine oxidase catalyzes the oxidation of xanthine to uric acid. This process generates excessive reactive oxygen species (ROS) that play an important role in atherogenesis. Recent studies show that LRR and PYD domains-containing protein 3 (NLRP3), a component of the inflammasome, may be involved in the formation of foam cells, a hallmark of atherosclerosis. This study was designed to study the role of various scavenger receptors and NLRP3 inflammasome in xanthine oxidase and uric acid-induced foam cell formation. Human vascular smooth muscle cells (VSMCs) and THP-1 macrophages were treated with xanthine oxidase or uric acid. Xanthine oxidase treatment (of both VSMCs and THP-1 cells) resulted in foam cell formation in concert with generation of ROS and expression of cluster of differentiation 36 (CD36) and oxidized low density lipoprotein (lectin-like) receptor 1 (LOX-1), but not of scavenger receptor A (SRA). Uric acid treatment resulted in foam cell formation, ROS generation and expression of CD36, but not of LOX-1 or SRA. Further, treatment of cells with xanthine oxidase, but not uric acid, activated NLRP3 and its downstream pro-inflammatory signals- caspase-1, interleukin (IL)-1β and IL-18. Blockade of LOX-1 or NLRP3 inflammasome with specific siRNAs reduced xanthine oxidase-induced foam cell formation, ROS generation and activation of NLRP3 and downstream signals. Xanthine oxidase induces foam cell formation in large part through activation of LOX-1 - NLRP3 pathway in both VSMCs and THP-1 cells, but uric acid-induced foam cell formation is exclusively through CD36 pathway. Further, LOX-1 activation is upstream of NLRP3 activation. Graphical Abstract Steps in the formation of foam cells in response to xanthine oxidase and uric acid. Xanthine oxidase stimulates LOX-1 expression on the cell membrane of macrophages and vascular smooth muscle cells (VSMCs) and increases generation of ROS, which activate NLRP3 inflammasome and downstream pro

Protein structural development of threadfin bream ( Nemipterus spp.) surimi gels induced by glucose oxidase.

PubMed

Wang, Lei; Fan, Daming; Fu, Lulu; Jiao, Xidong; Huang, Jianlian; Zhao, Jianxin; Yan, Bowen; Zhou, Wenguo; Zhang, Wenhai; Ye, Weijian; Zhang, Hao

2018-01-01

This study investigated the effect of glucose oxidase on the gel properties of threadfin bream surimi. The gel strength of surimi increased with the addition of 0.5‰ glucose oxidase after two-step heating. Based on the results of the chemical interactions, the hydrophobic interaction and disulfide bond of glucose oxidase-treated surimi samples increased compared with the control samples at the gelation temperature and gel modori temperature. The surface hydrophobicity of samples with glucose oxidase and glucose increased significantly ( p < 0.05) and total sulfhydryl groups decreased significantly ( p < 0.05). The analysis of Raman spectroscopy shows that the addition of glucose oxidase induced more α-helixes to turn into a more elongated random and flocculent structure. Glucose oxidase changes the secondary structure of the surimi protein, making more proteins depolarize and stretch and causing actomyosin to accumulate to each other, resulting in the formation of surimi gel.

Operational impact of using a vanadate oxidase method for direct bilirubin measurements at an academic medical center clinical laboratory.

PubMed

Dhungana, Neha; Morris, Cory; Krasowski, Matthew D

2017-08-01

The aim of this study was to compare the operational impact of using vanadate oxidase versus diazo direct bilirubin assays for an academic medical center patient population. Retrospective study was done over an approximately 3.5 year period. The main automated chemistry instrumentation was a Roche Diagnostics cobas 8000 line. The Roche Direct Bilirubin assay was compared to Diazyme Laboratories Direct Bilirubin Assay and Randox Laboratories Direct Bilirubin assay using manufacturer's guidelines for hemolysis index, lipemia index, and analytical measurement range (AMR). Retrospective data was analyzed for 47,333 serum/plasma specimens that had clinical orders for direct bilirubin. A total of 5943 specimens (12.6%) exceeded the hemolysis index limit for the Roche method compared to only 0.2% and 0.05% of specimens for the Diazyme and Randox methods, respectively. The impact was particularly large on patients less than 2 years old, for which 51.3% of specimens exceeded the hemolysis index for the Roche method. A total of 1671 specimens (3.5%) exceeded the lipemia index limit for the Roche method compared to less than 0.1% for the Randox method. Lastly, 988 (2.1%) of specimens had direct bilirubin concentrations exceeding the upper AMR limit of 10 mg/dL [171 µmol/L] for the Roche assay compared to less than 1% of specimens for the vanadate oxidase methods. Vanadate oxidase direct bilirubin methods offer advantages over diazo methods in terms of less interference by hemolysis and lipemia, as well as wider AMR. The advantages are particularly evident for neonatal and infant populations.

NADPH Oxidase as a Therapeutic Target for Oxalate Induced Injury in Kidneys

PubMed Central

Peck, Ammon B.; Khan, Saeed R.

2013-01-01

A major role of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family of enzymes is to catalyze the production of superoxides and other reactive oxygen species (ROS). These ROS, in turn, play a key role as messengers in cell signal transduction and cell cycling, but when they are produced in excess they can lead to oxidative stress (OS). Oxidative stress in the kidneys is now considered a major cause of renal injury and inflammation, giving rise to a variety of pathological disorders. In this review, we discuss the putative role of oxalate in producing oxidative stress via the production of reactive oxygen species by isoforms of NADPH oxidases expressed in different cellular locations of the kidneys. Most renal cells produce ROS, and recent data indicate a direct correlation between upregulated gene expressions of NADPH oxidase, ROS, and inflammation. Renal tissue expression of multiple NADPH oxidase isoforms most likely will impact the future use of different antioxidants and NADPH oxidase inhibitors to minimize OS and renal tissue injury in hyperoxaluria-induced kidney stone disease. PMID:23840917

[Experimental rationale for the parameters of a rapid method for oxidase activity determination].

PubMed

Butorina, N N

2010-01-01

Experimental rationale is provided for the parameters of a rapid (1-2-min) test to concurrently determine the oxidase activity of all bacteria grown on the membrane filter after water filtration. Oxidase reagents that are the aqueous solutions of tetramethyl-p-phenylenediamine dihydrochloride and demethyl-p-phenylenediamine dihydrochloride have been first ascertained to exert no effect on the viability and enzymatic activity of bacteria after one-hour contact. An algorithm has been improved for the rapid oxidase activity test: the allowable time for bacteria to contact oxidase reagents and procedures for minimizing the effect on bacterial biochemical activity following the contact. An accelerated method based on lactose medium with tergitol 7 and Endo agar has been devised to determine coliform bacteria, by applying the rapid oxidase test: the time of a final response is 18-24 hours. The method has been included into GOST 52426-2005.

Heterologous production and characterization of two glyoxal oxidases from Pycnoporus cinnabarinus

Treesearch

Marianne Daou; François Piumi; Daniel Cullen; Eric Record; Craig B. Faulds

2016-01-01

The genome of the white rot fungus Pycnoporus cinnabarinus includes a large number of genes encoding enzymes implicated in lignin degradation. Among these, three genes are predicted to encode glyoxal oxidase, an enzyme previously isolated from Phanerochaete chrysosporium. The glyoxal oxidase of P. chrysosporium...

Cloning and Analysis of the Alternative Oxidase Gene of Neurospora Crassa

PubMed Central

Li, Q.; Ritzel, R. G.; McLean, LLT.; McIntosh, L.; Ko, T.; Bertrand, H.; Nargang, F. E.

1996-01-01

Mitochondria of Neurospora crassa contain a cyanide-resistant alternative respiratory pathway in addition to the cytochrome pathway. The alternative oxidase is present only when electron flow through the cytochrome chain is restricted. Both genomic and cDNA copies for the alternative oxidase gene have been isolated and analyzed. The sequence of the predicted protein is homologous to that of other species. The mRNA for the alternative oxidase is scarce in wild-type cultures grown under normal conditions, but it is abundant in cultures grown in the presence of chloramphenicol, an inhibitor of mitochondrial protein synthesis, or in mutants deficient in mitochondrial cytochromes. Thus, induction of alternative oxidase appears to be at the transcriptional level. Restriction fragment length polymorphism mapping of the isolated gene demonstrated that it is located in a position corresponding to the aod-1 locus. Sequence analysis of mutant aod-1 alleles reveals mutations affecting the coding sequence of the alternative oxidase. The level of aod-1 mRNA in an aod-2 mutant strain that had been grown in the presence of chloramphenicol was reduced several fold relative to wild-type, supporting the hypothesis that the product of aod-2 is required for optimal expression of aod-1. PMID:8770590

Phospholipid alterations in cardiac sarcoplasmic reticulum induced by xanthine oxidase: contamination of commercial preparations of xanthine oxidase by phospholipase A/sub 2/

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gamache, D.A.; Kornberg, L.J.; Bartolf, M.

1986-05-01

Incubation of cardiac sarcoplasmic reticulum with xanthine oxidase alone at pH 7.0 resulted in a loss of lipid phosphorus that was potentiated by the addition of xanthine. Using autoclaved E.coli with 1-/sup 14/C-oleate in the 2-acyl position of membrane phospholipids, the authors demonstrate that many, but not all, commercial preparations of xanthine oxidase contain significant phospholipase A/sub 2/ (PLA/sub 2/) activity (64.3-545.6 nmols/min/mg). The PLA/sub 2/ was maximally active in the neutral-alkaline pH range, was Ca/sup 2 +/-dependent, and was unaffected by the addition of xanthine. PLA/sub 2/ activity was totally inhibited by 1mM EDTA whereas radical production by optimalmore » concentrations of xanthine/xanthine oxidase (X/XO) was unaffected by EDTA. Chromatographically purified xanthine oxidase (Sigma Grade III) contained high levels of PLA/sub 2/ activity (64.3 nmols/min/mg) compared to endogenous levels of neutral-active, Ca/sup 2 +/-dependent PLA/sub 2/ measured in various tissue homogenates (less than or equal to 0.5 nmols/ min/mg). Because X/XO mixtures are used extensively to study oxygen free radical-induced cell injury and membrane phospholipid alterations, the presence of a potent extracellular PLA/sub 2/ may have influenced previously published reports, and such studies should be interpreted cautiously.« less

A Biochemical Approach to Study the Role of the Terminal Oxidases in Aerobic Respiration in Shewanella oneidensis MR-1

PubMed Central

Le Laz, Sébastien; Kpebe, Arlette; Bauzan, Marielle; Lignon, Sabrina; Rousset, Marc; Brugna, Myriam

2014-01-01

The genome of the facultative anaerobic γ-proteobacterium Shewanella oneidensis MR-1 encodes for three terminal oxidases: a bd-type quinol oxidase and two heme-copper oxidases, a A-type cytochrome c oxidase and a cbb 3-type oxidase. In this study, we used a biochemical approach and directly measured oxidase activities coupled to mass-spectrometry analysis to investigate the physiological role of the three terminal oxidases under aerobic and microaerobic conditions. Our data revealed that the cbb 3-type oxidase is the major terminal oxidase under aerobic conditions while both cbb 3-type and bd-type oxidases are involved in respiration at low-O2 tensions. On the contrary, the low O2-affinity A-type cytochrome c oxidase was not detected in our experimental conditions even under aerobic conditions and would therefore not be required for aerobic respiration in S. oneidensis MR-1. In addition, the deduced amino acid sequence suggests that the A-type cytochrome c oxidase is a ccaa 3-type oxidase since an uncommon extra-C terminal domain contains two c-type heme binding motifs. The particularity of the aerobic respiratory pathway and the physiological implication of the presence of a ccaa 3-type oxidase in S. oneidensis MR-1 are discussed. PMID:24466040

Design, synthesis and molecular modeling of aloe-emodin derivatives as potent xanthine oxidase inhibitors.

PubMed

Shi, Da-Hua; Huang, Wei; Li, Chao; Liu, Yu-Wei; Wang, Shi-Fan

2014-03-21

A series of aloe-emodin derivatives were synthesized and evaluated as xanthine oxidase inhibitors. Among them, four aloe-emodin derivatives showed significant inhibitory activities against xanthine oxidase. The compound 4,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-carbaldehyde (A1) possessed the best xanthine oxidase inhibitory activity with IC50 of 2.79 μM. Lineweaver-Burk plot analysis revealed that A1 acted as a mixed-type inhibitor for xanthine oxidase. The docking study revealed that the molecule A1 had strong interactions with the active site of xanthine oxidase and this result was in agreement with kinetic study. Consequently, compound A1 is a new-type candidate for further development for the treatment of gout. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Lysyl Oxidase as a Serum Biomarker of Liver Fibrosis in Patients with Severe Obesity and Obstructive Sleep Apnea.

PubMed

Mesarwi, Omar A; Shin, Mi-Kyung; Drager, Luciano F; Bevans-Fonti, Shannon; Jun, Jonathan C; Putcha, Nirupama; Torbenson, Michael S; Pedrosa, Rodrigo P; Lorenzi-Filho, Geraldo; Steele, Kimberley E; Schweitzer, Michael A; Magnuson, Thomas H; Lidor, Anne O; Schwartz, Alan R; Polotsky, Vsevolod Y

2015-10-01

Obstructive sleep apnea (OSA) is associated with the progression of nonalcoholic fatty liver disease (NAFLD). We hypothesized that the hypoxia of OSA increases hepatic production of lysyl oxidase (LOX), an enzyme that cross-links collagen, and that LOX may serve as a biomarker of hepatic fibrosis. Thirty-five patients with severe obesity underwent liver biopsy, polysomnography, and serum LOX testing. A separate group with severe OSA had serum LOX measured before and after 3 mo of CPAP or no therapy, as did age-matched controls. LOX expression and secretion were measured in mouse hepatocytes following exposure to hypoxia. The Johns Hopkins Bayview Sleep Disorders Center, and the Hypertension Unit of the Heart Institute at the University of São Paulo Medical School. In the bariatric cohort, the apnea-hypopnea index was higher in patients with hepatic fibrosis than in those without fibrosis (42.7 ± 30.2 events/h, versus 16.2 ± 15.5 events/h; P = 0.002), as was serum LOX (84.64 ± 29.71 ng/mL, versus 45.46 ± 17.16 ng/mL; P < 0.001). In the sleep clinic sample, patients with severe OSA had higher baseline LOX than healthy controls (70.75 ng/mL versus 52.36 ng/mL, P = 0.046), and serum LOX decreased in patients with OSA on CPAP (mean decrease 20.49 ng/mL) but not in untreated patients (mean decrease 0.19 ng/mL). Hypoxic mouse hepatocytes demonstrated 5.9-fold increased LOX transcription (P = 0.046), and enhanced LOX protein secretion. The hypoxic stress of obstructive sleep apnea may increase circulating lysyl oxidase (LOX) levels. LOX may serve as a biomarker of liver fibrosis in patients with severe obesity and nonalcoholic fatty liver disease. © 2015 Associated Professional Sleep Societies, LLC.

Molecular mechanism of cell death induced by king cobra (Ophiophagus hannah) venom l-amino acid oxidase.

PubMed

Fung, Shin Yee; Lee, Mui Li; Tan, Nget Hong

2015-03-01

Snake venom LAAOs have been reported to exhibit a wide range of pharmacological activities, including cytotoxic, edema-inducing, platelet aggregation-inducing/platelet aggregation-inhibiting, bactericidal and antiviral activities. A heat-stable form of l-amino acid oxidase isolated from king cobra (Ophiophagus hannah) venom (OH-LAAO) has been shown to exhibit very potent cytotoxicity against human tumorigenic cells but not in their non-tumorigenic counterparts, and the cytotoxicity was due to the apoptosis-inducing effect of the enzyme. In this work, the molecular mechanism of cell death induced by OH-LAAO was investigated. The enzyme exerts its apoptosis-inducing effect presumably via both intrinsic and extrinsic pathways as suggested by the increase in caspase-8 and -9 activities. Oligonucleotide microarray analysis showed that the expression of a total of 178 genes was significantly altered as a result of oxidative stress induced by the hydrogen peroxide generated by the enzyme. Of the 178 genes, at least 27 genes are involved in apoptosis and cell death. These alterations of gene expression was presumably caused by the direct cytotoxic effect of H2O2 generated during the enzymatic reaction, as well as the non-specific oxidative modifications of signaling molecules that eventually lead to apoptosis and cell death. The very substantial up-regulation of cytochrome P450 genes may also contribute to the potent cytotoxic action of OH-LAAO by producing excessive reactive oxygen species (ROS). In conclusion, the potent apoptosis inducing activity of OH-LAAO was likely due to the direct cytotoxic effect of H2O2 generated during the enzymatic reaction, as well as the non-specific oxidation of signalling molecules. Copyright © 2015 Elsevier Ltd. All rights reserved.

Supplementary biochemical tests useful for the differentiation of oxidase positive staphylococci.

PubMed

Stepanović, Srdjan; Dakić, Ivana; Hauschild, Tomasz; Vuković, Dragana; Morrison, Donald; Jezek, Petr; Cirković, Ivana; Petrás, Petr

2007-06-01

Differentiation of the oxidase positive staphylococci, Staphylococcus sciuri, Staphylococcus lentus, Staphylococcus vitulinus and Staphylococcus fleurettii, based on tributyrin, urease, caseinase, gelatinase and DNase activity is described. These tests may be used for preliminary identification of oxidase positive isolates of staphylococci resulting in more accurate identification of these species.

NADPH oxidases: new kids on the block.

PubMed

Geiszt, Miklós

2006-07-15

Reactive oxygen species (ROS) play a pivotal role in many physiological processes including host defense, hormone biosynthesis, fertilization and cellular signaling. Altered production of ROS has been implicated in the development of immunodeficiency, hypothyroidism and cardiovascular pathologies. In the last few years, several enzymes were identified at the molecular level, which are now thought to be responsible for ROS production observed in diverse tissues. These enzymes show a high degree of homology to the phagocytic NADPH oxidase and are now designated the Nox family of NADPH oxidases. This review updates our knowledge on six new members of the Nox family: Nox1, Nox3, Nox4, Nox5, Duox1 and Duox2.

Monocyte and macrophage-targeted NADPH oxidase mediates antifungal host defense and regulation of acute inflammation in mice

PubMed Central

Grimm, Melissa J.; Vethanayagam, R. Robert; Almyroudis, Nikolaos G.; Dennis, Carly G.; Khan, A. Nazmul H.; D’Auria, Anthony; Singel, Kelly L.; Davidson, Bruce A.; Knight, Paul R.; Blackwell, Timothy S.; Hohl, Tobias M.; Mansour, Michael K.; Vyas, Jatin M.; Röhm, Marc; Urban, Constantin F.; Kelkka, Tiina; Holmdahl, Rikard; Segal, Brahm H.

2013-01-01

Chronic granulomatous disease, an inherited disorder of the NADPH oxidase in which phagocytes are defective in the generation of superoxide anion and downstream reactive oxidant species, is characterized by severe bacterial and fungal infections and excessive inflammation. Although NADPH oxidase isoforms exist in several lineages, reactive oxidant generation is greatest in neutrophils, where NADPH oxidase has been deemed vital for pathogen killing. In contrast, the function and importance of NADPH oxidase in macrophages are less clear. Therefore, we evaluated susceptibility to pulmonary aspergillosis in globally NADPH oxidase-deficient mice versus transgenic mice with monocyte/macrophage-targeted NADPH oxidase activity. We found that the lethal inoculum was more than 100-fold greater in transgenic versus globally NADPH oxidase-deficient mice. Consistent with these in vivo results, NADPH oxidase in mouse alveolar macrophages limited germination of phagocytosed Aspergillus fumigatus spores. Finally, globally NADPH oxidase-deficient mice developed exuberant neutrophilic lung inflammation and pro-inflammatory cytokine responses to zymosan, a fungal cell wall-derived product composed principally of particulate beta-glucans, whereas inflammation in transgenic and wildtype mice was mild and transient. Together, our studies identify a central role for monocyte/macrophage NADPH oxidase in controlling fungal infection and in limiting acute lung inflammation. PMID:23509361

Effect of mitoguazone on polyamine oxidase activity in rat liver.

PubMed

Ferioli, Maria Elena; Berselli, Debora; Caimi, Samuela

2004-12-01

Mitoguazone is a known inhibitor of polyamine biosynthesis through competitive inhibition of S-adenosylmethionine decarboxylase. A recent renewed interest in mitoguazone as an antineoplastic agent prompted us to investigate the effect of the drug on polyamine catabolism in rat liver, since the organ plays an important role in detoxification mechanisms. Thus, the purpose of this work was to evaluate the effect of in vivo mitoguazone administration on polyamine catabolic enzymes. In particular, our interest was directed to the changes in polyamine oxidase activity, since this enzyme has been recently confirmed to exert important functions that until now were underestimated. Mitoguazone administration induced hepatic polyamine oxidase activity starting at 4 h after administration, and the enzyme returned to basal levels 96 h after treatment. The changes in enzyme activity were accompanied by changes in putrescine concentrations, which increased starting at 4 h until 72 h after treatment. We also evaluated the activity of the newly identified spermine oxidase, which was not significantly changed by mitoguazone treatment. Therefore, we hypothesized that the enzyme involved in mitoguazone response of the liver is the polyamine oxidase, which acts on acetylated polyamines as substrate.

Oxidase-functionalized Fe(3)O(4) nanoparticles for fluorescence sensing of specific substrate.

PubMed

Liu, Cheng-Hao; Tseng, Wei-Lung

2011-10-03

This study reports the development of a reusable, single-step system for the detection of specific substrates using oxidase-functionalized Fe(3)O(4) nanoparticles (NPs) as a bienzyme system and using amplex ultrared (AU) as a fluorogenic substrate. In the presence of H(2)O(2), the reaction pH between Fe(3)O(4) NPs and AU was similar to the reaction of oxidase and the substrate. The catalytic activity of Fe(3)O(4) NPs with AU was nearly unchanged following modification with poly(diallyldimethylammonium chloride) (PDDA). Based on these features, we prepared a composite of PDDA-modified Fe(3)O(4) NPs and oxidase for the quantification of specific substrates through the H(2)O(2)-mediated oxidation of AU. By monitoring fluorescence intensity at 587 nm of oxidized AU, the minimum detectable concentrations of glucose, galactose, and choline were found to be 3, 2, and 20 μM using glucose oxidase-Fe(3)O(4), galactose oxidase-Fe(3)O(4), and choline oxidase-Fe(3)O(4) composites, respectively. The identification of glucose in blood was selected as the model to validate the applicability of this proposed method. Copyright © 2011 Elsevier B.V. All rights reserved.

A Mycobacterium tuberculosis Cytochrome bd Oxidase Mutant Is Hypersensitive to Bedaquiline

PubMed Central

Hartman, Travis E.

2014-01-01

ABSTRACT The new medicinal compound bedaquiline (BDQ) kills Mycobacterium tuberculosis by inhibiting F1Fo-ATP synthase. BDQ is bacteriostatic for 4 to 7 days and kills relatively slowly compared to other frontline tuberculosis (TB) drugs. Here we show that killing with BDQ can be improved significantly by inhibiting cytochrome bd oxidase, a non-proton-pumping terminal oxidase. BDQ was instantly bactericidal against a cytochrome bd oxidase null mutant of M. tuberculosis, and the rate of killing was increased by more than 50%. We propose that this exclusively bacterial enzyme should be a high-priority target for new drug discovery. PMID:25028424

Plasma amine oxidase activities in Norrie disease patients with an X-chromosomal deletion affecting monoamine oxidase.

PubMed

Murphy, D L; Sims, K B; Karoum, F; Garrick, N A; de la Chapelle, A; Sankila, E M; Norio, R; Breakefield, X O

1991-01-01

Two individuals with an X-chromosomal deletion were recently found to lack the genes encoding monoamine oxidase type A (MAO-A) and MAO-B. This abnormality was associated with almost total (90%) reductions in the oxidatively deaminated urinary metabolites of the MAO-A substrate, norepinephrine, and with marked (100-fold) increases in an MAO-B substrate, phenylethylamine, confirming systemic functional consequences of the genetic enzyme deficiency. However, urinary concentrations of the deaminated metabolites of dopamine and serotonin (5-HT) were essentially normal. To investigate other deaminating systems besides MAO-A and MAO-B that might produce these metabolites of dopamine and 5-HT, we examined plasma amine oxidase (AO) activity in these two patients and two additional patients with the same X-chromosomal deletion. Normal plasma AO activity was found in all four Norrie disease-deletion patients, in four patients with classic Norrie disease without a chromosomal deletion, and in family members of patients from both groups. Marked plasma amine metabolite abnormalities and essentially absent platelet MAO-B activity were found in all four Norrie disease-deletion patients, but in none of the other subjects in the two comparison groups. These results indicate that plasma AO is encoded by gene(s) independent of those for MAO-A and MAO-B, and raise the possibility that plasma AO, and perhaps the closely related tissue AO, benzylamine oxidase, as well as other atypical AOs or MAOs encoded independently from MAO-A and MAO-B may contribute to the oxidative deamination of dopamine and 5-HT in humans.

NADPH oxidase activation in neutrophils: Role of the Phosphorylation of its subunits.

PubMed

Belambri, Sahra A; Rolas, Loïc; Raad, Houssam; Hurtado-Nedelec, Margarita; Dang, Pham My-Chan; El-Benna, Jamel

2018-05-14

Neutrophils are key cells of innate immunity and during inflammation. Upon activation, they produce large amounts of superoxide anion (O 2 -. ) and ensuing reactive oxygen species (ROS) to kill phagocytized microbes. The enzyme responsible for O 2 -. production is called the phagocyte NADPH oxidase. This is a multicomponent enzyme system that becomes active after assembly of four cytosolic proteins (p47 phox , p67 phox , p40 phox and Rac2) with the transmembrane proteins (p22 phox and gp91 phox , which form the cytochrome b 558 ). gp91 phox represents the catalytic subunit of the NADPH oxidase and is also called NOX2. NADPH oxidase-derived ROS are essential for microbial killing and innate immunity; however, excessive ROS production induces tissue injury and prolonged inflammatory reactions that contribute to inflammatory diseases. Thus, NADPH oxidase activation must be tightly regulated in time and space in order to limit ROS production. NADPH oxidase activation is regulated by several processes such as phosphorylation of its components, exchange of GDP/GTP on Rac2 and binding of p47 phox and p40 phox to phospholipids. This review aims to provide new insights into the role of the phosphorylation of the NADPH oxidase components, i.e., gp91 phox , p22 phox , p47 phox , p67 phox and p40 phox , in the activation of this enzyme. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Safety assessment of bacterial choline oxidase protein introduced in transgenic crops for tolerance against abiotic stress.

PubMed

Singh, Abinav K; Singh, Bhanu P; Prasad, G B K S; Gaur, Shailendra N; Arora, Naveen

2008-12-24

Genetically modified crops have resistance to abiotic stress by introduction of choline oxidase protein. In the present study, the safety of choline oxidase protein derived from Arthrobacter globiformis was assessed for toxicity and allergenicity. The protein was stable at 90 degrees C for 1 h. Toxicity studies of choline oxidase in mice showed no significant difference (p > 0.05) from control in terms of growth, body weight, food consumption, and blood biochemical indices. Histology of gut tissue of mice fed protein showed normal gastric mucosal lining and villi in jejunum and ileum sections. Specific IgE in serum and IL-4 release in splenic culture supernatant were low in choline oxidase treated mice, comparable to control. Intravenous challenge with choline oxidase did not induce any adverse reaction, unlike ovalbumin group mice. Histology of lung tissues from choline oxidase sensitized mice showed normal airways, whereas ovalbumin-sensitized mice showed inflamed airways with eosinophilic infiltration and bronchoconstriction. ELISA carried out with food allergic patients' sera revealed no significant IgE affinity with choline oxidase. Also, choline oxidase did not show any symptoms of toxicity and allergenicity in mice.

Inactivation of 1-aminocyclopropane-1-carboxylate oxidase involves oxidative modifications.

PubMed

Barlow, J N; Zhang, Z; John, P; Baldwin, J E; Schofield, C J

1997-03-25

1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the final step in the biosynthesis of the plant signaling molecule ethylene. It is a member of the ferrous iron dependent family of oxidases and dioxygenases and is unusual in that it displays a very short half-life under catalytic conditions, typically less than 20 min, and a requirement for CO2 as an activator. The rates of inactivation of purified, recombinant ACC oxidase from tomato under various combinations of substrates and cofactors were measured. Inactivation was relatively slow in the presence of buffer alone (t1/2 > 1 h), but fast in the presence of ferrous iron and ascorbate (t1/2 approximately 10 min). The rate of iron/ascorbate-mediated inactivation was increased by the addition of ACC, unaffected by the addition of CO2 at saturation (supplied as bicarbonate) but decreased by the addition of catalase or ACC + CO2 at saturation (supplied as bicarbonate). Iron/ascorbate-mediated inactivation was accompanied by partial proteolysis as observed by SDS-PAGE analysis. The fragmentation pattern was altered when ACC was also included, suggesting that ACC can bind to ACC oxidase in the absence of bicarbonate. N-terminal sequencing of fragments resulted in identification of an internal cleavage site which we propose is proximate to active-site bound iron. Thus, ACC oxidase inactivates via relatively slow partial unfolding of the catalytically active conformation, oxidative damage mediated via hydrogen peroxide which is catalase protectable and oxidative damage to the active site which results in partial proteolysis and is not catalase protectable.

NADPH oxidases as novel pharmacologic targets against influenza A virus infection.

PubMed

Vlahos, Ross; Selemidis, Stavros

2014-12-01

Influenza A viruses represent a major global health care challenge, with imminent pandemics, emerging antiviral resistance, and long lag times for vaccine development, raising a pressing need for novel pharmacologic strategies that ideally target the pathology irrespective of the infecting strain. Reactive oxygen species (ROS) pervade all facets of cell biology with both detrimental and protective properties. Indeed, there is compelling evidence that activation of the NADPH oxidase 2 (NOX2) isoform of the NADPH oxidase family of ROS-producing enzymes promotes lung oxidative stress, inflammation, injury, and dysfunction resulting from influenza A viruses of low to high pathogenicity, as well as impeding virus clearance. By contrast, the dual oxidase isoforms produce ROS that provide vital protective antiviral effects for the host. In this review, we propose that inhibitors of NOX2 are better alternatives than broad-spectrum antioxidant approaches for treatment of influenza pathologies, for which clinical efficacy may have been limited owing to poor bioavailability and inadvertent removal of beneficial ROS. Finally, we briefly describe the current suite of NADPH oxidase inhibitors and the molecular features of the NADPH oxidase enzymes that could be exploited by drug discovery for development of more specific and novel inhibitors to prevent or treat disease caused by influenza. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

Biochemical, functional and structural characterization of Akbu-LAAO: a novel snake venom L-amino acid oxidase from Agkistrodon blomhoffii ussurensis.

PubMed

Sun, Ming-Zhong; Guo, Chunmei; Tian, Yuxiang; Chen, Duo; Greenaway, Frederick T; Liu, Shuqing

2010-04-01

An L-amino acid oxidase (Akbu-LAAO) was isolated from the venom of Agkistrodon blomhoffii ussurensis snake using DEAE Sephadex A-50 ion-exchange, Sephadex G-75 gel filtration, and high performance liquid chromatographies. The homogeneity and molecular mass of Akbu-LAAO were analyzed by SDS-PAGE and MALDI-TOF spectrometry. The sequences of ten peptides from Akbu-LAAO were established by HPLC-nESI-MS/MS analysis. Protein sequence alignment indicated that i) that Akbu-LAAO is a new snake venom LAAO, and ii) Akbu-LAAO shares homology with several LAAOs from the venoms of Calloselasma rhodost, Agkistrodon halys, Daboia russellii siamensis, and Trimeresurus stejnegeri. Akbu-LAAO is a homodimer with a molecular mass of approximately 124.4 kDa. It reacts optimally with its enzymatic substrate, Leu, at pH 4.7 with a K(m) of 2.1 mM. ICP-AES measurements showed that Akbu-LAAO contains four Zn(2+) per dimer that are unessential for the hydrolytic activity of the enzyme. The emission fluorescence intensity of Akbu-LAAO decreases by 61% on removal of Zn(2+) indicating that the zinc probably helps maintain the structural integrity of the enzyme. The addition of exogenous metal ions, including Mg(2+), Mn(2+), Ca(2+), Ce(3+), Nd(3+), Co(2+) and Tb(3+), increases the l-Leu hydrolytic activity of the enzyme. Akbu-LAAO shows apparent anti-aggregation effects on human and rabbit platelets. It exhibits a strong bacteriostasis effect on Staphylococcus aureus, eighteen fold that of cephalosporin C under the same conditions. Taken together, the biochemical, proteomic, structural and functional characterizations reveal that Akbu-LAAO is a novel LAAO with promise for biotechnological and medical applications. Copyright (c) 2010 Elsevier Masson SAS. All rights reserved.

Activation of monoamine oxidase isotypes by prolonged intake of aluminum in rat brain.

PubMed

Huh, Jae-Wan; Choi, Myung-Min; Lee, Jang Han; Yang, Seung-Ju; Kim, Mi Jung; Choi, Jene; Lee, Kwan Ho; Lee, Jong Eun; Cho, Sung-Woo

2005-10-01

Rats were fed 100 microM aluminum maltolate for one year in their drinking water. Brain aluminum contents have increased 4.2-fold in the aluminum-treated group, whereas no significant changes in the body weight, brain weight, and brain protein content were observed. Long-term aluminum feeding induced apoptosis as assessed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method and showed activatory effects on the catalytic efficiency (kcat/KM) of monoamine oxidase-A and monoamine oxidase-B up to 1.9- and 3.8-fold, respectively. The expression level of monoamine oxidase isotypes on the Western blot remained unchanged between the two groups, suggesting a change in post-translational regulation of the activities of monoamine oxidase isotypes by long-term aluminum feeding.

Role of diamine oxidase during the treatment of tumour-bearing mice with combinations of polyamine anti-metabolites.

PubMed Central

Kallio, A; Jänne, J

1983-01-01

Treatment of mice bearing L1210 leukaemia with 2-difluoromethylornithine, a specific inhibitor of ornithine decarboxylase (EC 4.1.1.17), produced a profound depletion of putrescine and spermidine in the tumour cells. Sequential combination of methylglyoxal bis(guanylhydrazone), an inhibitor of adenosylmethionine decarboxylase (EC 4.1.1.50), with difluoromethylornithine largely reversed the polyamine depletion and led to a marked accumulation of cadaverine in the tumour cells. Experiments carried out with the combination of difluoromethylornithine and aminoguanidine, a potent inhibitor of diamine oxidase (EC 1.4.3.6), indicated that the methylglyoxal bis(guanylhydrazone)-induced reversal of polyamine depletion was mediated by the known inhibition of diamine oxidase by the diguanidine. In spite of the normalization of the tumour cell polyamine pattern upon administration of methylglyoxal bis(guanylhydrazone) to difluoromethylornithine-treated animals, the combination of these two drugs produced a growth-inhibitory effect not achievable with either of the compounds alone. PMID:6411077

Simultaneous immobilization of glucose oxidase on the surface and cavity of hollow gold nanospheres as labels for highly sensitive electrochemical immunoassay of tumor marker.

PubMed

Song, Zhongju; Yuan, Ruo; Chai, Yaqin; Jiang, Wen; Su, Huilan; Che, Xin; Ran, Xiaoqi

2011-01-15

A novel tracer, glucose oxidase (GOD)-functionalized hollow gold nanospheres encapsulating glucose oxidase (Au(shell)@GOD), was designed to label the ferrocenemonocarboxylic-grafted secondary antibodies (Fc@Ab(2)) for highly sensitive detection of tumor marker using carboxyl group functionalized multiwall carbon nanotubes as platform. Initially, Au(shell)@GOD was synthesized specially by reverse micelle approach, and then the labeling of antibody and the preparation of GOD-functionalized Au(shell)@GOD were performed by one-pot assembly of Fc@Ab(2) and GOD on the surface of Au(shell)@GOD. The ferrocene used to label antibodies acted as a mediator of electron transfer between GOD and electrode surface. The high-content glucose oxidase in the tracer (on the surface and in the cavity) could significantly amplify the amperometric signal for sandwich-type immunoassay. Using carcinoembryonic antigen (CEA) as model analyte, the designed tracer showed linear range from 0.02 to 5.0 ng mL(-1) with the detection limit down to 6.7 pg mL(-1). The assay results of serum samples with the proposed method were in an acceptable agreement with the reference values. The new protocol showed acceptable stability and reproducibility, high sensitivity, and good precision, which could provide a promising potential for clinical screening and diagnosis of tumor disease. Copyright © 2010 Elsevier B.V. All rights reserved.

Purification, characterization and decolorization of bilirubin oxidase from Myrothecium verrucaria 3.2190

USDA-ARS?s Scientific Manuscript database

Myrothecium verrucaria 3.2190 is a nonligninolytic fungus that produces bilirubin oxidase. Both Myrothecium verrucaria and the extracellular bilirubin oxidase were tested for their ability to decolorize indigo carmine. The biosorption and biodegradation of the dye were detected during the process of...

Towards rational therapy with monoamine oxidase inhibitors.

PubMed

Tyrer, P

1976-04-01

A rational approach to the use of monoamine oxidase inhibitors (MAOIs) is outlined. Patients suitable for treatment cannot be classified adequately using conventional diagnostic labels. They include those with primary symptoms of hypochondriasis, agoraphobia and social phobias, irritability, somatic anxiety and anergia; those with primary depressed mood, guilt, ideas of reference and personality disorders seldom respond. There is great variation in the interval between the first administration of these drugs and clinical response, and this may account for the inconsistencies in published trials. The type of drug and its dose may affect rate of response, as may biochemical factors, including acetylator and monoamine oxidase status. To obtain maximum benefit, a course of therapy with MAOIs should last for several months.

In vivo low-level light therapy increases cytochrome oxidase in skeletal muscle.

PubMed

Hayworth, Christopher R; Rojas, Julio C; Padilla, Eimeira; Holmes, Genevieve M; Sheridan, Eva C; Gonzalez-Lima, F

2010-01-01

Low-level light therapy (LLLT) increases survival of cultured cells, improves behavioral recovery from neurodegeneration and speeds wound healing. These beneficial effects are thought to be mediated by upregulation of mitochondrial proteins, especially the respiratory enzyme cytochrome oxidase. However, the effects of in vivo LLLT on cytochrome oxidase in intact skeletal muscle have not been previously investigated. We used a sensitive method for enzyme histochemistry of cytochrome oxidase to examine the rat temporalis muscle 24 h after in vivo LLLT. The findings showed for the first time that in vivo LLLT induced a dose- and fiber type-dependent increase in cytochrome oxidase in muscle fibers. LLLT was particularly effective at enhancing the aerobic capacity of intermediate and red fibers. The findings suggest that LLLT may enhance the oxidative energy metabolic capacity of different types of muscle fibers, and that LLLT may be used to enhance the aerobic potential of skeletal muscle.

Mammalian monoamine-oxidizing enzymes, with special reference to benzylamine oxidase in human tissues.

PubMed

Lewinsohn, R

1984-01-01

A review is presented of the monoamine-oxidizing enzymes with special reference to the activity of benzylamine oxidase (BzAO) in human tissues. Methods of study of amine oxidases, properties (chiefly of BzAO) and some problems concerning substrate and inhibitor specificity and multiple forms of monoamine oxidase (MAO) are surveyed. The substrate specificity of human plasma BzAO is compared with that of amine-oxidizing enzymes in plasma or serum of other species. Correlations of plasma BzAO and platelet MAO activity with clinical findings are discussed. The distribution of amine oxidase activities in solid human tissues is reviewed, in particular BzAO in blood vessels and richly-vascularized tissues, as well as kinetic constants and altered patterns of activity of BzAO in human atherosclerosis. Activities of the amine oxidases in non-vascular smooth muscle, in cultured cells, and in various tissues related to human gestation, are discussed. The present knowledge of BzAO is discussed in terms of its possible clinical relevance to several human disease states, and the importance of the enzyme in the human body.

Herbivore-plant interactions: mixed-function oxidases and secondary plant substances.

PubMed

Brattsten, L B; Wilkinson, C F; Eisner, T

1977-06-17

The mixed-function oxidases of a polyphagous insect larva (the southern armyworm, Spodoptera eridania) were found to be induced by a diversity of secondary plant substances. The induction proceeds rapidly and in response to a small quantity of secondary substance. Following induction, the larva is less susceptible to dietary poisoning. It is argued that mixed-function oxidases play a major role in protecting herbivores against chemical stress from secondary plant substances.

Fluorescence ELISA based on glucose oxidase-mediated fluorescence quenching of quantum dots for highly sensitive detection of Hepatitis B.

PubMed

Wu, Yunqing; Zeng, Lifeng; Xiong, Ying; Leng, Yuankui; Wang, Hui; Xiong, Yonghua

2018-05-01

Herein, we present a novel sandwich fluorescence enzyme linked immunosorbent assay (ELISA) for highly sensitive detection of Hepatitis B virus surface antigen (HBsAg) based on glucose oxidase (GOx)-induced fluorescence quenching of mercaptopropionic acid-modified CdTe quantum dots (MPA-QDs). In this system, hydrogen peroxide (H 2 O 2 ) sensitive MPA-QDs was used as a signal output, and glucose oxidase (GOx) was used as label which can generate H 2 O 2 via catalytic oxidation of glucose. The proposed method showed dynamic linear detection of HBsAg both in the range of 47pgmL -1 ~ 380pgmL -1 and 0.75ngmL -1 ~ 12.12ngmL -1 . The detection limit of the proposed fluorescence ELISA was 1.16pgmL -1 , which was approximately 430-fold lower than that of horseradish peroxidase (HRP)-based conventional ELISA. The average recoveries for HBsAg-spiked serum samples ranged from 98.0% to 126.8% with the relative standard derivation below 10%, thus indicating acceptable precision and high reproducibility of the proposed fluorescence ELISA for HBsAg detection. Additionally, the developed method showed no false positive results analyzing 35 real HBsAg-negative serum samples, and exhibited excellent agreement (R 2 =0.9907) with a commercial time-resolved fluorescence immunoassay (TRFIA) kit for detecting 31 HBsAg-positive serum samples. In summary, the proposed method based on fluorescence quenching of H 2 O 2 sensitive QDs is considerably to be an excellent biodetection platform with ultrahigh sensitivity, good accuracy and excellent reliability. Copyright © 2018 Elsevier B.V. All rights reserved.

NADPH Oxidase-Driven Phagocyte Recruitment Controls Candida albicans Filamentous Growth and Prevents Mortality

PubMed Central

Brothers, Kimberly M.; Gratacap, Remi L.; Barker, Sarah E.; Newman, Zachary R.; Norum, Ashley; Wheeler, Robert T.

2013-01-01

Candida albicans is a human commensal and clinically important fungal pathogen that grows as both yeast and hyphal forms during human, mouse and zebrafish infection. Reactive oxygen species (ROS) produced by NADPH oxidases play diverse roles in immunity, including their long-appreciated function as microbicidal oxidants. Here we demonstrate a non-traditional mechanistic role of NADPH oxidase in promoting phagocyte chemotaxis and intracellular containment of fungi to limit filamentous growth. We exploit the transparent zebrafish model to show that failed NADPH oxidase-dependent phagocyte recruitment to C. albicans in the first four hours post-infection permits fungi to germinate extracellularly and kill the host. We combine chemical and genetic tools with high-resolution time-lapse microscopy to implicate both phagocyte oxidase and dual-specific oxidase in recruitment, suggesting that both myeloid and non-myeloid cells promote chemotaxis. We show that early non-invasive imaging provides a robust tool for prognosis, strongly connecting effective early immune response with survival. Finally, we demonstrate a new role of a key regulator of the yeast-to-hyphal switching program in phagocyte-mediated containment, suggesting that there are species-specific methods for modulation of NADPH oxidase-independent immune responses. These novel links between ROS-driven chemotaxis and fungal dimorphism expand our view of a key host defense mechanism and have important implications for pathogenesis. PMID:24098114

A Novel Colletotrichum graminicola Raffinose Oxidase in the AA5 Family

PubMed Central

Mollerup, Filip; Parikka, Kirsti; Koutaniemi, Sanna; Boer, Harry; Juvonen, Minna; Master, Emma; Tenkanen, Maija; Kruus, Kristiina

2017-01-01

ABSTRACT We describe here the identification and characterization of a copper radical oxidase from auxiliary activities family 5 (AA5_2) that was distinguished by showing preferential activity toward raffinose. Despite the biotechnological potential of carbohydrate oxidases from family AA5, very few members have been characterized. The gene encoding raffinose oxidase from Colletotrichum graminicola (CgRaOx; EC 1.1.3.−) was identified utilizing a bioinformatics approach based on the known modular structure of a characterized AA5_2 galactose oxidase. CgRaOx was expressed in Pichia pastoris, and the purified enzyme displayed the highest activity on the trisaccharide raffinose, whereas the activity on the disaccharide melibiose was three times lower and more than ten times lower activity was detected on d-galactose at a 300 mM substrate concentration. Thus, the substrate preference of CgRaOx was distinguished clearly from the substrate preferences of the known galactose oxidases. The site of oxidation for raffinose was studied by 1H nuclear magnetic resonance and mass spectrometry, and we confirmed that the hydroxyl group at the C-6 position was oxidized to an aldehyde and that in addition uronic acid was produced as a side product. A new electrospray ionization mass spectrometry method for the identification of C-6 oxidized products was developed, and the formation mechanism of the uronic acid was studied. CgRaOx presented a novel activity pattern in the AA5 family. IMPORTANCE Currently, there are only a few characterized members of the CAZy AA5 protein family. These enzymes are interesting from an application point of view because of their ability to utilize the cheap and abundant oxidant O2 without the requirement of complex cofactors such as FAD or NAD(P). Here, we present the identification and characterization of a novel AA5 member from Colletotrichum graminicola. As discussed in the present study, the bioinformatics approach using the modular structure of

Proposed structural basis of interaction of piperine and related compounds with monoamine oxidases.

PubMed

Rahman, Taufiq; Rahmatullah, Mohammed

2010-01-15

Several studies have revealed piperine and a few related compounds as potent inhibitors of monoamine oxidases without delineating the underlying mechanism. Using in silico modelling, we propose a structural basis of such activity by showing that these compounds can successfully dock into the inhibitor binding pockets of human monoamine oxidase isoforms with predicted affinities comparable to some known inhibitors. The results therefore suggest that piperine can be a promising lead for developing novel monoamine oxidase inhibitors. Copyright 2009 Elsevier Ltd. All rights reserved.

Targeting NADPH oxidase decreases oxidative stress in the transgenic sickle cell mouse penis.

PubMed

Musicki, Biljana; Liu, Tongyun; Sezen, Sena F; Burnett, Arthur L

2012-08-01

Sickle cell disease (SCD) is a state of chronic vasculopathy characterized by endothelial dysfunction and increased oxidative stress, but the sources and mechanisms responsible for reactive oxygen species (ROS) production in the penis are unknown. We evaluated whether SCD activates NADPH oxidase, induces endothelial nitric oxide synthase (eNOS) uncoupling, and decreases antioxidants in the SCD mouse penis. We further tested the hypothesis that targeting NADPH oxidase decreases oxidative stress in the SCD mouse penis. SCD transgenic (sickle) mice were used as an animal model of SCD. Hemizygous (hemi) mice served as controls. Mice received an NADPH oxidase inhibitor apocynin (10 mM in drinking water) or vehicle. Penes were excised at baseline for molecular studies. Markers of oxidative stress (4-hydroxy-2-nonenal [HNE]), sources of ROS (eNOS uncoupling and NADPH oxidase subunits p67(phox) , p47(phox) , and gp91(phox) ), and enzymatic antioxidants (superoxide dismutase [SOD]1, SOD2, catalase, and glutathione peroxidase-1 [GPx1]) were measured by Western blot in penes. Sources of ROS, oxidative stress, and enzymatic antioxidants in the SCD penis. Relative to hemi mice, SCD increased (P<0.05) protein expression of NADPH oxidase subunits p67(phox) , p47(phox) , and gp91(phox) , 4-HNE-modified proteins, induced eNOS uncoupling, and reduced Gpx1 expression in the penis. Apocynin treatment of sickle mice reversed (P<0.05) the abnormalities in protein expressions of p47(phox) , gp91(phox) (but not p67(phox) ) and 4-HNE, but only slightly (P>0.05) prevented eNOS uncoupling in the penis. Apocynin treatment of hemi mice did not affect any of these parameters. NADPH oxidase and eNOS uncoupling are sources of oxidative stress in the SCD penis; decreased GPx1 further contributes to oxidative stress. Inhibition of NADPH oxidase upregulation decreases oxidative stress, implying a major role for NADPH oxidase as a ROS source and a potential target for improving vascular function in

Targeting NADPH Oxidase Decreases Oxidative Stress in the Transgenic Sickle Cell Mouse Penis

PubMed Central

Musicki, Biljana; Liu, Tongyun; Sezen, Sena F.; Burnett, Arthur L.

2012-01-01

Introduction Sickle cell disease (SCD) is a state of chronic vasculopathy characterized by endothelial dysfunction and increased oxidative stress, but the sources and mechanisms responsible for reactive oxygen species (ROS) production in the penis are unknown. Aims We evaluated whether SCD activates NADPH oxidase, induces endothelial nitric oxide synthase (eNOS) uncoupling, and decreases antioxidants in the SCD mouse penis. We further tested the hypothesis that targeting NADPH oxidase decreases oxidative stress in the SCD mouse penis. Methods SCD transgenic (sickle) mice were used as an animal model of SCD. Hemizygous (hemi) mice served as controls. Mice received an NADPH oxidase inhibitor apocynin (10 mM in drinking water) or vehicle. Penes were excised at baseline for molecular studies. Markers of oxidative stress (4-hydroxy-2-nonenal [HNE]), sources of ROS (eNOS uncoupling and NADPH oxidase subunits p67phox, p47phox, and gp91phox), and enzymatic antioxidants (superoxide dismutase [SOD]1, SOD2, catalase, and glutathione peroxidase-1 [GPx1]) were measured by Western blot in penes. Main Outcome Measures Sources of ROS, oxidative stress, and enzymatic antioxidants in the SCD penis. Results Relative to hemi mice, SCD increased (P < 0.05) protein expression of NADPH oxidase subunits p67phox, p47phox, and gp91phox, 4-HNE-modified proteins, induced eNOS uncoupling, and reduced Gpx1 expression in the penis. Apocynin treatment of sickle mice reversed (P < 0.05) the abnormalities in protein expressions of p47phox, gp91phox (but not p67phox) and 4-HNE, but only slightly (P > 0.05) prevented eNOS uncoupling in the penis. Apocynin treatment of hemi mice did not affect any of these parameters. Conclusion NADPH oxidase and eNOS uncoupling are sources of oxidative stress in the SCD penis; decreased GPx1 further contributes to oxidative stress. Inhibition of NADPH oxidase upregulation decreases oxidative stress, implying a major role for NADPH oxidase as a ROS source and a

Continuous aryl alcohol oxidase production under growth-limited conditions using a trickle bed reactor.

PubMed

Pardo-Planas, Oscar; Atiyeh, Hasan K; Prade, Rolf A; Müller, Michael; Wilkins, Mark R

2018-05-01

An A. nidulans strain with a pyridoxine marker was used for continuous production of aryl alcohol oxidase (AAO) in a trickle bed reactor (TBR). Modified medium with reduced zinc, no copper, and 5 g/L ascorbic acid that reduced melanin production and increased AAO productivity under growth limited conditions was used. Two air flow rates, 0.11 L/min (0.1 vvm) and 1.1 L/min (1.0 vvm) were tested. More melanin formation and reduced protein productivity were observed with air flow rate of 1.1 L/min. Three random packings were used as support for the fungus inside the TBR column, two of which were hydrophobic and one which was hydrophilic, and three different dilution rates were tested. The use of GEA BCN 030 hydrophobic packing resulted in greater AAO yield and productivity than the other packings. Increasing dilution rates favored melanin formation and citric, lactic and succinic acid accumulation, which decreased AAO yield and productivity. Copyright © 2018 Elsevier Ltd. All rights reserved.

Multi-Copper Oxidases and Human Iron Metabolism

PubMed Central

Vashchenko, Ganna; MacGillivray, Ross T. A.

2013-01-01

Multi-copper oxidases (MCOs) are a small group of enzymes that oxidize their substrate with the concomitant reduction of dioxygen to two water molecules. Generally, multi-copper oxidases are promiscuous with regards to their reducing substrates and are capable of performing various functions in different species. To date, three multi-copper oxidases have been detected in humans—ceruloplasmin, hephaestin and zyklopen. Each of these enzymes has a high specificity towards iron with the resulting ferroxidase activity being associated with ferroportin, the only known iron exporter protein in humans. Ferroportin exports iron as Fe2+, but transferrin, the major iron transporter protein of blood, can bind only Fe3+ effectively. Iron oxidation in enterocytes is mediated mainly by hephaestin thus allowing dietary iron to enter the bloodstream. Zyklopen is involved in iron efflux from placental trophoblasts during iron transfer from mother to fetus. Release of iron from the liver relies on ferroportin and the ferroxidase activity of ceruloplasmin which is found in blood in a soluble form. Ceruloplasmin, hephaestin and zyklopen show distinctive expression patterns and have unique mechanisms for regulating their expression. These features of human multi-copper ferroxidases can serve as a basis for the precise control of iron efflux in different tissues. In this manuscript, we review the biochemical and biological properties of the three human MCOs and discuss their potential roles in human iron homeostasis. PMID:23807651

Function of the Pyruvate Oxidase-Lactate Oxidase Cascade in Interspecies Competition between Streptococcus oligofermentans and Streptococcus mutans

PubMed Central

Liu, Lei

2012-01-01

Complex interspecies interactions occur constantly between oral commensals and the opportunistic pathogen Streptococcus mutans in dental plaque. Previously, we showed that oral commensal Streptococcus oligofermentans possesses multiple enzymes for H2O2 production, especially lactate oxidase (Lox), allowing it to out-compete S. mutans. In this study, through extensive biochemical and genetic studies, we identified a pyruvate oxidase (pox) gene in S. oligofermentans. A pox deletion mutant completely lost Pox activity, while ectopically expressed pox restored activity. Pox was determined to produce most of the H2O2 in the earlier growth phase and log phase, while Lox mainly contributed to H2O2 production in stationary phase. Both pox and lox were expressed throughout the growth phase, while expression of the lox gene increased by about 2.5-fold when cells entered stationary phase. Since lactate accumulation occurred to a large degree in stationary phase, the differential Pox- and Lox-generated H2O2 can be attributed to differential gene expression and substrate availability. Interestingly, inactivation of pox causes a dramatic reduction in H2O2 production from lactate, suggesting a synergistic action of the two oxidases in converting lactate into H2O2. In an in vitro two-species biofilm experiment, the pox mutant of S. oligofermentans failed to inhibit S. mutans even though lox was active. In summary, S. oligofermentans develops a Pox-Lox synergy strategy to maximize its H2O2 formation so as to win the interspecies competition. PMID:22287002

The substrate oxidation mechanism of pyranose 2-oxidase and other related enzymes in the glucose-methanol-choline superfamily.

PubMed

Wongnate, Thanyaporn; Chaiyen, Pimchai

2013-07-01

Enzymes in the glucose-methanol-choline (GMC) oxidoreductase superfamily catalyze the oxidation of an alcohol moiety to the corresponding aldehyde. In this review, the current understanding of the sugar oxidation mechanism in the reaction of pyranose 2-oxidase (P2O) is highlighted and compared with that of other enzymes in the GMC family for which structural and mechanistic information is available, including glucose oxidase, choline oxidase, cholesterol oxidase, cellobiose dehydrogenase, aryl-alcohol oxidase, and pyridoxine 4-oxidase. Other enzymes in the family that have been newly discovered or for which less information is available are also discussed. A large primary kinetic isotope effect was observed for the flavin reduction when 2-d-D-glucose was used as a substrate, but no solvent kinetic isotope effect was detected for the flavin reduction step. The reaction of P2O is consistent with a hydride transfer mechanism in which there is stepwise formation of d-glucose alkoxide prior to the hydride transfer. Site-directed mutagenesis of P2O and pH-dependence studies indicated that His548 is a catalytic base that facilitates the deprotonation of C2-OH in D-glucose. This finding agrees with the current mechanistic model for aryl-alcohol oxidase, glucose oxidase, cellobiose dehydrogenase, methanol oxidase, and pyridoxine 4-oxidase, but is different from that of cholesterol oxidase and choline oxidase. Although all of the GMC enzymes share similar structural folding and use the hydride transfer mechanism for flavin reduction, they appear to have subtle differences in the fine-tuned details of how they catalyze substrate oxidation. © 2013 The Authors Journal compilation © 2013 FEBS.

Direct comparison of gluco-oligosaccharide oxidase variants and glucose oxidase: substrate range and H2O2 stability.

PubMed

Vuong, Thu V; Foumani, Maryam; MacCormick, Benjamin; Kwan, Rachel; Master, Emma R

2016-11-21

Glucose oxidase (GO) activity is generally restricted to glucose and is susceptible to inactivation by H 2 O 2 . By comparison, the Y300A variant of gluco-oligosaccharide oxidase (GOOX) from Sarocladium strictum showed broader substrate range and higher H 2 O 2 stability. Specifically, Y300A exhibited up to 40 times higher activity on all tested sugars except glucose, compared to GO. Moreover, fusion of the Y300A variant to a family 22 carbohydrate binding module from Clostridium thermocellum (CtCBM22A) nearly doubled its catalytic efficiency on glucose, while retaining significant activity on oligosaccharides. In the presence of 200 mM of H 2 O 2 , the recombinant CtCBM22A_Y300A retained 80% of activity on glucose and 100% of activity on cellobiose, the preferred substrate for this enzyme. By contrast, a commercial glucose oxidase reported to contain ≤0.1 units catalase/ mg protein, retained 60% activity on glucose under the same conditions. GOOX variants appear to undergo a different mechanism of inactivation, as a loss of histidine instead of methionine was observed after H 2 O 2 incubation. The addition of CtCBM22A also promoted functional binding of the fusion enzyme to xylan, facilitating its simultaneous purification and immobilization using edible oat spelt xylan, which might benefit the usage of this enzyme preparation in food and baking applications.

Steady-state kinetics of substrate binding and iron release in tomato ACC oxidase.

PubMed

Thrower, J S; Blalock, R; Klinman, J P

2001-08-14

1-Aminocyclopropane-1-carboxylate oxidase (ACC oxidase) catalyzes the last step in the biosynthetic pathway of the plant hormone, ethylene. This unusual reaction results in the oxidative ring cleavage of 1-aminocyclopropane carboxylate (ACC) into ethylene, cyanide, and CO2 and requires ferrous ion, ascorbate, and molecular oxygen for catalysis. A new purification procedure and assay method have been developed for tomato ACC oxidase that result in greatly increased enzymatic activity. This method allowed us to determine the rate of iron release from the enzyme and the effect of the activator, CO2, on this rate. Initial velocity studies support an ordered kinetic mechanism where ACC binds first followed by O2; ascorbate can bind after O2 or possibly before ACC. This kinetic mechanism differs from one recently proposed for the ACC oxidase from avocado.

Neovascularization in an arterio-venous loop-containing tissue engineering chamber: role of NADPH oxidase

PubMed Central

Jiang, F; Zhang, G; Hashimoto, I; Kumar, B S; Bortolotto, S; Morrison, W A; Dusting, G J

2008-01-01

Using an in vivo arterio-venous loop-containing tissue-engineering chamber, we have created a variety of vascularized tissue blocks, including functional myocardium. The viability of the transplanted cells is limited by the rate of neovascularization in the chamber. A Nox2-containing nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is thought to have a critical role in ischaemic angiogenesis. In this study we investigated whether NADPH oxidase is involved in the neovascularization process in the tissue-engineering chamber. New blood vessels originating from the venous and the arterial ends of the loop could be identified after 3 days, and the vessel density (by lectin staining) peaked after 7 days and was maintained for at least 14 days. This was accompanied by granulation tissue formation and concomitant increase in the mRNA level of Nox4 NADPH oxidase. Although the total level of Nox2 mRNA in the chamber tissue decreased from day 3 to day 7, immunohistochemistry identified a strong expression of Nox2 in the endothelial cells of the new vessels. In human microvascular endothelial cells, the NADPH oxidase inhibitor apocynin reduced NADPH oxidase activity and inhibited the angiogenic responses in vitro. Local treatment with the NADPH oxidase inhibitors apocynin or gp91ds-tat peptide significantly suppressed the vessel growth in the chamber. In conclusion, NADPH oxidase-dependent redox signalling is important for neovascularization in this novel tissue-engineering chamber in vivo, and boosting this signalling might be a new approach to extending vascularization and tissue growth. PMID:19012731

Insights into proton translocation in cbb3 oxidase from MD simulations.

PubMed

Carvalheda, Catarina A; Pisliakov, Andrei V

2017-05-01

Heme-copper oxidases are membrane protein complexes that catalyse the final step of the aerobic respiration, namely the reduction of oxygen to water. The energy released during catalysis is coupled to the active translocation of protons across the membrane, which contributes to the establishment of an electrochemical gradient that is used for ATP synthesis. The distinctive C-type (or cbb 3 ) cytochrome c oxidases, which are mostly present in proteobacteria, exhibit a number of unique structural and functional features, including high catalytic activity at low oxygen concentrations. At the moment, the functioning mechanism of C-type oxidases, in particular the proton transfer/pumping mechanism presumably via a single proton channel, is still poorly understood. In this work we used all-atom molecular dynamics simulations and continuum electrostatics calculations to obtain atomic-level insights into the hydration and dynamics of a cbb 3 oxidase. We provide the details of the water dynamics and proton transfer pathways for both the "chemical" and "pumped" protons, and show that formation of protonic connections is strongly affected by the protonation state of key residues, namely H243, E323 and H337. Copyright © 2017 Elsevier B.V. All rights reserved.

Expression Studies of Gibberellin Oxidases in Developing Pumpkin Seeds1

PubMed Central

Frisse, Andrea; Pimenta, Maria João; Lange, Theo

2003-01-01

Two cDNA clones, 3-ox and 2-ox, have been isolated from developing pumpkin (Cucurbita maxima) embryos that show significant amino acid homology to gibberellin (GA) 3-oxidases and 2-oxidases, respectively. Recombinant fusion protein of clone 3-ox converted GA12-aldehyde, GA12, GA15, GA24, GA25, and GA9 to GA14-aldehyde, GA14, GA37, GA36, GA13, and GA4, respectively. Recombinant 2-ox protein oxidized GA9, GA4, and GA1 to GA51, GA34, and GA8, respectively. Previously cloned GA 7-oxidase revealed additional 3β-hydroxylation activity of GA12. Transcripts of this gene were identified in endosperm and embryo of the developing seed by quantitative reverse transcriptase-polymerase chain reaction and localized in protoderm, root apical meristem, and quiescent center by in situ hybridization. mRNA of the previously cloned GA 20-oxidase from pumpkin seeds was localized in endosperm and in tissues of protoderm, ground meristem, and cotyledons of the embryo. However, transcripts of the recently cloned GA 20-oxidase from pumpkin seedlings were found all over the embryo, and in tissues of the inner seed coat at the micropylar end. Previously cloned GA 2β,3β-hydroxylase mRNA molecules were specifically identified in endosperm tissue. Finally, mRNA molecules of the 3-ox and 2-ox genes were found in the embryo only. 3-ox transcripts were localized in tissues of cotyledons, protoderm, and inner cell layers of the root apical meristem, and 2-ox transcripts were found in all tissues of the embryo except the root tips. These results indicate tissue-specific GA-biosynthetic pathways operating within the developing seed. PMID:12644672

NADPH Oxidase-Dependent Signaling in Endothelial Cells: Role in Physiology and Pathophysiology

PubMed Central

Ushio-Fukai, Masuko; Malik, Asrar B.

2009-01-01

Abstract Reactive oxygen species (ROS) including superoxide (O2·−) and hydrogen peroxide (H2O2) are produced endogenously in response to cytokines, growth factors; G-protein coupled receptors, and shear stress in endothelial cells (ECs). ROS function as signaling molecules to mediate various biological responses such as gene expression, cell proliferation, migration, angiogenesis, apoptosis, and senescence in ECs. Signal transduction activated by ROS, “oxidant signaling,” has received intense investigation. Excess amount of ROS contribute to various pathophysiologies, including endothelial dysfunction, atherosclerosis, hypertension, diabetes, and acute respiratory distress syndrome (ARDS). The major source of ROS in EC is a NADPH oxidase. The prototype phagaocytic NADPH oxidase is composed of membrane-bound gp91phox and p22hox, as well as cytosolic subunits such as p47phox, p67phox and small GTPase Rac. In ECs, in addition to all the components of phagocytic NADPH oxidases, homologues of gp91phox (Nox2) including Nox1, Nox4, and Nox5 are expressed. The aim of this review is to provide an overview of the emerging area of ROS derived from NADPH oxidase and oxidant signaling in ECs linked to physiological and pathophysiological functions. Understanding these mechanisms may provide insight into the NADPH oxidase and oxidant signaling components as potential therapeutic targets. Antioxid. Redox Signal. 11, 791–810. PMID:18783313

Honey as an apitherapic product: its inhibitory effect on urease and xanthine oxidase.

PubMed

Sahin, Huseyin

2016-01-01

The aim of this study was to evaluate new natural inhibitor sources for the enzymes urease and xanthine oxidase (XO). Chestnut, oak and polyfloral honey extracts were used to determine inhibition effects of both enzymes. In addition to investigate inhibition, the antioxidant capacities of these honeys were determined using total phenolic content (TPC), ferric reducing antioxidant power (FRAP), and DPPH radical scavenging activity assays. Due to their high phenolic content, chestnut and oak honeys are found to be a powerful source for inhibition of both enzymes. Especially, oak honeys were efficient for urease inhibition with 0.012-0.021 g/mL IC50 values, and also chestnut honeys were powerful for XO inhibition with 0.028-0.039 g/mL IC50 values. Regular daily consumption of these honeys can prevent gastric ulcers deriving from Helicobacter pylori and pathological disorders mediated by reactive oxygen species.

Ectopic expression of GA 2-oxidase 6 from rapeseed (Brassica napus L.) causes dwarfism, late flowering and enhanced chlorophyll accumulation in Arabidopsis thaliana.

PubMed

Yan, Jindong; Liao, Xiaoying; He, Reqing; Zhong, Ming; Feng, Panpan; Li, Xinmei; Tang, Dongying; Liu, Xuanming; Zhao, Xiaoying

2017-02-01

Gibberellins (GAs) are endogenous hormones that play an important role in higher plant growth and development. GA2-oxidase (GA2ox) promotes catabolism and inactivation of bioactive GAs or their precursors. In this study, we identified the GA2-oxidase gene, BnGA2ox6, and found it to be highly expressed in the silique and flower. Overexpression of BnGA2ox6 in Arabidopsis resulted in GA-deficiency symptoms, including inhibited elongation of the hypocotyl and stem, delayed seed germination, and late flowering. BnGA2ox6 overexpression reduced silique growth, but had no effect on seed development. Additionally, BnGA2ox6 overexpression enhanced chlorophyll b and total chlorophyll accumulation, and downregulated mRNA expression levels of the CHL1 and RCCR genes, which are involved in the chlorophyll degradation. These findings suggest that BnGA2ox6 regulates plant hight, silique development, flowering and chlorophyll accumulation in transgenic Arabidopsis. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Comparative analysis of lysyl oxidase (like) family members in pulmonary fibrosis.

PubMed

Aumiller, Verena; Strobel, Benjamin; Romeike, Merrit; Schuler, Michael; Stierstorfer, Birgit E; Kreuz, Sebastian

2017-03-10

Extracellular matrix (ECM) composition and stiffness are major driving forces for the development and persistence of fibrotic diseases. Lysyl oxidase (LOX) and LOX-like (LOXL) proteins play crucial roles in ECM remodeling due to their collagen crosslinking and intracellular functions. Here, we systematically investigated LOX/L expression in primary fibroblasts and epithelial cells under fibrotic conditions, Bleomycin (BLM) induced lung fibrosis and in human IPF tissue. Basal expression of all LOX/L family members was detected in epithelial cells and at higher levels in fibroblasts. Various pro-fibrotic stimuli broadly induced LOX/L expression in fibroblasts, whereas specific induction of LOXL2 and partially LOX was observed in epithelial cells. Immunohistochemical analysis of lung tissue from 14 IPF patients and healthy donors revealed strong induction of LOX and LOXL2 in bronchial and alveolar epithelium as well as fibroblastic foci. Using siRNA experiments we observed that LOXL2 and LOXL3 were crucial for fibroblast-to-myofibroblast transition (FMT). As FMT could only be reconstituted with an enzymatically active LOXL2 variant, we conclude that LOXL2 enzymatic function is crucial for fibroblast transdifferentiation. In summary, our study provides a comprehensive analysis of the LOX/L family in fibrotic lung disease and indicates prominent roles for LOXL2/3 in fibroblast activation and LOX/LOXL2 in IPF.

Increased xanthine oxidase during labour--implications for oxidative stress.

PubMed

Many, A; Roberts, J M

1997-11-01

Xanthine dehydrogenase/oxidase (XDH/XO) produces uric acid. When in the oxidase form, this production is coupled with the generation of free radicals. Hypoxia-reperfusion enhances conversion of XDH to XO. Since the placenta is exposed to short periods of hypoxia reperfusion during labour, 17 placentae of pregnancy terminated by elective caesarean section and five placentae of pregnancies terminated by caesarean section during labour were examined for XDH/XO activity. It was found that XO activity was higher in the placentae of labouring women (P = 0.003), which suggests that labour enhances conversion of XDH to XO, facilitating free radical production.

A Conserved Steroid Binding Site in Cytochrome c Oxidase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qin, Ling; Mills, Denise A.; Buhrow, Leann

2010-09-02

Micromolar concentrations of the bile salt deoxycholate are shown to rescue the activity of an inactive mutant, E101A, in the K proton pathway of Rhodobacter sphaeroides cytochrome c oxidase. A crystal structure of the wild-type enzyme reveals, as predicted, deoxycholate bound with its carboxyl group at the entrance of the K path. Since cholate is a known potent inhibitor of bovine oxidase and is seen in a similar position in the bovine structure, the crystallographically defined, conserved steroid binding site could reveal a regulatory site for steroids or structurally related molecules that act on the essential K proton path.

Nox4 NADPH oxidase mediates oxidative stress and apoptosis caused by TNF-α in cerebral vascular endothelial cells

PubMed Central

Basuroy, Shyamali; Bhattacharya, Sujoy; Leffler, Charles W.; Parfenova, Helena

2009-01-01

Inflammatory brain disease may damage cerebral vascular endothelium leading to cerebral blood flow dysregulation. The proinflammatory cytokine TNF-α causes oxidative stress and apoptosis in cerebral microvascular endothelial cells (CMVEC) from newborn pigs. We investigated contribution of major cellular sources of reactive oxygen species to endothelial inflammatory response. Nitric oxide synthase and xanthine oxidase inhibitors (Nω-nitro-l-arginine and allopurinol) had no effect, while mitochondrial electron transport inhibitors (CCCP, 2-thenoyltrifluoroacetone, and rotenone) attenuated TNF-α-induced superoxide (O2•−) and apoptosis. NADPH oxidase inhibitors (diphenylene iodonium and apocynin) greatly reduced TNF-α-evoked O2•− generation and apoptosis. TNF-α rapidly increased NADPH oxidase activity in CMVEC. Nox4, the cell-specific catalytic subunit of NADPH oxidase, is highly expressed in CMVEC, contributes to basal O2•− production, and accounts for a burst of oxidative stress in response to TNF-α. Nox4 small interfering RNA, but not Nox2, knockdown prevented oxidative stress and apoptosis caused by TNF-α in CMVEC. Nox4 is colocalized with HO-2, the constitutive isoform of heme oxygenase (HO), which is critical for endothelial protection against TNF-α toxicity. The products of HO activity, bilirubin and carbon monoxide (CO, as a CO-releasing molecule, CORM-A1), inhibited Nox4-generated O2•− and apoptosis caused by TNF-α stimulation. We conclude that Nox4 is the primary source of inflammation- and TNF-α-induced oxidative stress leading to apoptosis in brain endothelial cells. The ability of CO and bilirubin to combat TNF-α-induced oxidative stress by inhibiting Nox4 activity and/or by O2•− scavenging, taken together with close intracellular compartmentalization of HO-2 and Nox4 in cerebral vascular endothelium, may contribute to HO-2 cytoprotection against inflammatory cerebrovascular disease. PMID:19118162

Production of a new D-amino acid oxidase from the fungus Fusarium oxysporum.

PubMed

Gabler, M; Fischer, L

1999-08-01

The fungus Fusarium oxysporum produced a D-amino acid oxidase (EC 1. 4.3.3) in a medium containing glucose as the carbon and energy source and ammonium sulfate as the nitrogen source. The specific D-amino acid oxidase activity was increased up to 12.5-fold with various D-amino acids or their corresponding derivatives as inducers. The best inducers were D-alanine (2.7 microkat/g of dry biomass) and D-3-aminobutyric acid (2.6 microkat/g of dry biomass). The addition of zinc ions was necessary to permit the induction of peroxisomal D-amino acid oxidase. Bioreactor cultivations were performed on a 50-liter scale, yielding a volumetric D-amino acid oxidase activity of 17 microkat liter(-1) with D-alanine as an inducer. Under oxygen limitation, the volumetric activity was increased threefold to 54 microkat liter(-1) (3,240 U liter(-1)).

Isolation and bioelectrochemical characterization of novel fungal sources with oxidasic activity applied in situ for the cathodic oxygen reduction in microbial fuel cells.

PubMed

Morant, Kyriale Vasconcelos; da Silva, Paulo Henrique; de Campos-Takaki, Galba Maria; Hernández, Camilo Enrique La Rotta

2014-11-01

Brazilian filamentous fungi Rhizopus sp. (SIS-31), Aspergillus sp. (SIS-18) and Penicillium sp. (SIS-21), sources of oxidases were isolated from Caatinga's soils and applied during the in situ cathodic oxygen reduction in fuel cells. All strains were cultivated in submerged cultures using an optimized saline medium enriched with 10 g L(-1) of glucose, 3.0 g L(-1) of peptone and 0.0005 g L(-1) of CuSO4 as enzyme inducer. Parameters of oxidase activity, glucose consumption and microbial growth were evaluated. In-cell experiments evaluated by chronoamperometry were performed and two different electrode compositions were also compared. Maximum current densities of 125.7, 98.7 and 11.5 μA cm(-2) were observed before 24 h and coulombic efficiencies of 56.5, 46.5 and 23.8% were obtained for SIS-31, SIS-21 and SIS-18, respectively. Conversely, maximum power outputs of 328.73, 288.80 and 197.77 mW m(-3) were observed for SIS-18, SIS-21 and SIS-31, respectively. This work provides the primary experimental evidences that fungi isolated from the Caatinga region in Brazil can serve as efficient biocatalysts during the oxygen reduction in air-cathodes to improve electricity generation in MFCs. Copyright © 2014 Elsevier Inc. All rights reserved.

Reconstituted high-density lipoprotein suppresses leukocyte NADPH oxidase activation by disrupting lipid rafts.

PubMed

Peshavariya, Hitesh; Dusting, Gregory J; Di Bartolo, Belinda; Rye, Kerry-Anne; Barter, Philip J; Jiang, Fan

2009-08-01

Reconstituted discoidal high-density lipoprotein (rHDL) has potent vascular protective actions. Native HDL suppresses cellular generation of reactive oxygen species, whereas this antioxidant effect of rHDL is less clear. This study examined the effects of rHDL on NADPH oxidase, a major source of cellular superoxide generation, in both leukocytes and human umbilical vein endothelial cells. Superoxide was measured with lucigenin-enhanced chemiluminescence. Expression of NADPH oxidase sub-units was determined by real-time PCR. Pre-treatment of HL-60 cells with rHDL (10 and 25 microM) for 1 h significantly reduced phorbol 12-myristate 13-acetate-stimulated superoxide production. Treatment with rHDL for up to 24 h did not change the mRNA expression of NADPH oxidase sub-units. In HL-60 cells, depletion of cholesterol from the plasma membrane by methyl-beta-cyclodextrin mimicked the effect of rHDL, whereas cholesterol repletion blunted the effects of rHDL. Treatment with rHDL induced disruption of the lipid raft structures and blunted PMA-induced redistribution of p47phox into lipid rafts. In contrast, treatment of endothelial cells with rHDL for up to 18 h had no effect on either basal or tumour necrosis factor-alpha-stimulated NADPH oxidase activity, but markedly suppressed the cytokine-induced expression of proinflammatory adhesion molecules. The results suggest that rHDL inhibits NADPH oxidase activation in leukocytes, probably by interrupting the assembly of NADPH oxidase sub-units at the lipid rafts. This effect may contribute to the vascular protective actions of rHDL against inflammation-mediated oxidative damage.

An updated patent review: xanthine oxidase inhibitors for the treatment of hyperuricemia and gout (2011-2015).

PubMed

Ojha, Ritu; Singh, Jagjeet; Ojha, Anu; Singh, Harbinder; Sharma, Sahil; Nepali, Kunal

2017-03-01

Xanthine oxidase (XO) is a versatile molybdoflavoprotein, widely distributed, occurring in milk, kidney, lung, heart, and vascular endothelium. Catalysis by XO to produce uric acid and reactive oxygen species leads to many diseases. Anti hyperuricemic therapy by xanthine oxidase inhibitors has been mainly employed for the treatment of gout. Area covered: This review covers the patent literature (2011-2015) and also presents the interesting strategies/rational approaches employed for the design of xanthine oxidase inhibitors reported recently. Expert opinion: Recent literature indicates that various non purine scaffolds have been extensively investigated for xanthine oxidase inhibition. The significant potential endowed by heteroaryl based compounds, in particularly fused heterocycles clearly highlights their clinical promise and the need for detailed investigation. Studies by various research groups have also revealed that the flavone framework is open for isosteric replacements and structural modifications for yielding potent non purine xanthine oxidase inhibitors. In addition, various plant extracts recently reported to possess significant xanthine oxidase inhibitory potential presents enough promise to initiate a screening program for the identification of other plant extracts and phytoconstituents possessing inhibitory potential towards the enzyme.

NADPH Oxidase versus Mitochondria-Derived ROS in Glucose-Induced Apoptosis of Pericytes in Early Diabetic Retinopathy

PubMed Central

Mustapha, Nik M.; Tarr, Joanna M.; Kohner, Eva M.; Chibber, Rakesh

2010-01-01

Objectives. Using apocynin (inhibitor of NADPH oxidase), and Mitoquinol 10 nitrate (MitoQ; mitochondrial-targeted antioxidant), we addressed the importance of mitochondria versus NADPH oxidase-derived ROS in glucose-induced apoptosis of pericytes. Methods. NADPH oxidase was localised using Western blot analysis and cytochrome C reduction assay. Apoptosis was detected by measuring caspase-3 activity. Intracellular glucose concentration, ROS formation and Nε-(carboxymethyl) lysine (CML) content were measured using Amplex Red assay kit, dihydroethidium (DHE), and competitive immunoabsorbant enzyme-linked assay (ELISA), respectively. Results. NADPH oxidase was localised in the cytoplasm of pericytes suggesting ROS production within intracellular compartments. High glucose (25 mM) significantly increased apoptosis, intracellular glucose concentration, and CML content. Apoptosis was associated with increased gp91phox expression, activity of NADPH oxidase, and intracellular ROS production. Apocynin and not MitoQ significantly blunted the generation of ROS, formation of intracellular CML and apoptosis. Conclusions. NADPH oxidase and not mitochondria-derived ROS is responsible for the accelerated apoptosis of pericytes in diabetic retinopathy. PMID:20652059

Roothairless5, which functions in maize (Zea mays L.) root hair initiation and elongation encodes a monocot-specific NADPH oxidase.

PubMed

Nestler, Josefine; Liu, Sanzhen; Wen, Tsui-Jung; Paschold, Anja; Marcon, Caroline; Tang, Ho Man; Li, Delin; Li, Li; Meeley, Robert B; Sakai, Hajime; Bruce, Wesley; Schnable, Patrick S; Hochholdinger, Frank

2014-09-01

Root hairs are instrumental for nutrient uptake in monocot cereals. The maize (Zea mays L.) roothairless5 (rth5) mutant displays defects in root hair initiation and elongation manifested by a reduced density and length of root hairs. Map-based cloning revealed that the rth5 gene encodes a monocot-specific NADPH oxidase. RNA-Seq, in situ hybridization and qRT-PCR experiments demonstrated that the rth5 gene displays preferential expression in root hairs but also accumulates to low levels in other tissues. Immunolocalization detected RTH5 proteins in the epidermis of the elongation and differentiation zone of primary roots. Because superoxide and hydrogen peroxide levels are reduced in the tips of growing rth5 mutant root hairs as compared with wild-type, and Reactive oxygen species (ROS) is known to be involved in tip growth, we hypothesize that the RTH5 protein is responsible for establishing the high levels of ROS in the tips of growing root hairs required for elongation. Consistent with this hypothesis, a comparative RNA-Seq analysis of 6-day-old rth5 versus wild-type primary roots revealed significant over-representation of only two gene ontology (GO) classes related to the biological functions (i.e. oxidation/reduction and carbohydrate metabolism) among 893 differentially expressed genes (FDR <5%). Within these two classes the subgroups 'response to oxidative stress' and 'cellulose biosynthesis' were most prominently represented. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

Genetics Home Reference: isolated sulfite oxidase deficiency

MedlinePlus

... Metabolic Disorders (CLIMB) March of Dimes: Amino Acid Metabolism Disorders The Compassionate Friends GeneReviews (1 link) Isolated Sulfite Oxidase Deficiency ClinicalTrials.gov (1 link) ClinicalTrials.gov Scientific Articles on PubMed (1 link) PubMed OMIM (1 link) ...

PKC delta and NADPH oxidase in retinoic acid-induced neuroblastoma cell differentiation.

PubMed

Nitti, Mariapaola; Furfaro, Anna Lisa; Cevasco, Claudia; Traverso, Nicola; Marinari, Umberto Maria; Pronzato, Maria Adelaide; Domenicotti, Cinzia

2010-05-01

The role of reactive oxygen species (ROS) in the regulation of signal transduction processes has been well established in many cell types and recently the fine tuning of redox signalling in neurons received increasing attention. With regard to this, the involvement of NADPH oxidase (NOX) in neuronal pathophysiology has been proposed but deserves more investigation. In the present study, we used SH-SY5Y neuroblastoma cells to analyse the role of NADPH oxidase in retinoic acid (RA)-induced differentiation, pointing out the involvement of protein kinase C (PKC) delta in the activation of NOX. Retinoic acid induces neuronal differentiation as revealed by the increased expression of MAP2, the decreased cell doubling rate, and the gain in neuronal morphological features and these events are accompanied by the increased expression level of PKC delta and p67(phox), one of the components of NADPH oxidase. Using DPI to inhibit NOX activity we show that retinoic acid acts through this enzyme to induce morphological changes linked to the differentiation. Moreover, using rottlerin to inhibit PKC delta or transfection experiments to overexpress it, we show that retinoic acid acts through this enzyme to induce MAP2 expression and to increase p67(phox) membrane translocation leading to NADPH oxidase activation. These findings identify the activation of PKC delta and NADPH oxidase as crucial steps in RA-induced neuroblastoma cell differentiation. 2010 Elsevier Inc. All rights reserved.

Structural insights into electron transfer in caa3-type cytochrome oxidase

PubMed Central

Lyons, Joseph A.; Aragão, David; Slattery, Orla; Pisliakov, Andrei V.; Soulimane, Tewfik; Caffrey, Martin

2012-01-01

Summary Paragraph Cytochrome c oxidase is a member of the heme copper oxidase superfamily (HCO)1. HCOs function as the terminal enzymes in the respiratory chain of mitochondria and aerobic prokaryotes, coupling molecular oxygen reduction to transmembrane proton pumping. Integral to the enzyme’s function is the transfer of electrons from cytochrome c to the oxidase via a transient association of the two proteins. Electron entry and exit are proposed to occur from the same site on cytochrome c2–4. Here we report the crystal structure of the caa3-type cytochrome oxidase from Thermus thermophilus, which has a covalently tethered cytochrome c domain. Crystals were grown in a bicontinuous mesophase using a synthetic short-chain monoacylglycerol as the hosting lipid. From the electron density map, at 2.36 Å resolution, a novel integral membrane subunit and a native glycoglycerophospholipid embedded in the complex were identified. Contrary to previous electron transfer mechanisms observed for soluble cytochrome c, the structure reveals the architecture of the electron transfer complex for the fused cupredoxin/cytochrome c domain which implicates different sites on cytochrome c for electron entry and exit. Support for an alternative to the classical proton gate characteristic of this HCO class is presented. PMID:22763450

Congruence between PM H+-ATPase and NADPH oxidase during root growth: a necessary probability.

PubMed

Majumdar, Arkajo; Kar, Rup Kumar

2018-07-01

Plasma membrane (PM) H + -ATPase and NADPH oxidase (NOX) are two key enzymes responsible for cell wall relaxation during elongation growth through apoplastic acidification and production of ˙OH radical via O 2 ˙ - , respectively. Our experiments revealed a putative feed-forward loop between these enzymes in growing roots of Vigna radiata (L.) Wilczek seedlings. Thus, NOX activity was found to be dependent on proton gradient generated across PM by H + -ATPase as evident from pharmacological experiments using carbonyl cyanide m-chlorophenylhydrazone (CCCP; protonophore) and sodium ortho-vanadate (PM H + -ATPase inhibitor). Conversely, H + -ATPase activity retarded in response to different ROS scavengers [CuCl 2 , N, N' -dimethylthiourea (DMTU) and catalase] and NOX inhibitors [ZnCl 2 and diphenyleneiodonium (DPI)], while H 2 O 2 promoted PM H + -ATPase activity at lower concentrations. Repressing effects of Ca +2 antagonists (La +3 and EGTA) on the activity of both the enzymes indicate its possible mediation. Since, unlike animal NOX, the plant versions do not possess proton channel activity, harmonized functioning of PM H + -ATPase and NOX appears to be justified. Plasma membrane NADPH oxidase and H + -ATPase are functionally synchronized and they work cooperatively to maintain the membrane electrical balance while mediating plant cell growth through wall relaxation.

Crystal Structures of Intermediates in the Nitroalkane Oxidase Reaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heroux, A.; Bozinovski, D; Valley, M

2009-01-01

The flavoenzyme nitroalkane oxidase is a member of the acyl-CoA dehydrogenase superfamily. Nitroalkane oxidase catalyzes the oxidation of neutral nitroalkanes to nitrite and the corresponding aldehydes or ketones. Crystal structures to 2.2 {angstrom} resolution or better of enzyme complexes with bound substrates and of a trapped substrate-flavin adduct are described. The D402N enzyme has no detectable activity with neutral nitroalkanes. The structure of the D402N enzyme crystallized in the presence of 1-nitrohexane or 1-nitrooctane shows the presence of the substrate in the binding site. The aliphatic chain of the substrate extends into a tunnel leading to the enzyme surface. Themore » oxygens of the substrate nitro group interact both with amino acid residues and with the 2'-hydroxyl of the FAD. When nitroalkane oxidase oxidizes nitroalkanes in the presence of cyanide, an electrophilic flavin imine intermediate can be trapped (Valley, M. P., Tichy, S. E., and Fitzpatrick, P. F. (2005) J. Am. Chem. Soc. 127, 2062-2066). The structure of the enzyme trapped with cyanide during oxidation of 1-nitrohexane shows the presence of the modified flavin. A continuous hydrogen bond network connects the nitrogen of the CN-hexyl-FAD through the FAD 2'-hydroxyl to a chain of water molecules extending to the protein surface. Together, our complementary approaches provide strong evidence that the flavin cofactor is in the appropriate oxidation state and correlates well with the putative intermediate state observed within each of the crystal structures. Consequently, these results provide important structural descriptions of several steps along the nitroalkane oxidase reaction cycle.« less

A gene encoding the plant-like alternative oxidase is present in Phytomonas but absent in Leishmania spp.

PubMed

Van Hellemond, J J; Simons, B; Millenaar, F F; Tielens, A G

1998-01-01

The constituents of the respiratory chain are believed to differ among the trypanosomatids; bloodstream stages of African trypanosomes and Phytomonas promastigotes oxidize ubiquinol by a ubiquinol:oxygen oxidoreductase, also known as alternative oxidase, whereas Leishmania spp. oxidize ubiquinol via a classic cytochrome-containing respiratory chain. The molecular basis for this elementary difference in ubiquinol oxidation by the mitochondrial electron-transport chain in distinct trypanosomatids was investigated. The presence of a gene encoding the plant-like alternative oxidase could be demonstrated in Phytomonas and Trypanosoma brucei, trypanosomatids that are known to contain alternative oxidase activity. Our results further demonstrated that Leishmania spp. lack a gene encoding the plant-like alternative oxidase, and therefore, all stages of Leishmania spp. will lack the alternative oxidase protein. The observed fundamental differences between the respiratory chains of distinct members of the trypanosomatid family are thus caused by the presence or absence of a gene encoding the plant-like alternative oxidase.

Androgen receptor and monoamine oxidase polymorphism in wild bonobos

PubMed Central

Garai, Cintia; Furuichi, Takeshi; Kawamoto, Yoshi; Ryu, Heungjin; Inoue-Murayama, Miho

2014-01-01

Androgen receptor gene (AR), monoamine oxidase A gene (MAOA) and monoamine oxidase B gene (MAOB) have been found to have associations with behavioral traits, such as aggressiveness, and disorders in humans. However, the extent to which similar genetic effects might influence the behavior of wild apes is unclear. We examined the loci AR glutamine repeat (ARQ), AR glycine repeat (ARG), MAOA intron 2 dinucleotide repeat (MAin2) and MAOB intron 2 dinucleotide repeat (MBin2) in 32 wild bonobos, Pan paniscus, and compared them with those of chimpanzees, Pan troglodytes, and humans. We found that bonobos were polymorphic on the four loci examined. Both loci MAin2 and MBin2 in bonobos showed a higher diversity than in chimpanzees. Because monoamine oxidase influences aggressiveness, the differences between the polymorphisms of MAin2 and MBin2 in bonobos and chimpanzees may be associated with the differences in aggression between the two species. In order to understand the evolution of these loci and AR, MAOA and MAOB in humans and non-human primates, it would be useful to conduct future studies focusing on the potential association between aggressiveness, and other personality traits, and polymorphisms documented in bonobos. PMID:25606465

Ultrafine carbon particles promote rotenone-induced dopamine neuronal loss through activating microglial NADPH oxidase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Yinxi; Liu, Dan; Zhang, Huifeng

Background: Atmospheric ultrafine particles (UFPs) and pesticide rotenone were considered as potential environmental risk factors for Parkinson's disease (PD). However, whether and how UFPs alone and in combination with rotenone affect the pathogenesis of PD remains largely unknown. Methods: Ultrafine carbon black (ufCB, a surrogate of UFPs) and rotenone were used individually or in combination to determine their roles in chronic dopaminergic (DA) loss in neuron-glia, and neuron-enriched, mix-glia cultures. Immunochemistry using antibody against tyrosine hydroxylase was performed to detect DA neuronal loss. Measurement of extracellular superoxide and intracellular reactive oxygen species (ROS) were performed to examine activation of NADPHmore » oxidase. Genetic deletion and pharmacological inhibition of NADPH oxidase and MAC-1 receptor in microglia were employed to examine their role in DA neuronal loss triggered by ufCB and rotenone. Results: In rodent midbrain neuron-glia cultures, ufCB and rotenone alone caused neuronal death in a dose-dependent manner. In particularly, ufCB at doses of 50 and 100 μg/cm{sup 2} induced significant loss of DA neurons. More importantly, nontoxic doses of ufCB (10 μg/cm{sup 2}) and rotenone (2 nM) induced synergistic toxicity to DA neurons. Microglial activation was essential in this process. Furthermore, superoxide production from microglial NADPH oxidase was critical in ufCB/rotenone-induced neurotoxicity. Studies in mix-glia cultures showed that ufCB treatment activated microglial NADPH oxidase to induce superoxide production. Firstly, ufCB enhanced the expression of NADPH oxidase subunits (gp91{sup phox}, p47{sup phox} and p40{sup phox}); secondly, ufCB was recognized by microglial surface MAC-1 receptor and consequently promoted rotenone-induced p47{sup phox} and p67{sup phox} translocation assembling active NADPH oxidase. Conclusion: ufCB and rotenone worked in synergy to activate NADPH oxidase in microglia, leading to

Analysis of cellulase and polyphenol oxidase production by southern pine beetle associated fungi

Treesearch

Abduvali Valiev; Zumrut B. Ogel; Dier D. Klepzig

2009-01-01

In this study, the production of extracellular enzymes by fungi associated with southern pine beetle was investigated for the first time. Cellulase and polyphenol oxidase production were analyzed for three beetle associated fungi. Only the mutualistic symbiont Entomocorticium sp. A was found to produce cellulases and polyphenol oxidase....

Inhibition of the NADPH oxidase regulates HO-1 expression in chronic myeloid leukemia

PubMed Central

Singh, Melissa M.; Irwin, Mary E.; Gao, Yin; Ban, Kechen; Shi, Ping; Arlinghaus, Ralph B.; Amin, Hesham M.; Chandra, Joya

2011-01-01

Background Patients with blast crisis phase chronic myelogeneous leukemia (CML) have poor response to tyrosine kinase inhibitors designed to inhibit the BCR-ABL1 oncogene. Recent work has shown that heme oxygenase 1 (HO-1) expression is increased in BCR-ABL1 expressing cells and that inhibition of HO-1 in CML leads to reduced cellular growth suggesting HO-1 may be a plausible target for therapy. Here we sought to clarify the mechanism of HO-1 overexpression and the role of the NADPH oxidase as a contributor to this mechanism in CML. Methods HO-1 expression was evaluated in CML bone marrow specimens from patients in various stages of disease, in a transplant based model for CML and in CML cell lines. Chemical and genetic inhibition of the NADPH oxidase was carried out in CML cells. Results Blast crisis CML patient specimens displayed higher levels of HO-1 staining than chronic or accelerated phase. HO-1 upregulation in BCR-ABL1 expressing cells was suppressed by diphenyliodonium (DPI), a chemical inhibitor of the NADPH oxidase. Targeting the NADPH oxidase through RNAi to Rac1, a dominant negative Rac1 construct or an inhibitor of Rac1 activity also blunted HO-1 protein expression. Moreover, inhibition of the NADPH oxidase by RNAi directed towards p47phox similarly abrogated HO-1 levels. Conclusion BCR-ABL1 expression upregulates HO-1, a survival factor for CML cells. This upregulation is more pronounced in blast crisis CML relative to early stage disease and is mediated by the NADPH oxidase components Rac1 and p47phox. Expression of p47phox is increased in BCR-ABL1 expressing cells. PMID:22139798

Functional Assembly of Soluble and Membrane Recombinant Proteins of Mammalian NADPH Oxidase Complex.

PubMed

Souabni, Hajer; Ezzine, Aymen; Bizouarn, Tania; Baciou, Laura

2017-01-01

Activation of phagocyte cells from an innate immune system is associated with a massive consumption of molecular oxygen to generate highly reactive oxygen species (ROS) as microbial weapons. This is achieved by a multiprotein complex, the so-called NADPH oxidase. The activity of phagocyte NADPH oxidase relies on an assembly of more than five proteins, among them the membrane heterodimer named flavocytochrome b 558 (Cytb 558 ), constituted by the tight association of the gp91 phox (also named Nox2) and p22 phox proteins. The Cytb 558 is the membrane catalytic core of the NADPH oxidase complex, through which the reducing equivalent provided by NADPH is transferred via the associated prosthetic groups (one flavin and two hemes) to reduce dioxygen into superoxide anion. The other major proteins (p47 phox , p67 phox , p40 phox , Rac) requisite for the complex activity are cytosolic proteins. Thus, the NADPH oxidase functioning relies on a synergic multi-partner assembly that in vivo can be hardly studied at the molecular level due to the cell complexity. Thus, a cell-free assay method has been developed to study the NADPH oxidase activity that allows measuring and eventually quantifying the ROS generation based on optical techniques following reduction of cytochrome c. This setup is a valuable tool for the identification of protein interactions, of crucial components and additives for a functional enzyme. Recently, this method was improved by the engineering and the production of a complete recombinant NADPH oxidase complex using the combination of purified proteins expressed in bacterial and yeast host cells. The reconstitution into artificial membrane leads to a fully controllable system that permits fine functional studies.

NADPH oxidases in the arbuscular mycorrhizal symbiosis.

PubMed

Belmondo, Simone; Calcagno, Cristina; Genre, Andrea; Puppo, Alain; Pauly, Nicolas; Lanfranco, Luisa

2016-01-01

Plant NADPH oxidases are the major source of reactive oxygen species (ROS) that plays key roles as both signal and stressor in several plant processes, including defense responses against pathogens. ROS accumulation in root cells during arbuscular mycorrhiza (AM) development has raised the interest in understanding how ROS-mediated defense programs are modulated during the establishment of this mutualistic interaction. We have recently analyzed the expression pattern of 5 NADPH oxidase (also called RBOH) encoding genes in Medicago truncatula, showing that only one of them (MtRbohE) is specifically upregulated in arbuscule-containing cells. In line with this result, RNAi silencing of MtRbohE generated a strong alteration in root colonization, with a significant reduction in the number of arbusculated cells. On this basis, we propose that MtRBOHE-mediated ROS production plays a crucial role in the intracellular accommodation of arbuscules.

NADPH oxidases in the arbuscular mycorrhizal symbiosis

PubMed Central

Belmondo, Simone; Calcagno, Cristina; Genre, Andrea; Puppo, Alain; Pauly, Nicolas; Lanfranco, Luisa

2016-01-01

ABSTRACT Plant NADPH oxidases are the major source of reactive oxygen species (ROS) that plays key roles as both signal and stressor in several plant processes, including defense responses against pathogens. ROS accumulation in root cells during arbuscular mycorrhiza (AM) development has raised the interest in understanding how ROS-mediated defense programs are modulated during the establishment of this mutualistic interaction. We have recently analyzed the expression pattern of 5 NADPH oxidase (also called RBOH) encoding genes in Medicago truncatula, showing that only one of them (MtRbohE) is specifically upregulated in arbuscule-containing cells. In line with this result, RNAi silencing of MtRbohE generated a strong alteration in root colonization, with a significant reduction in the number of arbusculated cells. On this basis, we propose that MtRBOHE-mediated ROS production plays a crucial role in the intracellular accommodation of arbuscules. PMID:27018627

Xanthine oxidase biosensor for monitoring meat spoilage

NASA Astrophysics Data System (ADS)

Vanegas, D. C.; Gomes, C.; McLamore, E. S.

2014-05-01

In this study, we have designed an electrochemical biosensor for real-time detection of specific biomarkers of bacterial metabolism related to meat spoilage (hypoxanthine and xanthine). The selective biosensor was developed by assembling a `sandwich' of nanomaterials and enzymes on a platinum-iridium electrode (1.6 mm tip diameter). The materials deposited on the sensor tip include amorphous platinum nanoclusters (i.e. Pt black), reduced graphene oxide, nanoceria, and xanthine oxidase. Xanthine oxidase was encapsulated in laponite hydrogel and used for the biorecognition of hypoxanthine and xanthine (two molecules involved in the rotting of meat by spoilage microorganisms). The developed biosensor demonstrated good electrochemical performance toward xanthine with sensitivity of 2.14 +/- 1.48 μA/mM, response time of 5.2 +/- 1.5 sec, lower detection limit of 150 +/- 39 nM, and retained at least 88% of its activity after 7 days of continuous use.

Biosensor for the enantioselective analysis of the thyroid hormones (+)-3,3',5-triiodo-L-thyronine (T3) and (+)-3,3',5,5'-tetraiodo-L-thyronine (T4).

PubMed

Aboul-Enein, Hassan V; Stefan, Raluca-Ioana; Litescu, Simona; Radu, Gabriel Lucian

2002-01-01

An amperometric biosensor based on L-aminoacid oxidase is proposed for enantioselective assay of (+)-3,3',5-triiodo-L-thyronine (L-T3) and (+)-3,3',5,5'-tetraiodo-L-thyronine (L-T4), due to the fact that only the L enantiomer has the hormonal activity. The construction of the amperometric biosensor is simple and reproducible. The analytical information obtained from enantioselective analysis are reliable. The RSD <1% assured by using the amperometric biosensors for L enantiomers assay as raw materials, and from tablets, demonstrated their suitability for the analysis of T3 and T4 at ppb concentration levels.

Oxoferryl-porphyrin radical catalytic intermediate in cytochrome bd oxidases protects cells from formation of reactive oxygen species.

PubMed

Paulus, Angela; Rossius, Sebastiaan Gijsbertus Hendrik; Dijk, Madelon; de Vries, Simon

2012-03-16

The quinol-linked cytochrome bd oxidases are terminal oxidases in respiration. These oxidases harbor a low spin heme b(558) that donates electrons to a binuclear heme b(595)/heme d center. The reaction with O(2) and subsequent catalytic steps of the Escherichia coli cytochrome bd-I oxidase were investigated by means of ultra-fast freeze-quench trapping followed by EPR and UV-visible spectroscopy. After the initial binding of O(2), the O-O bond is heterolytically cleaved to yield a kinetically competent heme d oxoferryl porphyrin π-cation radical intermediate (compound I) magnetically interacting with heme b(595). Compound I accumulates to 0.75-0.85 per enzyme in agreement with its much higher rate of formation (~20,000 s(-1)) compared with its rate of decay (~1,900 s(-1)). Compound I is next converted to a short lived heme d oxoferryl intermediate (compound II) in a phase kinetically matched to the oxidation of heme b(558) before completion of the reaction. The results indicate that cytochrome bd oxidases like the heme-copper oxidases break the O-O bond in a single four-electron transfer without a peroxide intermediate. However, in cytochrome bd oxidases, the fourth electron is donated by the porphyrin moiety rather than by a nearby amino acid. The production of reactive oxygen species by the cytochrome bd oxidase was below the detection level of 1 per 1000 turnovers. We propose that the two classes of terminal oxidases have mechanistically converged to enzymes in which the O-O bond is broken in a single four-electron transfer reaction to safeguard the cell from the formation of reactive oxygen species.

Proton gradients produced by glucose oxidase microcapsules containing motor F0F1-ATPase for continuous ATP biosynthesis.

PubMed

Duan, Li; Qi, Wei; Yan, Xuehai; He, Qiang; Cui, Yue; Wang, Kewei; Li, Dongxiang; Li, Junbai

2009-01-15

Glucose oxidase (GOD) microcapsules held together by cross-linker, glutaraldehyde (GA), are fabricated by the layer-by-layer (LbL) assembly technique. The lipid bilayer containing CF(0)F(1)-ATPase was coated on the outer shell of GOD microcapsules. Driven under the proton gradients produced by catalysis of GOD microcapsules for glucose, ATP is synthesized from ADP and inorganic phosphate catalyzed by the ATPase rotary catalysis. The results show here that ATPase reconstituted on the GOD microcapsules retains its catalytic activity.

Immunological comparison of sulfite oxidase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pollock, V.; Barber, M.J.

1991-03-11

Polyclonal antibodies (rabbit), elicited against FPLC-purified chicken and rat liver sulfite oxidase (SO), have been examined for inhibition and binding to purified chicken (C), rat (R), bovine (B), alligator (A) and shark (S) liver enzymes. Anti-CSO IgG cross-reacted with all five enzymes, with varying affinities, in the order CSO=ASO{gt}RSO{gt}BSO{gt}SSO. Anti-ROS IgG also cross-reacted with all five enzymes in the order RSO{gt}CSO=ASO{gt}BSO{gt}SSO. Anti-CSO IgG inhibited sulfite:cyt. c reductase (S:CR), sulfite:ferricyanide reductase (S:FR) and sulfite:dichlorophenolindophenol reductase (S:DR) activities of CSO to different extents (S:CR{gt}S:FR=S:DR). Similar differential inhibition was found for anti-ROS IgG and RSO S:CR, S:FR and S:DR activities. Anti-CSO IgG inhibitedmore » S:CR activities in the order CSO=ASO{much gt}SSO{gt}BSO. RSO was uninhibited. For anti-RSO IgG the inhibition order was RSO{gt}SSO{gt}BSO{gt}ASO. CSO was uninhibited. Anti-CSO and RSO IgGs partially inhibited Chlorella nitrate reductase (NR). Minor cross-reactivity was found for xanthine oxidase. Common antigenic determinants for all five SO's and NR are indicated.« less

Glutamate Excitotoxicity Linked to Spermine Oxidase Overexpression.

PubMed

Pietropaoli, Stefano; Leonetti, Alessia; Cervetto, Chiara; Venturini, Arianna; Mastrantonio, Roberta; Baroli, Giulia; Persichini, Tiziana; Colasanti, Marco; Maura, Guido; Marcoli, Manuela; Mariottini, Paolo; Cervelli, Manuela

2018-02-03

Excitotoxic stress has been associated with several different neurological disorders, and it is one of the main causes of neuronal degeneration and death. To identify new potential proteins that could represent key factors in excitotoxic stress and to study the relationship between polyamine catabolism and excitotoxic damage, a novel transgenic mouse line overexpressing spermine oxidase enzyme in the neocortex (Dach-SMOX) has been engineered. These transgenic mice are more susceptible to excitotoxic injury and display a higher oxidative stress, highlighted by 8-Oxo-2'-deoxyguanosine increase and activation of defense mechanisms, as demonstrated by the increase of nuclear factor erythroid 2-related factor 2 (Nrf-2) in the nucleus. In Dach-SMOX astrocytes and neurons, an alteration of the phosphorylated and non-phosphorylated subunits of glutamate receptors increases the kainic acid response in these mice. Moreover, a decrease in excitatory amino acid transporters and an increase in the system x c - transporter, a Nrf-2 target, was observed. Sulfasalazine, a system x c - transporter inhibitor, was shown to revert the increased susceptibility of Dach-SMOX mice treated with kainic acid. We demonstrated that astrocytes play a crucial role in this process: neuronal spermine oxidase overexpression resulted in an alteration of glutamate excitability, in glutamate uptake and efflux in astrocytes involved in the synapse. Considering the involvement of oxidative stress in many neurodegenerative diseases, Dach-SMOX transgenic mouse can be considered as a suitable in vivo genetic model to study the involvement of spermine oxidase in excitotoxicity, which can be considered as a possible therapeutic target.

Design, synthesis and biological evaluation of novel xanthine oxidase inhibitors bearing a 2-arylbenzo[b]furan scaffold.

PubMed

Tang, Hong-Jin; Li, Wei; Zhou, Mei; Peng, Li-Ying; Wang, Jin-Xin; Li, Jia-Huang; Chen, Jun

2018-05-10

Xanthine oxidase, which catalyzes the oxidative reaction of hypoxanthine and xanthine into uric acid, is a key enzyme to the pathogenesis of hyperuricemia and gout. In this study, for the purpose of discovering novel xanthine oxidase (XO) inhibitors, a series of 2-arylbenzo[b]furan derivatives (3a-3d, 4a-4o and 6a-6d) were designed and synthesized. All these compounds were evaluated their xanthine oxidase inhibitory and antioxidant activities by using in vitro enzymatic assay and cellular model. The results showed that a majority of the designed compounds exhibited potent xanthine oxidase inhibitory effects and antioxidant activities, and compound 4a emerged as the most potent xanthine oxidase inhibitor (IC 50  = 4.45 μM). Steady-state kinetic measurements of the inhibitor 4a with the bovine milk xanthine oxidase indicated a mixed type inhibition with 3.52 μM K i and 13.14 μM K is , respectively. The structure-activity relationship analyses have also been presented. Compound 4a exhibited the potent hypouricemic effect in the potassium oxonate-induced hyperuricemic mice model. A molecular docking study of compound 4a was performed to gain an insight into its binding mode with xanthine oxidase. These results highlight the identification of a new class of xanthine oxidase inhibitors that have potential to be more efficacious in treatment of gout. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

A decade of crystallization drops: Crystallization of the cbb3 cytochrome c oxidase from Pseudomonas stutzeri

PubMed Central

Buschmann, Sabine; Richers, Sebastian; Ermler, Ulrich; Michel, Hartmut

2014-01-01

The cbb3 cytochrome c oxidases are distant members of the superfamily of heme copper oxidases. These terminal oxidases couple O2 reduction with proton transport across the plasma membrane and, as a part of the respiratory chain, contribute to the generation of an electrochemical proton gradient. Compared with other structurally characterized members of the heme copper oxidases, the recently determined cbb3 oxidase structure at 3.2 Å resolution revealed significant differences in the electron supply system, the proton conducting pathways and the coupling of O2 reduction to proton translocation. In this paper, we present a detailed report on the key steps for structure determination. Improvement of the protein quality was achieved by optimization of the number of lipids attached to the protein as well as the separation of two cbb3 oxidase isoenzymes. The exchange of n-dodecyl-β-d-maltoside for a precisely defined mixture of two α-maltosides and decanoylsucrose as well as the choice of the crystallization method had a most profound impact on crystal quality. This report highlights problems frequently encountered in membrane protein crystallization and offers meaningful approaches to improve crystal quality. PMID:24488923

A decade of crystallization drops: crystallization of the cbb3 cytochrome c oxidase from Pseudomonas stutzeri.

PubMed

Buschmann, Sabine; Richers, Sebastian; Ermler, Ulrich; Michel, Hartmut

2014-04-01

The cbb3 cytochrome c oxidases are distant members of the superfamily of heme copper oxidases. These terminal oxidases couple O2 reduction with proton transport across the plasma membrane and, as a part of the respiratory chain, contribute to the generation of an electrochemical proton gradient. Compared with other structurally characterized members of the heme copper oxidases, the recently determined cbb3 oxidase structure at 3.2 Å resolution revealed significant differences in the electron supply system, the proton conducting pathways and the coupling of O2 reduction to proton translocation. In this paper, we present a detailed report on the key steps for structure determination. Improvement of the protein quality was achieved by optimization of the number of lipids attached to the protein as well as the separation of two cbb3 oxidase isoenzymes. The exchange of n-dodecyl-β-D-maltoside for a precisely defined mixture of two α-maltosides and decanoylsucrose as well as the choice of the crystallization method had a most profound impact on crystal quality. This report highlights problems frequently encountered in membrane protein crystallization and offers meaningful approaches to improve crystal quality. © 2014 The Protein Society.

Age-related ultrastructural and monoamine oxidase changes in the rat optic nerve.

PubMed

Taurone, S; Ripandelli, G; Minni, A; Lattanzi, R; Miglietta, S; Pepe, N; Fumagalli, L; Micera, A; Pastore, F S; Artico, M

2016-01-01

The aim of this paper is to study the morphology and the distribution of the monoamine oxidase enzymatic system in the optic nerve of 4 month-old Wistar (young) and 28 month-old Wistar (old) rats. The optic nerve was harvested from 20 young and old rats. The segment of optic nerve was divided longitudinally into two pieces, each 0.1 mm in length. The first piece was used for transmission electron microscopy. The second piece was stained with histochemical reaction for monoamine oxidase. The agerelated changes in the optic nerve of rats include micro-anatomical details, ultrastructure and monoamine oxidase histochemical staining. A strong decrease of the thin nerve fibers and a swelling of the thick ones can be observed in optic nerve fibers of old rats. Increased monoamine oxidase histochemical staining of the optic nerve of aged rats is well demonstrated. The increase of meningeal shealth and the decrease of thin nerve fibers of the optic nerve in old rats are well documented. Morphological, ultrastructural and histochemical changes observed in optic nerve fibers of the old rats show a close relation with aging.

Abnormal kinetic behavior of cytochrome oxidase in a case of Leigh disease.

PubMed Central

Glerum, M; Robinson, B H; Spratt, C; Wilson, J; Patrick, D

1987-01-01

Cultured skin fibroblasts from a child with fatal lacticacidemia displayed an abnormally high lactate:pyruvate ratio of 77:1, compared with control values of 22:1-27:1. When protease-treated isolated mitochondria were used, activity of the respiratory-chain enzymes was found to be approximately 60% of normal, and adenosine triphosphate synthesis was found to be normal with all substrates tested. In mitochondria prepared by means of digitonin treatment, adenosine triphosphate synthesis was depressed with all substrates tested, suggesting a defect in the operation of the cytochrome oxidase complex. In disrupted whole cells from the patient, cytochrome oxidase activity was 56% of the activity in the control cell line with the lowest activity. In the presence of a twofold excess of oxidized cytochrome c, patient cells showed 31% of the activity in controls. Cytochrome oxidase activity in both sonicated whole-cell preparations and in sonicated mitochondria displayed abnormal kinetics with regard to the substrate-reduced cytochrome c, which was particularly evident in the presence of excess oxidized cytochrome c. We believe that kinetically abnormal cytochrome oxidase complex is responsible for the biochemical and clinical abnormalities present in this patient. PMID:2821802

Genetics Home Reference: cytochrome c oxidase deficiency

MedlinePlus

... are caused by mutations in genes found within nuclear DNA; however, in some rare instances, mutations in genes located within mtDNA cause this condition. The genes associated with cytochrome c oxidase deficiency are involved in energy production in mitochondria through a process called oxidative ...

Antioxidant and anti-inflammatory effects of Scoparia dulcis L.

PubMed

Coulibaly, Ahmed Y; Kiendrebeogo, Martin; Kehoe, Patrick G; Sombie, Pierre A E D; Lamien, Charles E; Millogo, Jeanne F; Nacoulma, Odile G

2011-12-01

Different extracts were obtained from Scoparia dulcis L. (Scrophulariaceae) by successive extraction with hexane, chloroform, and methanol. These extracts exhibited significant antioxidant capacity in various antioxidant models mediated (xantine oxidase and lipoxygenase) or not mediated (2,2-diphenyl-picrylhydrazyl, ferric-reducing antioxidant power, β-carotene bleaching, lipid peroxidation) by enzymes. The antioxidant activity of the extracts was related to their phytochemical composition in terms of polyphenol and carotenoid contents. The chloroform extract was richest in phytochemicals and had the highest antioxidant activity in the different antioxidant systems. All the extracts exhibited less than 50% inhibition on xanthine oxidase but more than 50% inhibition on lipid peroxidation and lipoxygenase. The extracts strongly inhibited lipid peroxidation mediated by lipoxygenase.

NADPH Oxidase Inhibition Improves Neurological Outcomes in Surgically-Induced Brain Injury

PubMed Central

Lo, Wendy; Bravo, Thomas; Jadhav, Vikram; Zhang, John H.; Tang, Jiping

2007-01-01

Neurosurgical procedures can result in brain injury by various means including direct trauma, hemorrhage, retractor stretch, and electrocautery. This surgically-induced brain injury (SBI) can cause post-operative complications such as brain edema. By creating a mouse model of SBI, we tested whether NADPH oxidase, an important reactive oxygen species producing enzyme, is involved in SBI using transgenic mice lacking gp91phox subunit of NADPH oxidase (gp91phox KO) and apocynin, a specific inhibitor of NADPH oxidase. Neurological function and brain edema were evaluated at 24 hours post-SBI in gp91phox KO and wild-type littermates grouped into SBI and sham-surgery groups. Alternatively, mice were grouped into vehicle- and apocynin-treated (5mg/kg, i.p. 30 minutes before SBI) groups. Oxidative stress indicated by lipid peroxidation (LPO) was measured at 3 and 24 hours post SBI. The gp91phox KO mice, but not the apocynin-treated mice showed significantly improved neurological scores. Brain edema was observed in both gp91phox KO and wild-type groups after SBI; however, there was no significant difference between these two groups. Brain edema was also not affected by apocynin-pretreatment. LPO levels were significantly higher in SBI group in both gp91phox KO and wild-type groups as compared to sham group. A trend, although without statistical significance, was noted towards attenuation of LPO in the gp91phox KO animals as compared to wild-type group. LPO levels were significantly attenuated at 3 hours post-SBI by apocynin pretreatment but not at 24 hours post-SBI. These results suggest that chronic and acute inhibition of NADPH oxidase activity does not reduce brain edema after SBI. Long-term inhibition of NADPH oxidase, however improves neurological functions after SBI. PMID:17317004

Physiological consequences of starvation in Pseudomonas putida: degradation of intracellular protein and loss of activity of the inducible enzymes of L-arginine catabolism.

PubMed

Fan, C L; Rodwell, V W

1975-12-01

We investigated the degradation of radioisotopically labeled intracellular protein in starved, intact cells of Pseudomonas putida P2 (ATCC 25571) and the regulation of this process. Intracellular protein isotopically labeled with L-[4,5-3H]leucine during log-phase growth at 30 C is degraded at rates of 1 to 2%/h in log-phase cells and 7 to 9%/h in starved cells. Rifampin, chloramphenicol, and tosyllysine chloromethylketone lower the rate of protein degradation by starved cells. Addition to starved cells of a nutrient upon which the culture is induced for growth rapidly lowers the rate of protein degradation from 7 to 9%/h to less than 1.5%/h. A nutrient that is oxidized but that cannot immediately support growth also lowers the rate of starvation-induced protein degradation. Proteolytic activity of cell extracts requires a divalent metal ion and may be inhibited up to 60% by tosyllysine chloromethylketone or p-toluenesulfonyl fluoride. Rifampin and chloramphenicol have no effect. In contrast to intact cells, extracts of growing or starving cells degrade protein at equivalent rates. We also investigated the stabilities of the inducible transport system and of four inducible intracellular enzymes of L-arginine catabolism. These include: the membrane-associated, L-arginine-specific transport system; L-arginine oxidase (oxidase); alpha-ketoarginine decarboxylase (decarboxylase); gamma-guanidinobutyraldehyde dehydrogenase ( dehydrogenase); and gamma-guanidinobutyrate amidinohydrolase (hydrolase). In starved cells, the rates of loss of activities were: transport and dehydrogenase activities, stable; oxidase and decarboxylase activities, 20 to 30%/h; hydrolase activity, 5 to 8%/h. Chloramphenicol decreases the rate of loss of oxidase, decarboxylase, and hydrolase activity, whereas p-toluenesulfonyl fluoride lowers the rate of loss of decarboxylase but not of oxidase or hydrolase activity. Addition to starved cells of a nutrient for which they are already induced for

Gene cloning and heterologous expression of pyranose 2-oxidase from the brown-rot fungus, Gloeophyllum trabeum

Treesearch

Diane Dietrich; Casey Crooks

2009-01-01

A pyranose 2-oxidase gene from the brown-rot basidiomycete Gloeophyllum trabeum was isolated using homology-based degenerate PCR. The gene structure was determined and compared to that of several pyranose 2-oxidases cloned from white-rot fungi. The G. trabeum pyranose 2-oxidase gene consists of 16 coding exons with canonical promoter CAAT and TATA elements in the 5’UTR...

New recombinant cyclohexylamine oxidase variants for deracemization of secondary amines by orthogonally assaying designed mutants with structurally diverse substrates

NASA Astrophysics Data System (ADS)

Li, Guangyue; Yao, Peiyuan; Cong, Peiqian; Ren, Jie; Wang, Lei; Feng, Jinhui; Lau, Peter C. K.; Wu, Qiaqing; Zhu, Dunming

2016-05-01

To further expand the substrate range of the cyclohexylamine oxidase (CHAO) from Brevibacterium oxydans, a library of diverse mutants was created and assayed toward a group of structurally diverse substrates. Among them, mutants T198A and M226A exhibited enhanced activity relative to wt CHAO for most (S)-enantiomers of primary amines and some secondary amines. While mutants T198I, L199I, L199F, M226I and M226T were more active than wt CHAO toward the primary amines, mutants T198F, L199T, Y321A, Y321T, Y321I and Y321F enhanced the enzyme activity toward the secondary amines. In particular, mutant Y321I displayed an enhanced catalytic efficiency toward 1-(4-methoxybenzyl)-1, 2, 3, 4, 5, 6, 7, 8-octahydroisoquinoline (13). Whereas a double mutant, Y321I/M226T, acted on (S)-N-(prop-2-yn-1-yl)-2, 3-dihydro-1H-inden-1-amine [(S)-8]. Since (R)-8 is an irreversible inhibitor of monoamine oxidase and (S)-13 is an intermediate of dextromethorphan, a cough suppressant drug, deracemizations of 8 and 13 were carried out with crude enzyme extracts of the respective mutants. This resulted in 51% and 78% isolated yields of (R)-8 and (S)-13, respectively, each with high enantiomeric excess (93% and 99% ee). The results demonstrated the application potential of the evolved CHAO mutants in drug synthesis requiring chiral secondary amines.

New recombinant cyclohexylamine oxidase variants for deracemization of secondary amines by orthogonally assaying designed mutants with structurally diverse substrates

PubMed Central

Li, Guangyue; Yao, Peiyuan; Cong, Peiqian; Ren, Jie; Wang, Lei; Feng, Jinhui; Lau, Peter C.K.; Wu, Qiaqing; Zhu, Dunming

2016-01-01

To further expand the substrate range of the cyclohexylamine oxidase (CHAO) from Brevibacterium oxydans, a library of diverse mutants was created and assayed toward a group of structurally diverse substrates. Among them, mutants T198A and M226A exhibited enhanced activity relative to wt CHAO for most (S)-enantiomers of primary amines and some secondary amines. While mutants T198I, L199I, L199F, M226I and M226T were more active than wt CHAO toward the primary amines, mutants T198F, L199T, Y321A, Y321T, Y321I and Y321F enhanced the enzyme activity toward the secondary amines. In particular, mutant Y321I displayed an enhanced catalytic efficiency toward 1-(4-methoxybenzyl)-1, 2, 3, 4, 5, 6, 7, 8-octahydroisoquinoline (13). Whereas a double mutant, Y321I/M226T, acted on (S)-N-(prop-2-yn-1-yl)-2, 3-dihydro-1H-inden-1-amine [(S)-8]. Since (R)-8 is an irreversible inhibitor of monoamine oxidase and (S)-13 is an intermediate of dextromethorphan, a cough suppressant drug, deracemizations of 8 and 13 were carried out with crude enzyme extracts of the respective mutants. This resulted in 51% and 78% isolated yields of (R)-8 and (S)-13, respectively, each with high enantiomeric excess (93% and 99% ee). The results demonstrated the application potential of the evolved CHAO mutants in drug synthesis requiring chiral secondary amines. PMID:27138090

Electron spin resonance characterization of vascular xanthine and NAD(P)H oxidase activity in patients with coronary artery disease: relation to endothelium-dependent vasodilation.

PubMed

Spiekermann, Stephan; Landmesser, Ulf; Dikalov, Sergey; Bredt, Martin; Gamez, Graciela; Tatge, Helma; Reepschläger, Nina; Hornig, Burkhard; Drexler, Helmut; Harrison, David G

2003-03-18

Increased inactivation of nitric oxide by superoxide (O2*-) contributes to endothelial dysfunction in patients with coronary disease (CAD). We therefore characterized the vascular activities of xanthine oxidase and NAD(P)H oxidase, 2 major O2*--producing enzyme systems, and their relationship with flow-dependent, endothelium-mediated vasodilation (FDD) in patients with CAD. Xanthine- and NAD(P)H-mediated O*.- formation was determined in coronary arteries from 10 patients with CAD and 10 controls by using electron spin resonance spectroscopy. Furthermore, activity of endothelium-bound xanthine oxidase in vivo and FDD of the radial artery were determined in 21 patients with CAD and 10 controls. FDD was measured before and after infusion of the antioxidant vitamin C (25 mg/min i.a.) to determine the portion of FDD inhibited by radicals. In coronary arteries from patients with CAD, xanthine- and NAD(P)H-mediated O2*- formation was increased compared with controls (xanthine: 12+/-2 versus 7+/-1 nmol O2*-/ microg protein; NADH: 11+/-1 versus 7+/-1 nmol O2*-/ microg protein; and NADPH: 12+/-2 versus 9+/-1 nmol O2*-/ microg protein; each P<0.05). Endothelium-bound xanthine oxidase activity was increased by >200% in patients with CAD (25+/-4 versus 9+/-1 nmol O2*-/ microL plasma per min; P<0.05) and correlated inversely with FDD (r=-0.55; P<0.05) and positively with the effect of vitamin C on FDD (r=0.54; P<0.05). The present study represents the first electron spin resonance measurements of xanthine and NAD(P)H oxidase activity in human coronary arteries and supports the concept that increased activities of both enzymes contribute to increased vascular oxidant stress in patients with CAD. Furthermore, the present study suggests that increased xanthine oxidase activity contributes to endothelial dysfunction in patients with CAD and may thereby promote the atherosclerotic process.

d-Alanine Oxidase from Escherichia coli: Localization and Induction by l-Alanine

PubMed Central

Raunio, R. P.; Jenkins, W. T.

1973-01-01

Dialyzed membranes of Escherichia coli prepared by an ethylenediaminetetraacetic acid-lysozyme method catalyze the oxidation of both l-alanine and d-alanine. The specific activities for the oxidations of both d-alanine and l-alanine are increased fivefold when the cells are grown in the presence of either l-alanine or dl-alanine, but are increased only slightly when grown in the presence of d-alanine. In the dl-alanine-induced system, the specific activities for the oxidations of some other d-amino acids are also raised. dl-alanine also induces two other alanine catabolizing enzymes, alanine dehydrogenase and alanine-glutamate aminotransferase which are found in the “soluble” fraction of lysozyme-treated cells. The oxidations of both l-alanine and d-alanine were associated with the membranes of induced cells. After the membranes were disintegrated by sonic treatment, both l-alanine and d-alanine oxidation catalysts sedimented in a sucrose density gradient together with d-lactate and l-lactate dehydrogenases, apparently as a single multienzyme complex. PMID:4146872

Polyamine oxidase activity in rats treated with mitoguazone: specific and permanent decrease in thymus.

PubMed

Ferioli, M E; Armanni, A

2003-01-01

To extend the knowledge on the role of polyamine oxidase in thymus physiology, we evaluated the in vivo effect of the polyamine biosynthetic pathway inhibitor mitoguazone. The drug markedly and permanently decreased the enzyme activity in the organ, in which the level of putrescine also decreased at the later times observed. A byproduct of the reaction catalyzed by polyamine oxidase is hydrogen peroxide, a well known inducer of apoptosis. The decrease in polyamine oxidase activity, with the consequent decrease in hydrogen peroxide production, is correlated with a positive effect on thymus physiology. Since mitoguazone has been successfully employed in patients with AIDS-related diseases, in which the reconstitution of the immune function is a favorable prognostic index, we hypothesized that mitoguazone may have the thymus as target organ, and that the decrease in polyamine oxidase activity may have a role in the positive effect of the drug.

Resveratrol protects vascular endothelial cells from high glucose-induced apoptosis through inhibition of NADPH oxidase activation-driven oxidative stress.

PubMed

Chen, Feng; Qian, Li-Hua; Deng, Bo; Liu, Zhi-Min; Zhao, Ying; Le, Ying-Ying

2013-09-01

Hyperglycemia-induced oxidative stress has been implicated in diabetic vascular complications in which NADPH oxidase is a major source of reactive oxygen species (ROS) generation. Resveratrol is a naturally occurring polyphenol, which has vasoprotective effects in diabetic animal models and inhibits high glucose (HG)-induced oxidative stress in endothelial cells. We aimed to examine whether HG-induced NADPH oxidase activation and ROS production contribute to glucotoxicity to endothelial cells and the effect of resveratrol on glucotoxicity. Using a murine brain microvascular endothelial cell line bEnd3, we found that NADPH oxidase inhibitor (apocynin) and resveratrol both inhibited HG-induced endothelial cell apoptosis. HG-induced elevation of NADPH oxidase activity and production of ROS were inhibited by apocynin, suggesting that HG induces endothelial cell apoptosis through NADPH oxidase-mediated ROS production. Mechanistic studies revealed that HG upregulated NADPH oxidase subunit Nox1 but not Nox2, Nox4, and p22(phox) expression through NF-κB activation, which resulted in elevation of NADPH oxidase activity and consequent ROS production. Resveratrol prevented HG-induced endothelial cell apoptosis through inhibiting HG-induced NF-κB activation, NADPH oxidase activity elevation, and ROS production. HG induces endothelial cell apoptosis through NF-κB/NADPH oxidase/ROS pathway, which was inhibited by resveratrol. Our findings provide new potential therapeutic targets against brain vascular complications of diabetes. © 2013 John Wiley & Sons Ltd.

Oxoferryl-Porphyrin Radical Catalytic Intermediate in Cytochrome bd Oxidases Protects Cells from Formation of Reactive Oxygen Species*

PubMed Central

Paulus, Angela; Rossius, Sebastiaan Gijsbertus Hendrik; Dijk, Madelon; de Vries, Simon

2012-01-01

The quinol-linked cytochrome bd oxidases are terminal oxidases in respiration. These oxidases harbor a low spin heme b558 that donates electrons to a binuclear heme b595/heme d center. The reaction with O2 and subsequent catalytic steps of the Escherichia coli cytochrome bd-I oxidase were investigated by means of ultra-fast freeze-quench trapping followed by EPR and UV-visible spectroscopy. After the initial binding of O2, the O–O bond is heterolytically cleaved to yield a kinetically competent heme d oxoferryl porphyrin π-cation radical intermediate (compound I) magnetically interacting with heme b595. Compound I accumulates to 0.75–0.85 per enzyme in agreement with its much higher rate of formation (∼20,000 s−1) compared with its rate of decay (∼1,900 s−1). Compound I is next converted to a short lived heme d oxoferryl intermediate (compound II) in a phase kinetically matched to the oxidation of heme b558 before completion of the reaction. The results indicate that cytochrome bd oxidases like the heme-copper oxidases break the O–O bond in a single four-electron transfer without a peroxide intermediate. However, in cytochrome bd oxidases, the fourth electron is donated by the porphyrin moiety rather than by a nearby amino acid. The production of reactive oxygen species by the cytochrome bd oxidase was below the detection level of 1 per 1000 turnovers. We propose that the two classes of terminal oxidases have mechanistically converged to enzymes in which the O–O bond is broken in a single four-electron transfer reaction to safeguard the cell from the formation of reactive oxygen species. PMID:22287551

Structure-Based Alteration of Substrate Specificity and Catalytic Activity of Sulfite Oxidase from Sulfite Oxidation to Nitrate Reduction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qiu, James A.; Wilson, Heather L.; Rajagopalan, K.V.

Eukaryotic sulfite oxidase is a dimeric protein that contains the molybdenum cofactor and catalyzes the metabolically essential conversion of sulfite to sulfate as the terminal step in the metabolism of cysteine and methionine. Nitrate reductase is an evolutionarily related molybdoprotein in lower organisms that is essential for growth on nitrate. In this study, we describe human and chicken sulfite oxidase variants in which the active site has been modified to alter substrate specificity and activity from sulfite oxidation to nitrate reduction. On the basis of sequence alignments and the known crystal structure of chicken sulfite oxidase, two residues are conservedmore » in nitrate reductases that align with residues in the active site of sulfite oxidase. On the basis of the crystal structure of yeast nitrate reductase, both positions were mutated in human sulfite oxidase and chicken sulfite oxidase. The resulting double-mutant variants demonstrated a marked decrease in sulfite oxidase activity but gained nitrate reductase activity. An additional methionine residue in the active site was proposed to be important in nitrate catalysis, and therefore, the triple variant was also produced. The nitrate reducing ability of the human sulfite oxidase triple mutant was nearly 3-fold greater than that of the double mutant. To obtain detailed structural data for the active site of these variants, we introduced the analogous mutations into chicken sulfite oxidase to perform crystallographic analysis. The crystal structures of the Mo domains of the double and triple mutants were determined to 2.4 and 2.1 {angstrom} resolution, respectively.« less

Role of Valine 464 in the Flavin Oxidation Reaction Catalyzed by Choline Oxidase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Finnegan, Steffan; Agniswamy, Johnson; Weber, Irene T.

2010-11-03

The oxidation of reduced flavin cofactors by oxygen is a very important reaction that is central to the chemical versatility of hundreds of flavoproteins classified as monooxygenases and oxidases. These enzymes are characterized by bimolecular rate constants {ge} 10{sup 5} M{sup -1} s{sup -1} and produce water and hydrogen peroxide, respectively. A hydrophobic cavity close to the reactive flavin C(4a) atom has been previously identified in the 3D structure of monooxygenases but not in flavoprotein oxidases. In the present study, we have investigated by X-ray crystallography, mutagenesis, steady-state, and rapid reaction approaches the role of Val464, which is <6 {angstrom}more » from the flavin C(4a) atom in choline oxidase. The 3D structure of the Val464Ala enzyme was essentially identical to that of the wild-type enzyme as shown by X-ray crystallography. Time-resolved anaerobic substrate reduction of the enzymes showed that replacement of Val464 with alanine or threonine did not affect the reductive half-reaction. Steady-state and rapid kinetics as well as enzyme-monitored turnovers indicated that the oxidative half-reaction in the Ala464 and Thr464 enzymes was decreased by 50-fold with respect to the wild-type enzyme. We propose that the side chain of Val464 in choline oxidase provides a nonpolar site that is required to guide oxygen in proximity of the C(4a) atom of the flavin, where it will subsequently react via electrostatic catalysis. Visual analysis of available structures suggests that analogous nonpolar sites are likely present in most flavoprotein oxidases. Mechanistic considerations provide rationalization for the differences between sites in monooxygenases and oxidases.« less

Hypouricaemic action of mangiferin results from metabolite norathyriol via inhibiting xanthine oxidase activity.

PubMed

Niu, Yanfen; Liu, Jia; Liu, Hai-Yang; Gao, Li-Hui; Feng, Guo-Hua; Liu, Xu; Li, Ling

2016-09-01

Context Mangiferin has been reported to possess a potential hypouricaemic effect. However, the pharmacokinetic studies in rats showed that its oral bioavailability was only 1.2%, suggesting that mangiferin metabolites might exert the action. Objective The hypouricaemic effect and the xanthine oxidase inhibition of mangiferin and norathyriol, a mangiferin metabolite, were investigated. Inhibition of norathyriol analogues (compounds 3-9) toward xanthine oxidase was also evaluated. Materials and methods For a dose-dependent study, mangiferin (1.5-6.0 mg/kg) and norathyriol (0.92-3.7 mg/kg) were administered intragastrically to mice twice daily for five times. For a time-course study, mice received mangiferin and norathyriol both at a single dose of 7.1 μmol/kg. In vitro, inhibition of test compounds (2.4-2.4 mM) against xanthine oxidase activity was evaluated by the spectrophotometrical method. The inhibition type was identified from Lineweaver-Burk plots. Results Norathyriol (0.92, 1.85 and 3.7 mg/kg) dose dependently decreased the serum urate levels by 27.0, 33.6 and 37.4%, respectively. The action was more potent than that of mangiferin at the low dose, but was equivalent at the higher doses. Additionally, the hypouricaemic action of them exhibited a time dependence. In vitro, norathyriol markedly inhibited the xanthine oxidase activities, with the IC50 value of 44.6 μM, but mangiferin did not. The kinetic studies showed that norathyriol was an uncompetitive inhibitor by Lineweaver-Burk plots. The structure-activity relationships exhibited that three hydroxyl groups in norathyriol at the C-1, C-3 and C-6 positions were essential for maintaining xanthine oxidase inhibition. Discussion and conclusion Norathyriol was responsible for the hypouricaemic effect of mangiferin via inhibiting xanthine oxidase activity.

Biochemical Conservation and Evolution of Germacrene A Oxidase in Asteraceae*

PubMed Central

Nguyen, Don Trinh; Göpfert, Jens Christian; Ikezawa, Nobuhiro; MacNevin, Gillian; Kathiresan, Meena; Conrad, Jürgen; Spring, Otmar; Ro, Dae-Kyun

2010-01-01

Sesquiterpene lactones are characteristic natural products in Asteraceae, which constitutes ∼8% of all plant species. Despite their physiological and pharmaceutical importance, the biochemistry and evolution of sesquiterpene lactones remain unexplored. Here we show that germacrene A oxidase (GAO), evolutionarily conserved in all major subfamilies of Asteraceae, catalyzes three consecutive oxidations of germacrene A to yield germacrene A acid. Furthermore, it is also capable of oxidizing non-natural substrate amorphadiene. Co-expression of lettuce GAO with germacrene synthase in engineered yeast synthesized aberrant products, costic acids and ilicic acid, in an acidic condition. However, cultivation in a neutral condition allowed the de novo synthesis of a single novel compound that was identified as germacrene A acid by gas and liquid chromatography and NMR analyses. To trace the evolutionary lineage of GAO in Asteraceae, homologous genes were further isolated from the representative species of three major subfamilies of Asteraceae (sunflower, chicory, and costus from Asteroideae, Cichorioideae, and Carduoideae, respectively) and also from the phylogenetically basal species, Barnadesia spinosa, from Barnadesioideae. The recombinant GAOs from these genes clearly showed germacrene A oxidase activities, suggesting that GAO activity is widely conserved in Asteraceae including the basal lineage. All GAOs could catalyze the three-step oxidation of non-natural substrate amorphadiene to artemisinic acid, whereas amorphadiene oxidase diverged from GAO displayed negligible activity for germacrene A oxidation. The observed amorphadiene oxidase activity in GAOs suggests that the catalytic plasticity is embedded in ancestral GAO enzymes that may contribute to the chemical and catalytic diversity in nature. PMID:20351109

Ascorbic Acid Metabolism in Pea Seedlings. A Comparison of d-Glucosone, l-Sorbosone, and l-Galactono-1,4-Lactone as Ascorbate Precursors1

PubMed Central

Pallanca, Jane E.; Smirnoff, Nicholas

1999-01-01

l-Ascorbic acid (AsA) accumulates in pea (Pisum sativum L.) seedlings during germination, with the most rapid phase of accumulation coinciding with radicle emergence. Monodehydroascorbate reductase and dehydroascorbic acid reductase were active in the embryonic axes before AsA accumulation started, whereas AsA oxidase and AsA peroxidase activities increased in parallel with AsA. Excised embryonic axes were used to investigate the osone pathway of AsA biosynthesis, in which d-glucosone and l-sorbosone are the proposed intermediates. [U-14C]Glucosone was incorporated into AsA and inhibited the incorporation of [U-14C]glucose (Glc) into AsA. A higher d-glucosone concentration (5 mm) inhibited AsA accumulation. l-Sorbosone did not affect AsA pool size but caused a small inhibition in the incorporation of [U-14C]Glc into AsA. Oxidase and dehydrogenase activities capable of converting Glc or Glc-6-phosphate to glucosone were not detected in embryonic axis extracts. The osones are therefore unlikely to be physiological intermediates of AsA biosynthesis. l-Galactono-1,4-lactone, recently proposed as the AsA precursor (G.L. Wheeler, M.A. Jones, N. Smirnoff [1998] Nature 393: 365–369), was readily converted to AsA by pea embryonic axes. Although l-galactono-1,4-lactone did not inhibit [14C]Glc incorporation into AsA, this does not mean that it is not a precursor, because competition between endogenous and exogenous pools was minimized by its very small pool size and rapid metabolism. PMID:10364396

Hypoglycemic neuronal death is triggered by glucose reperfusion and activation of neuronal NADPH oxidase

PubMed Central

Suh, Sang Won; Gum, Elizabeth T.; Hamby, Aaron M.; Chan, Pak H.; Swanson, Raymond A.

2007-01-01

Hypoglycemic coma and brain injury are potential complications of insulin therapy. Certain neurons in the hippocampus and cerebral cortex are uniquely vulnerable to hypoglycemic cell death, and oxidative stress is a key event in this cell death process. Here we show that hypoglycemia-induced oxidative stress and neuronal death are attributable primarily to the activation of neuronal NADPH oxidase during glucose reperfusion. Superoxide production and neuronal death were blocked by the NADPH oxidase inhibitor apocynin in both cell culture and in vivo models of insulin-induced hypoglycemia. Superoxide production and neuronal death were also blocked in studies using mice or cultured neurons deficient in the p47phox subunit of NADPH oxidase. Chelation of zinc with calcium disodium EDTA blocked both the assembly of the neuronal NADPH oxidase complex and superoxide production. Inhibition of the hexose monophosphate shunt, which utilizes glucose to regenerate NADPH, also prevented superoxide formation and neuronal death, suggesting a mechanism linking glucose reperfusion to superoxide formation. Moreover, the degree of superoxide production and neuronal death increased with increasing glucose concentrations during the reperfusion period. These results suggest that high blood glucose concentrations following hypoglycemic coma can initiate neuronal death by a mechanism involving extracellular zinc release and activation of neuronal NADPH oxidase. PMID:17404617

Overexpression of 20-Oxidase Confers a Gibberellin-Overproduction Phenotype in Arabidopsis

PubMed Central

Huang, Shihshieh; Raman, Anuradha S.; Ream, Joel E.; Fujiwara, Hideji; Cerny, R. Eric; Brown, Sherri M.

1998-01-01

In the gibberellin (GA) biosynthesis pathway, 20-oxidase catalyzes the oxidation and elimination of carbon-20 to give rise to C19-GAs. All bioactive GAs are C19-GAs. We have overexpressed a cDNA encoding 20-oxidase isolated from Arabidopsis seedlings in transgenic Arabidopsis plants. These transgenic plants display a phenotype that may be attributed to the overproduction of GA. The phenotype includes a longer hypocotyl, lighter-green leaves, increased stem elongation, earlier flowering, and decreased seed dormancy. However, the fertility of the transgenic plants is not affected. Increased levels of endogenous GA1, GA9, and GA20 were detected in seedlings of the transgenic line examined. GA4, which is thought to be the predominantly active GA in Arabidopsis, was not present at increased levels in this line. These results suggest that the overexpression of this 20-oxidase increases the levels of some endogenous GAs in transgenic seedlings, which causes the GA-overproduction phenotype. PMID:9808721

Conformational lock and dissociative thermal inactivation of lentil seedling amine oxidase.

PubMed

Moosavi-Nejad, S Zahra; Moosavi-Movahedi, Ali-Akbar; Rezaei-Tavirani, Mostafa; Floris, Giovanni; Medda, Rosaria

2003-03-31

The kinetics of thermal inactivation of copper-containing amine oxidase from lentil seedlings were studied in a 100 mM potassium phosphate buffer, pH 7, using putrescine as the substrate. The temperature range was between 47-60 degrees C. The thermal inactivation curves were not linear at 52 and 57 degrees C; three linear phases were shown. The first phase gave some information about the number of dimeric forms of the enzyme that were induced by the higher temperatures using the "conformational lock" pertaining theory to oligomeric enzyme. The "conformational lock" caused two additional dimeric forms of the enzyme when the temperature increased to 57 degrees C. The second and third phases were interpreted according to a dissociative thermal inactivation model. These phases showed that lentil amine oxidase was reversibly-dissociated before the irreversible thermal inactivation. Although lentil amine oxidase is not a thermostable enzyme, its dimeric structure can form "conformational lock," conferring a structural tolerance to the enzyme against heat stress.

A Mycobacterium tuberculosis cytochrome bd oxidase mutant is hypersensitive to bedaquiline.

PubMed

Berney, Michael; Hartman, Travis E; Jacobs, William R

2014-07-15

The new medicinal compound bedaquiline (BDQ) kills Mycobacterium tuberculosis by inhibiting F1Fo-ATP synthase. BDQ is bacteriostatic for 4 to 7 days and kills relatively slowly compared to other frontline tuberculosis (TB) drugs. Here we show that killing with BDQ can be improved significantly by inhibiting cytochrome bd oxidase, a non-proton-pumping terminal oxidase. BDQ was instantly bactericidal against a cytochrome bd oxidase null mutant of M. tuberculosis, and the rate of killing was increased by more than 50%. We propose that this exclusively bacterial enzyme should be a high-priority target for new drug discovery. Importance: A major drawback of current TB chemotherapy is its long duration. New drug regimens with rapid killing kinetics are desperately needed. Our study demonstrates that inhibition of a nonessential bacterial enzyme greatly improves the efficacy of the latest TB drug bedaquiline and emphasizes that screening for compounds with synergistic killing mechanisms is a promising strategy. Copyright © 2014 Berney et al.

The glucose oxidase-peroxidase assay for glucose

USDA-ARS?s Scientific Manuscript database

The glucose oxidase-peroxidase assay for glucose has served as a very specific, sensitive, and repeatable assay for detection of glucose in biological samples. It has been used successfully for analysis of glucose in samples from blood and urine, to analysis of glucose released from starch or glycog...

Effect of ionic detergents, nonionic detergents, and chaotropic agents on polyphenol oxidase activity from dormant saffron (Crocus sativus L.) corms.

PubMed

Saeidian, Shahriar; Keyhani, Ezzatollah; Keyhani, Jacqueline

2007-05-02

Polyphenol oxidase (PPO; EC 1.14.18.1) catalyzes the hydroxylation of monophenols to o-diphenols (cresolase activity) and the oxidation of o-diphenols to o-quinones (catecholase activity), leading to browning in plants and produce. Further interest in the enzyme has been triggered by the active role that it plays in plant defense systems. PPO can be found in latent forms and is activated in vitro by various agents including urea, detergents, and proteases. The activation of PPO from several sources by sodium dodecyl sulfate (SDS) has been extensively investigated, but reports on the effect of other detergents or on the differential effect of detergents on each of PPO's activities are scarce. In addition, investigations on the enzyme in other plant parts besides fruits and vegetables are also scarce. Here, the effect of various detergents and chaotropic agents on PPO from dormant saffron (Crocus sativus L.) corm extract was investigated. SDS and sarkosyl activated the cresolase activity, while only SDS activated the catecholase activity. All other detergents tested, in milli- or micromolar concentrations, inhibited the cresolase activity but barely affected the catecholase activity. In contrast, urea and guanidine-HCl drastically inhibited the catecholase activity but moderately inhibited the cresolase activity. The same effects were obtained on the partially purified enzyme. Results identified a PPO, present in dormant corms, which was activated only by anionic detergents and was inhibited by other reputed activating agents such as urea. Results also emphasized the differences in structure and accessibility of the active sites for cresolase and catecholase activities.

Expanding the substrate scope of chitooligosaccharide oxidase from Fusarium graminearum by structure-inspired mutagenesis.

PubMed

Ferrari, Alessandro R; Lee, Misun; Fraaije, Marco W

2015-06-01

Chitooligosaccharide oxidase from Fusarium graminearum (ChitO) oxidizes N-acetyl-D-glucosamine (GlcNAc) and its oligomers with high efficiency at the C1-hydroxyl moiety while it shows poor or no activity with other carbohydrates. By sequence and structural comparison with other known carbohydrate oxidases (glucooligosaccharide oxidase from Acremonium strictum and lactose oxidase from Microdochium nivale) eleven mutants were designed to redirect the catalytic scope of ChitO for improved oxidation of lactose, cellobiose and maltose. The catalytic properties of the most interesting mutants were further improved by combining single mutations. This has resulted in the creation of a set of ChitO variants that display totally different substrate tolerances. One ChitO variant shows a dramatic improvement in catalytic efficiency towards oxidation of glucose, cellobiose, lactose, and maltose. We also describe a ChitO variant with the highest catalytic efficiency in GlcNAc oxidation so far reported in the literature. © 2015 Wiley Periodicals, Inc.

Regulation of superoxide anion production by NADPH oxidase in monocytes/macrophages: contributions to atherosclerosis.

PubMed

Cathcart, Martha K

2004-01-01

Monocyte extravasation into the vessel wall has been shown to be a critical step in the development of atherosclerosis. Upon activation, monocytes produce a burst of superoxide anion due to activation of the NADPH oxidase enzyme complex. Monocyte-derived superoxide anion contributes to oxidant stress in inflammatory sites, is required for monocyte-mediated LDL oxidation, and alters basic cell functions such as adhesion and proliferation. We hypothesize that monocyte-derived superoxide anion production contributes to atherosclerotic lesion formation. In this brief review, we summarize our current understanding of the signal transduction pathways regulating NADPH oxidase activation and related superoxide anion production in activated human monocytes. Novel pathways are identified that may serve as future targets for therapeutic intervention in this pathogenic process. The contributions of superoxide anion and NADPH oxidase to atherogenesis are discussed. Future experiments are needed to clarify the exact role of NADPH oxidase-derived superoxide anion in atherogenesis, particularly that derived from monocytes.

Expression of novel rice gibberellin 2-oxidase gene is under homeostatic regulation by biologically active gibberellins.

PubMed

Sakai, Miho; Sakamoto, Tomoaki; Saito, Tamio; Matsuoka, Makoto; Tanaka, Hiroshi; Kobayashi, Masatomo

2003-04-01

We have cloned two genes for gibberellin (GA) 2-oxidase from rice ( Oryza sativa L.). Expression of OsGA2ox2 was not observed. The other gene, OsGA2ox3, was expressed in every tissue examined and was enhanced by the application of biologically active GA. Recombinant OsGA2ox3 protein catalyzed the metabolism of GA(1) to GA(8) and GA(20) to GA(29)-catabolite. These results indicate that OsGA2ox3 is involved in the homeostatic regulation of the endogenous level of biologically active GA in rice.

Interrupted reperfusion reduces the activation of NADPH oxidase after cerebral I/R injury.

PubMed

Shen, Jia; Bai, Xiao-Yin; Qin, Yuan; Jin, Wei-Wei; Zhou, Jing-Yin; Zhou, Ji-Ping; Yan, Ying-Gang; Wang, Qiong; Bruce, Iain C; Chen, Jiang-Hua; Xia, Qiang

2011-06-15

Interrupted reperfusion reduces ischemia/reperfusion (I/R) injury. This study was designed to determine whether NADPH oxidase participates in the neural protection against global I/R injury after interrupted reperfusion. Mice were randomly divided into five groups: sham (sham-operated), I/R (20-min global I/R), RR (I/R+interrupted reperfusion), Apo (I/R+apocynin administration), and RR+Apo. Behavioral tests (pole test, beam walking, and Morris water maze) and Nissl staining were undertaken in all five groups; superoxide levels, expression of gp91(phox) and p47(phox), p47(phox) translocation, and Rac1 activation were measured in the sham, I/R, and RR groups. The motor coordination, bradykinesia, and spatial learning and memory, as well as the neuron survival rates, were better in the RR, Apo, and RR+Apo groups than in the I/R group. The NADPH oxidase-dependent superoxide levels, p47(phox) and gp91(phox) expression, p47(phox) translocation, and Rac1 activation were lower in the RR group than in the I/R group. In conclusion, the neural protective effect of interrupted reperfusion is at least partly mediated by decreasing the expression and assembly of NADPH oxidase and the levels of NADPH oxidase-derived superoxide. The most striking reduction Rac1-GTP in the RR group suggests that interrupted reperfusion also acts on the activation of assembled NADPH oxidase by reducing the availability of Rac1-GTP. Copyright © 2011 Elsevier Inc. All rights reserved.

Absence of Proton Channels in COS-7 Cells Expressing Functional NADPH Oxidase Components

PubMed Central

Morgan, Deri; Cherny, Vladimir V.; Price, Marianne O.; Dinauer, Mary C.; DeCoursey, Thomas E.

2002-01-01

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is an enzyme of phagocytes that produces bactericidal superoxide anion (O2 −) via an electrogenic process. Proton efflux compensates for the charge movement across the cell membrane. The proton channel responsible for the H+ efflux was thought to be contained within the gp91phox subunit of NADPH oxidase, but recent data do not support this idea (DeCoursey, T.E., V.V. Cherny, D. Morgan, B.Z. Katz, and M.C. Dinauer. 2001. J. Biol. Chem. 276:36063–36066). In this study, we investigated electrophysiological properties and superoxide production of COS-7 cells transfected with all NADPH oxidase components required for enzyme function (COSphox). The 7D5 antibody, which detects an extracellular epitope of the gp91phox protein, labeled 96–98% of COSphox cells. NADPH oxidase was functional because COSphox (but not COSWT) cells stimulated by phorbol myristate acetate (PMA) or arachidonic acid (AA) produced superoxide anion. No proton currents were detected in either wild-type COS-7 cells (COSWT) or COSphox cells studied at pHo 7.0 and pHi 5.5 or 7.0. Anion currents that decayed at voltages positive to 40 mV were the only currents observed. PMA or AA did not elicit detectable H+ current in COSWT or COSphox cells. Therefore, gp91phox does not function as a proton channel in unstimulated cells or in activated cells with a demonstrably functional oxidase. PMID:12034764

Phenol contents, oxidase activities, and the resistance of coffee to the leaf miner Leucoptera coffeella.

PubMed

Ramiro, Daniel Alves; Guerreiro-Filho, Oliveiro; Mazzafera, Paulo

2006-09-01

We examined the role of phenolic compounds, and the enzymes peroxidase and polyphenol oxidase, in the expression of resistance of coffee plants to Leucoptera coffeella (Lepidoptera: Lyonetiidae). The concentrations of total soluble phenols and chlorogenic acid (5-caffeoylquinic acid), and the activities of the oxidative enzymes peroxidase (POD) and polyphenol oxidase (PPO), were estimated in leaves of Coffea arabica, C. racemosa, and progenies of crosses between these species, which have different levels of resistance, before and after attack by this insect. The results indicate that phenols do not play a central role in resistance to the coffee leaf miner. Differences were detected between the parental species in terms of total soluble phenol concentrations and activities of the oxidative enzymes. However, resistant and susceptible hybrid plants did not differ in any of these characteristics. Significant induction of chlorogenic acid and PPO was only found in C. racemosa, the parental donator of the resistance genes against L. coffeella. High-performance liquid chromatography (HPLC) analysis also showed qualitative similarity between hybrids and the susceptible C. arabica. These results suggest that the phenolic content and activities of POD and PPO in response to the attack by the leaf miner may not be a strong evidence of their participation in direct defensive mechanisms.

An improved amperometric L-lactate biosensor based on covalent immobilization of microbial lactate oxidase onto carboxylated multiwalled carbon nanotubes/copper nanoparticles/polyaniline modified pencil graphite electrode.

PubMed

Dagar, Kusum; Pundir, C S

2017-01-01

An improved amperometric l-lactate biosensor was constructed based on covalent immobilization of lactate oxidase (LOx) from Pediococcus species onto carboxylated multiwalled carbon nanotubes (cMWCNT)/copper nanoparticles (CuNPs)/polyaniline (PANI) hybrid film electrodeposited on the surface of a pencil graphite electrode (PGE). The enzyme electrode was characterized by cyclic voltammetry (CV), scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy and electrochemical impedance spectroscopy (EIS), while CuNPs synthesized by chemical reduction method, were characterized by transmission electron microscopy (TEM), UV spectrascopy and X-ray diffraction (XRD). The biosensor showed maximum response within 5s at pH 8.0 in 0.05M sodium phosphate buffer and 37°C, when operated at 20mVs -1 . The biosensor had a detection limit of 0.25μM with a wide working range between 1μM-2500μM. The biosensor was employed for measurement of l-lactic acid level in plasma of apparently healthy and diseased persons. Analytical recovery of added lactic acid in plasma was 95.5%. Within- and between-batch coefficients of variations were 6.24% and 4.19% respectively. There was a good correlation (R 2 =0.97) between plasma lactate values as measured by standard enzymatic spectrophotometric method and the present biosensor. The working enzyme electrode was used 180 times over a period of 140 days, when stored at 4°C. Copyright © 2016. Published by Elsevier Inc.

Myeloperoxidase amplified high glucose-induced endothelial dysfunction in vasculature: Role of NADPH oxidase and hypochlorous acid.

PubMed

Tian, Rong; Ding, Yun; Peng, Yi-Yuan; Lu, Naihao

2017-03-11

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived reactive oxygen species (ROS) such as superoxide and hydrogen peroxide (H 2 O 2 ), have emerged as important molecules in the pathogenesis of diabetic endothelial dysfunction. Additionally, neutrophils-derived myeloperoxidase (MPO) and MPO-catalyzed hypochlorous acid (HOCl) play important roles in the vascular injury. However, it is unknown whether MPO can use vascular-derived ROS to induce diabetic endothelial dysfunction. In the present study, we demonstrated that NADPH oxidase was the main source of ROS formation in high glucose-cultured human umbilical vein endothelial cells (HUVECs), and played a critical role in high glucose-induced endothelial dysfunction such as cell apoptosis, loss of cell viability and reduction of nitric oxide (NO). However, the addition of MPO could amplify the high glucose-induced endothelial dysfunction which was inhibited by the presence of apocynin (NADPH oxidase inhibitor), catalase (H 2 O 2 scavenger), or methionine (HOCl scavenger), demonstrating the contribution of NADPH oxidase-H 2 O 2 -MPO-HOCl pathway in the MPO/high glucose-induced vascular injury. In high glucose-incubated rat aortas, MPO also exacerbated the NADPH oxidase-induced impairment of endothelium-dependent relaxation. Consistent with these in vitro data, in diabetic rat aortas, both MPO expresion and NADPH oxidase activity were increased while the endothelial function was simultaneously impaired. The results suggested that vascular-bound MPO could amplify high glucose-induced vascular injury in diabetes. MPO-NADPH oxidase-HOCl may represent an important pathogenic pathway in diabetic vascular diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

Mannitol oxidase and polyol dehydrogenases in the digestive gland of gastropods: Correlations with phylogeny and diet

PubMed Central

Amaral-de-Carvalho, Diogo; Oliveira, Elsa; Alves, Ângela; Costa, Vítor; Calado, Gonçalo

2018-01-01

Mannitol oxidase and polyol dehydrogenases are enzymes that convert polyalcohols into sugars. Mannitol oxidase was previously investigated in terrestrial snails and slugs, being also present in a few aquatic gastropods. However, the overall distribution of this enzyme in the Gastropoda was not known. Polyol dehydrogenases are also poorly studied in gastropods and other mollusks. In this study, polyalcohol oxidase and dehydrogenase activities were assayed in the digestive gland of 26 species of gastropods, representing the clades Patellogastropoda, Neritimorpha, Vetigastropoda, Caenogastropoda and Heterobranchia. Marine, freshwater and terrestrial species, including herbivores and carnivores were analyzed. Ultrastructural observations were undertake in species possessing mannitol oxidase, in order to investigate the correlation between this enzyme and the presence of tubular structures known to be associated with it. Mannitol oxidase activity was detected in the digestive gland of herbivores from the clades Caenogastropoda and Heterobranchia, but not in any carnivores or in herbivores from the clades Patellogastropoda, Neritimorpha and Vetigastropoda. In most of the species used in this study, dehydrogenase activities were detected using both D-mannitol and D-sorbitol as substrates. Nevertheless, in some carnivores these activities were not detected with both polyalcohols. Ultrastructural observations revealed tubular structures in digestive gland cells of some species having mannitol oxidase activity, but they were not observed in others. Based on our results, we suggest that mannitol oxidase first occurred in a herbivorous or omnivorous ancestor of Apogastropoda, the clade formed by caenogastropods and heterobranchs, being subsequently lost in those species that shifted towards a carnivorous diet. PMID:29529078

Exploiting the synthetic lethality between terminal respiratory oxidases to kill Mycobacterium tuberculosis and clear host infection

PubMed Central

Kalia, Nitin P.; Hasenoehrl, Erik J.; Ab Rahman, Nurlilah B.; Koh, Vanessa H.; Ang, Michelle L. T.; Sajorda, Dannah R.; Hards, Kiel; Grüber, Gerhard; Alonso, Sylvie; Cook, Gregory M.; Berney, Michael; Pethe, Kevin

2017-01-01

The recent discovery of small molecules targeting the cytochrome bc1:aa3 in Mycobacterium tuberculosis triggered interest in the terminal respiratory oxidases for antituberculosis drug development. The mycobacterial cytochrome bc1:aa3 consists of a menaquinone:cytochrome c reductase (bc1) and a cytochrome aa3-type oxidase. The clinical-stage drug candidate Q203 interferes with the function of the subunit b of the menaquinone:cytochrome c reductase. Despite the affinity of Q203 for the bc1:aa3 complex, the drug is only bacteriostatic and does not kill drug-tolerant persisters. This raises the possibility that the alternate terminal bd-type oxidase (cytochrome bd oxidase) is capable of maintaining a membrane potential and menaquinol oxidation in the presence of Q203. Here, we show that the electron flow through the cytochrome bd oxidase is sufficient to maintain respiration and ATP synthesis at a level high enough to protect M. tuberculosis from Q203-induced bacterial death. Upon genetic deletion of the cytochrome bd oxidase-encoding genes cydAB, Q203 inhibited mycobacterial respiration completely, became bactericidal, killed drug-tolerant mycobacterial persisters, and rapidly cleared M. tuberculosis infection in vivo. These results indicate a synthetic lethal interaction between the two terminal respiratory oxidases that can be exploited for anti-TB drug development. Our findings should be considered in the clinical development of drugs targeting the cytochrome bc1:aa3, as well as for the development of a drug combination targeting oxidative phosphorylation in M. tuberculosis. PMID:28652330

The Intimate and Controversial Relationship between Voltage Gated Proton Channels and the Phagocyte NADPH Oxidase

PubMed Central

DeCoursey, Thomas E.

2016-01-01

Summary One of the most fascinating and exciting periods in my scientific career entailed dissecting the symbiotic relationship between two membrane transporters, the NADPH oxidase complex and voltage gated proton channels (HV1). By the time I entered this field, there had already been substantial progress toward understanding NADPH oxidase, but HV1 were known only to a tiny handful of cognoscenti around the world. Having identified the first proton currents in mammalian cells in 1991, I needed to find a clear function for these molecules if the work was to become fundable. The then-recent discoveries of Henderson, Chappell, and colleagues in 1987–1988 that led them to hypothesize interactions of both molecules during the respiratory burst of phagocytes provided an excellent opportunity. In a nutshell, both transporters function by moving electrical charge across the membrane: NADPH oxidase moves electrons and HV1 moves protons. The consequences of electrogenic NADPH oxidase activity on both membrane potential and pH strongly self-limit this enzyme. Fortunately, both consequences specifically activate HV1, and HV1 activity counteracts both consequences, a kind of yin-yang relationship. Notwithstanding a decade starting in 1995 when many believed the opposite, these are two separate molecules that function independently despite their being functionally interdependent in phagocytes. The relationship between NADPH oxidase and HV1 has become a paradigm that somewhat surprisingly has now extended well beyond the phagocyte NADPH oxidase -- an industrial strength producer of reactive oxygen species (ROS) -- to myriad other cells that produce orders of magnitude less ROS for signaling purposes. These cells with their seven NADPH oxidase (NOX) isoforms provide a vast realm of mechanistic obscurity that will occupy future studies for years to come. PMID:27558336

The intimate and controversial relationship between voltage-gated proton channels and the phagocyte NADPH oxidase.

PubMed

DeCoursey, Thomas E

2016-09-01

One of the most fascinating and exciting periods in my scientific career entailed dissecting the symbiotic relationship between two membrane transporters, the Nicotinamide adenine dinucleotide phosphate reduced form (NADPH) oxidase complex and voltage-gated proton channels (HV 1). By the time I entered this field, there had already been substantial progress toward understanding NADPH oxidase, but HV 1 were known only to a tiny handful of cognoscenti around the world. Having identified the first proton currents in mammalian cells in 1991, I needed to find a clear function for these molecules if the work was to become fundable. The then-recent discoveries of Henderson, Chappell, and colleagues in 1987-1988 that led them to hypothesize interactions of both molecules during the respiratory burst of phagocytes provided an excellent opportunity. In a nutshell, both transporters function by moving electrical charge across the membrane: NADPH oxidase moves electrons and HV 1 moves protons. The consequences of electrogenic NADPH oxidase activity on both membrane potential and pH strongly self-limit this enzyme. Fortunately, both consequences specifically activate HV 1, and HV 1 activity counteracts both consequences, a kind of yin-yang relationship. Notwithstanding a decade starting in 1995 when many believed the opposite, these are two separate molecules that function independently despite their being functionally interdependent in phagocytes. The relationship between NADPH oxidase and HV 1 has become a paradigm that somewhat surprisingly has now extended well beyond the phagocyte NADPH oxidase - an industrial strength producer of reactive oxygen species (ROS) - to myriad other cells that produce orders of magnitude less ROS for signaling purposes. These cells with their seven NADPH oxidase (NOX) isoforms provide a vast realm of mechanistic obscurity that will occupy future studies for years to come. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Production of Dwarf Lettuce by Overexpressing a Pumpkin Gibberellin 20-Oxidase Gene

PubMed Central

Niki, Tomoya; Nishijima, Takaaki; Nakayama, Masayoshi; Hisamatsu, Tamotsu; Oyama-Okubo, Naomi; Yamazaki, Hiroko; Hedden, Peter; Lange, Theo; Mander, Lewis N.; Koshioka, Masaji

2001-01-01

We investigated the effect of overexpressing a pumpkin gibberellin (GA) 20-oxidase gene encoding an enzyme that forms predominantly biologically inactive products on GA biosynthesis and plant morphology in transgenic lettuce (Lactuca sativa cv Vanguard) plants. Lettuce was transformed with the pumpkin GA 20-oxidase gene downstream of a strong constitutive promoter cassette (El2–35S-Ω). The transgenic plants in which the pumpkin gene was detected by polymerase chain reaction were dwarfed in the T2 generation, whereas transformants with a normal growth phenotype did not contain the transgene. The result of Southern-blot analysis showed that the transgene was integrated as a single copy; the plants segregated three dwarfs to one normal in the T2 generation, indicating that the transgene was stable and dominant. The endogenous levels of GA1 and GA4 were reduced in the dwarfs, whereas large amounts of GA17 and GA25, which are inactive products of the pumpkin GA 20-oxidase, accumulated in these lines. These results indicate that a functional pumpkin GA 20-oxidase is expressed in the transgenic lettuce, resulting in a diversion of the normal pathway of GA biosynthesis to inactive products. Furthermore, this technique may be useful for controlling plant stature in other agricultural and horticultural species. PMID:11457947

Ectopic expression of pumpkin gibberellin oxidases alters gibberellin biosynthesis and development of transgenic Arabidopsis plants.

PubMed

Radi, Abeer; Lange, Theo; Niki, Tomoya; Koshioka, Masaji; Lange, Maria João Pimenta

2006-02-01

Immature pumpkin (Cucurbita maxima) seeds contain gibberellin (GA) oxidases with unique catalytic properties resulting in GAs of unknown function for plant growth and development. Overexpression of pumpkin GA 7-oxidase (CmGA7ox) in Arabidopsis (Arabidopsis thaliana) resulted in seedlings with elongated roots, taller plants that flower earlier with only a little increase in bioactive GA4 levels compared to control plants. In the same way, overexpression of the pumpkin GA 3-oxidase1 (CmGA3ox1) resulted in a GA overdose phenotype with increased levels of endogenous GA4. This indicates that, in Arabidopsis, 7-oxidation and 3-oxidation are rate-limiting steps in GA plant hormone biosynthesis that control plant development. With an opposite effect, overexpression of pumpkin seed-specific GA 20-oxidase1 (CmGA20ox1) in Arabidopsis resulted in dwarfed plants that flower late with reduced levels of GA4 and increased levels of physiological inactive GA17 and GA25 and unexpected GA34 levels. Severe dwarfed plants were obtained by overexpression of the pumpkin GA 2-oxidase1 (CmGA2ox1) in Arabidopsis. This dramatic change in phenotype was accompanied by a considerable decrease in the levels of bioactive GA4 and an increase in the corresponding inactivation product GA34 in comparison to control plants. In this study, we demonstrate the potential of four pumpkin GA oxidase-encoding genes to modulate the GA plant hormone pool and alter plant stature and development.

Purification and characterization of l,(l/d)-aminopeptidase from Guinea pig serum.

PubMed

Krstanović, Marina; Brgles, Marija; Halassy, Beata; Frkanec, Ruza; Vrdoljak, Anto; Branović, Karmen; Tomasić, Jelka; Benedetti, Fabio

2006-01-01

Mammalian sera contain enzymes that catalyze the hydrolytic degradation of peptidoglycans and molecules of related structure and are relevant for the metabolism of peptidoglycans. We now report on a novel L,(L/D)-aminopeptidase found in human and mammalian sera. The enzyme hydrolyses the pentapeptide L-Ala-D-iso-Gln-meso-DAP(omegaNH(2))-D-Ala-D-Ala yielding the free L-alanine and the respective tetrapeptide (K(M) 18 mM). L,(L/D)-aminopeptidase from guinea pig serum was highly purified in four chromatographic steps, up to 700-fold. Molecular weight of the enzyme was estimated by HPLC to be approximately 175,000. The configuration of alanine obtained by hydrolysis of the pentapeptide was determined by oxidation with L-amino acid oxidase. The amino acids sequence in the respective tetrapeptide was deduced from the results of mass spectrometry. The novel L,(L/D)-aminopeptidase also hydrolyzed alanine-4-nitroanilide (K(M)=0.6 mM) and several peptides comprising L-amino acids. Peptides containing D-amino acid at the amino end and L-Asp-L-Asp were not the substrates for this enzyme. The purified enzyme also exhibited enkephalin degrading activity, hydrolyzing enkephalins comprising L,L- and L,D-peptide bonds. The enzyme was inhibited strongly by metal chelating agents, bestatin and amastatin.

Identification and characterization of a novel flavin-containing spermine oxidase of mammalian cell origin.

PubMed Central

Vujcic, Slavoljub; Diegelman, Paula; Bacchi, Cyrus J; Kramer, Debora L; Porter, Carl W

2002-01-01

During polyamine catabolism, spermine and spermidine are first acetylated by spermidine/spermine N(1)-acetyltransferase (SSAT) and subsequently oxidized by polyamine oxidase (PAO) to produce spermidine and putrescine, respectively. In attempting to clone the PAO involved in this back-conversion pathway, we encountered an oxidase that preferentially cleaves spermine in the absence of prior acetylation by SSAT. A BLAST search using maize PAO sequences identified homologous mammalian cDNAs derived from human hepatoma and mouse mammary carcinoma: the encoded proteins differed by 20 amino acids. When either cDNA was transiently transfected into HEK-293 cells, intracellular spermine pools decreased by 75% while spermidine and N (1)-acetylspermidine pools increased, suggesting that spermine was selectively and directly oxidized by the enzyme. Substrate specificity using lysates of oxidase-transfected HEK-293 cells revealed that the newly identified oxidase strongly favoured spermine over N (1)-acetylspermine and that it failed to act on N (1)-acetylspermidine, spermidine or the preferred PAO substrate, N (1), N (12)-diacetylspermine. The PAO inhibitor, MDL-72,527, only partially blocked oxidation of spermine while a previously reported PAO substrate, N (1)-( n -octanesulphonyl)spermine, potently inhibited the reaction. Overall, the data indicate that the enzyme represents a novel mammalian oxidase which, on the basis of substrate specificity, we have designated spermine oxidase in order to distinguish it from the PAO involved in polyamine back-conversion. The identification of an enzyme capable of directly oxidizing spermine to spermidine has important implications for understanding polyamine homoeostasis and for interpreting metabolic and cellular responses to clinically relevant polyamine analogues and inhibitors. PMID:12141946

Identification and biochemical characterization of polyamine oxidases in amphioxus: Implications for emergence of vertebrate-specific spermine and acetylpolyamine oxidases.

PubMed

Wang, Huihui; Liu, Baobao; Li, Hongyan; Zhang, Shicui

2016-01-10

Polyamine oxidases (PAOs) have been identified in a wide variety of animals, as well as in fungi and plant. Generally, plant PAOs oxidize spermine (Spm), spermidine (Spd) and their acetylated derivatives, N(1)-acetylspermine (N(1)-Aspm) and N(1)-acetylspermidine (N(1)-Aspd), while yeast PAOs oxidize Spm, N(1)-Aspm and N(1)-Aspd, but not Spd. By contrast, two different enzymes, namely spermine oxidase (SMO) and acetylpolyamine oxidase (APAO), specifically catalyze the oxidation of Spm and N(1)-Aspm/N(1)-Aspd, respectively. However, our knowledge on the biochemical and structural characterization of PAOs remains rather limited, and their evolutionary history is still enigmatic. In this study, two amphioxus (Branchiostoma japonicum) PAO genes, named Bjpao1 and Bjpao2, were cloned and characterized. Both Bjpao1 and Bjpao2 displayed distinct tissue-specific expression patterns. Notably, rBjPAO1 oxidized both spermine and spermidine, but not N(1)-acetylspermine, whereas rBjPAO2 oxidizes both spermidine and N(1)-acetylspermine, but not spermine. To understand structure-function relationship, the enzymatic activities of mutant BjPAOs that were generated by site-directed mutagenesis and expressed in E. coli were examined, The results indicate that the residues H64, K301 and T460 in rBjPAO1, and H69, K315 and T467 in rBjPAO2 were all involved in substrate binding and enzyme catalytic activity to some extent. Based on our results and those of others, a model depicting the divergent evolution and functional specialization of vertebrate SMO and APAO genes is proposed. Copyright © 2015 Elsevier B.V. All rights reserved.

Polyphenol oxidase and peroxidase expression in four pineapple varieties (Ananas comosus L.) after a chilling injury.

PubMed

Raimbault, Astrid-Kim; Marie-Alphonsine, Paul-Alex; Horry, Jean-Pierre; Francois-Haugrin, Madlyn; Romuald, Karell; Soler, Alain

2011-01-12

Pineapple internal browning (IB) is a chilling injury that produces enzymatic browning associated with flesh translucency. Pineapple biodiversity allowed the investigation of how polyphenol oxidase (PPO) and peroxidase (POD) activities with their different isoforms are involved in the IB mechanism. Fruits of four varieties that expressed IB symptoms differently, Smooth Cayenne (SCay) and the hybrids MD2, Flhoran 41 (Flh 41), and Flhoran 53 (Flh 53), were stressed by cold. The susceptible varieties showed classical brown spots but different patterns of IB, whereas MD2 and controls showed no IB. Enzymatic activities were measured on fruit protein extracts and PPO and POD isoforms separated on mini-gels (PhastSystem). Only PPO activity was significantly enhanced in the presence of IB. Up to six PPO isoforms were identified in the susceptible varieties. PPO was barely detectable in the nonsusceptible variety MD2 and in controls. The number of PPO isoforms and the total PPO activity after chilling are varietal characteristics.

Salicylic Acid Regulation of Respiration in Higher Plants: Alternative Oxidase Expression.

PubMed Central

Rhoads, DM; McIntosh, L

1992-01-01

Alternative respiratory pathway capacity increases during the development of the thermogenic appendix of a voodoo lily inflorescence. The levels of the alternative oxidase proteins increased dramatically between D-4 (4 days prior to the day of anthesis) and D-3 and continued to increase until the day of anthesis (D-day). The level of salicylic acid (SA) in the appendix is very low early on D-1, but increases to a high level in the evening of D-1. Thermogenesis occurs after a few hours of light on D-day. Therefore, the initial accumulation of the alternative oxidase proteins precedes the increase in SA by 3 days, indicating that other regulators may be involved. A 1.6-kb transcript encoding the alternative oxidase precursor protein accumulated to a high level in the appendix tissue by D-1. Application of SA to immature appendix tissue caused an increase in alternative pathway capacity and a dramatic accumulation of the alternative oxidase proteins and the 1.6-kb transcript. Time course experiments showed that the increase in capacity, protein levels, and transcript level corresponded precisely. The response to SA was blocked by cycloheximide or actinomycin D, indicating that de novo transcription and translation are required. However, nuclear, in vitro transcription assays indicated that the accumulation of the 1.6-kb transcript did not result from a simple increase in the rate of transcription of aox1. PMID:12297672

The hemibiotrophic cacao pathogen Moniliophthora perniciosa depends on a mitochondrial alternative oxidase for biotrophic development

USDA-ARS?s Scientific Manuscript database

The mitochondrial alternative oxidase (AOX) is a non-energy conserving ubiquinol oxidase found in most fungal genomes studied to date. With the development of fungicides containing cytochrome-dependent respiratory chain (CRC) inhibitors, a strong interest in studying AOX functions in phytopathogenic...

Combined effect of loss of the caa3 oxidase and Crp regulation drives Shewanella to thrive in redox-stratified environments.

PubMed

Zhou, Guangqi; Yin, Jianhua; Chen, Haijiang; Hua, Yijie; Sun, Linlin; Gao, Haichun

2013-09-01

Shewanella species are a group of facultative Gram-negative microorganisms with remarkable respiration abilities that allow the use of a diverse array of terminal electron acceptors (EA). Like most bacteria, S. oneidensis possesses multiple terminal oxidases, including two heme-copper oxidases (caa3- and cbb3-type) and a bd-type quinol oxidase. As aerobic respiration is energetically favored, mechanisms underlying the fact that these microorganisms thrive in redox-stratified environments remain vastly unexplored. In this work, we discovered that the cbb3-type oxidase is the predominant system for respiration of oxygen (O2), especially when O2 is abundant. Under microaerobic conditions, the bd-type quinol oxidase has a significant role in addition to the cbb3-type oxidase. In contrast, multiple lines of evidence suggest that under test conditions the caa3-type oxidase, an analog to the mitochondrial enzyme, has no physiological significance, likely because of its extremely low expression. In addition, expression of both cbb3- and bd-type oxidases is under direct control of Crp (cAMP receptor protein) but not the well-established redox regulator Fnr (fumarate nitrate regulator) of canonical systems typified in Escherichia coli. These data, collectively, suggest that adaptation of S. oneidensis to redox-stratified environments is likely due to functional loss of the caa3-type oxidase and switch of the regulatory system for respiration.

IRON REGULATES XANTHINE OXIDASE ACTIVITY IN THE LUNG

EPA Science Inventory

The iron chelator deferoxamine has been reported to inhibit both xanthine oxidase (XO) and xanthine dehydrogenase activity, but the relationship of this effect to the availability of iron in the cellular and tissue environment remains unexplored. XO and total xanthine oxidoreduct...

Molecular mechanisms of hypertension: role of Nox family NADPH oxidases.

PubMed

Sedeek, Mona; Hébert, Richard L; Kennedy, Chris R; Burns, Kevin D; Touyz, Rhian M

2009-03-01

Molecular mechanisms contributing to the pathoetiology of hypertension are complex, involving many interacting systems such as signaling through G protein-coupled receptors, the renin-angiotensin system, vascular inflammation and remodeling, vascular senescence and aging and developmental programming, as highlighted in the current issue of the journal. Common to these systems is NADPH oxidase-derived reactive oxygen species (ROS). This editorial highlights current concepts relating to the production of ROS in hypertension and focuses on the Nox family NADPH oxidases, major sources of free radicals in the cardiovascular and renal systems. ROS play a major role as intracellular signaling molecules to regulate normal biological cellular responses. In pathological conditions, loss of redox homeostasis contributes to vascular oxidative damage. Recent evidence indicates that specific enzymes, the Nox family of NADPH oxidases, have the sole function of generating ROS in a highly regulated fashion in physiological conditions, and that in disease states, hyperactivation of Noxes contributes to oxidative stress and consequent cardiovascular and renal injury. The Nox family comprises seven members, Nox1-Nox7. Nox1, Nox2 (gp91phox-containing NADPH oxidase), Nox4 and Nox5 have been identified in the cardiovascular-renal systems and have been implicated in the pathophysiology of cardiovascular and renal disease. Noxes, which are differentially regulated in hypertension, are major sources of cardiovascular and renal oxidative stress. This has evoked considerable interest because of the possibilities that therapies targeted against specific Nox isoforms to decrease ROS generation or to increase nitric oxide availability or both may be useful in minimizing vascular injury and renal dysfunction, and thereby prevent or regress target organ damage associated with hypertension.

High-level expression of the Penicillium notatum glucose oxidase gene in Pichia pastoris using codon optimization.

PubMed

Gao, Zhaowei; Li, Zhuofu; Zhang, Yuhong; Huang, Huoqing; Li, Mu; Zhou, Liwei; Tang, Yunming; Yao, Bin; Zhang, Wei

2012-03-01

The glucose oxidase (GOD) gene from Penicillium notatum was expressed in Pichia pastoris. The 1,815 bp gene, god-w, encodes 604 amino acids. Recombinant GOD-w had optimal activity at 35-40°C and pH 6.2 and was stable, from pH 3 to 7 maintaining >75% maximum activity after incubation at 50°C for 1 h. GOD-w worked as well as commercial GODs to improve bread making. To achieve high-level expression of recombinant GOD in P. pastoris, 272 nucleotides involving 228 residues were mutated, consistent with the codon bias of P. pastoris. The optimized recombinant GOD-m yielded 615 U ml(-1) (2.5 g protein l(-1)) in a 3 l fermentor--410% higher than GOD-w (148 U ml(-1)), and thus is a low-cost alternative for the bread baking industry.

Growth hormone and drug metabolism. Acute effects on microsomal mixed-function oxidase activities in rat liver.

PubMed Central

Wilson, J T; Spelsberg, T C

1976-01-01

Adult male rats were subjected either to sham operation or to hypophysectomy and adrenalectomy and maintained for a total of 10 days before treatment with growth hormone. Results of the early effects of growth hormone on the activities of the mixed-function oxidases in rat liver over a 96h period after growth-hormone treatment are presented. 2. Hypophysectomy and adrenalectomy result in decreased body and liver weight and decreased drug metabolism (mixed-function oxidases). Concentrations of electron-transport-system components are also decreased. 3. In the hypophysectomized/adrenalectomized rats, growth hormone decreases the activities of the liver mixed-function oxidases and the cytochrome P-450 and cytochrome c reductases, as well as decreasing the concentration of cytochrome P-450 compared with that of control rats. Similar but less dramatic results are obtained with sham-operated rats. 4. It is concluded that whereas growth hormone enhances liver growth, including induction of many enzyme activities, it results in a decrease in mixed-function oxidase activity. Apparently, mixed-function oxidase activity decreases in liver when growth (mitogenesis) increases. PMID:938458

Immunological and molecular comparison of polyphenol oxidase in Rosaceae fruit trees.

PubMed

Haruta, M; Murata, M; Kadokura, H; Homma, S

1999-03-01

An antibody raised against apple polyphenol oxidase (PPO) cross-reacted with PPOs from Japanese pear (Pyrus pyrifolia), pear (Pyrus communis), peach (Prunus persica), Chinese quince (Pseudocydonia sinensis) and Japanese loquat (Eriobotrya japonica). Core fragments (681 bp) of the corresponding PPO genes were amplified and characterized. The deduced protein sequences showed identities of 85.3 to 97.5%. Chlorogenic acid oxidase activity of these PPOs showed higher activities when assayed at pH 4 than at pH 6. These results indicate that PPOs in Rosaceae plants are structurally and enzymatically similar.

A multicopper oxidase contributes to the copper tolerance of Brucella melitensis 16M.

PubMed

Wu, Tonglei; Wang, Shaohua; Wang, Zhen; Peng, Xiaowei; Lu, Yanli; Wu, Qingmin

2015-06-01

Copper is a potent antimicrobial agent. Multiple mechanisms of copper tolerance are utilized by some pathogenic bacteria. BMEII0580, which is significantly similar to the multicopper oxidase from Escherichia coli, was predicted to be the probable blue copper protein YacK precursor in Brucella melitensis 16M, and was designated as Brucella multicopper oxidase (BmcO). A bioinformatics analysis indicated that the typical motifs of multicopper oxidases are present in BmcO. BmcO, the expression of which was up-regulated by copper, could catalyze the oxidation of 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), dimethoxyphenol (DMP) and para-phenylenediamine (pPD), which are widely used as substrates for multicopper oxidase. Additionally, BmcO exhibited ferroxidase activity, which indicated that it might play an important role in the Fe(2+) uptake of B. melitensis. Importantly, the mutant strain 16MΔbmcO was more sensitive to copper than the wild-type strain B. melitensis 16M as well as its complementation strain 16MΔbmcO(bmcO). The infection assays of cells showed that similar bacterial numbers of B. melitensis 16M, 16MΔbmcO and 16MΔbmcO(bmcO) strains were recovered from the infected macrophages. This result indicated that BmcO was not essential for B. melitensis intracellular growth. In conclusion, our results confirm that BmcO is a multicopper oxidase and contributes to the copper tolerance of B. melitensis 16M. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Absence of proton channels in COS-7 cells expressing functional NADPH oxidase components.

PubMed

Morgan, Deri; Cherny, Vladimir V; Price, Marianne O; Dinauer, Mary C; DeCoursey, Thomas E

2002-06-01

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is an enzyme of phagocytes that produces bactericidal superoxide anion (O(2)(-)) via an electrogenic process. Proton efflux compensates for the charge movement across the cell membrane. The proton channel responsible for the H(+) efflux was thought to be contained within the gp91(phox) subunit of NADPH oxidase, but recent data do not support this idea (DeCoursey, T.E., V.V. Cherny, D. Morgan, B.Z. Katz, and M.C. Dinauer. 2001. J. Biol. Chem. 276:36063-36066). In this study, we investigated electrophysiological properties and superoxide production of COS-7 cells transfected with all NADPH oxidase components required for enzyme function (COS(phox)). The 7D5 antibody, which detects an extracellular epitope of the gp91(phox) protein, labeled 96-98% of COS(phox) cells. NADPH oxidase was functional because COS(phox) (but not COS(WT)) cells stimulated by phorbol myristate acetate (PMA) or arachidonic acid (AA) produced superoxide anion. No proton currents were detected in either wild-type COS-7 cells (COS(WT)) or COS(phox) cells studied at pH(o) 7.0 and pH(i) 5.5 or 7.0. Anion currents that decayed at voltages positive to 40 mV were the only currents observed. PMA or AA did not elicit detectable H(+) current in COS(WT) or COS(phox) cells. Therefore, gp91(phox) does not function as a proton channel in unstimulated cells or in activated cells with a demonstrably functional oxidase.

Isolation and characterization of a monoamine oxidase B selective inhibitor from tobacco smoke.

PubMed

Khalil, Ashraf A; Davies, Bruce; Castagnoli, Neal

2006-05-15

It is well established that tobacco smokers have reduced levels of monoamine oxidase activities both in the brain and peripheral organs. Furthermore, extensive evidence suggests that smokers are less prone to develop Parkinson's disease. These facts, plus the observation that inhibition of monoamine oxidase B protects against the parkinsonian inducing effects of the nigrostriatal neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, have prompted studies to identify monoamine oxidase inhibitors in the tobacco plant and tobacco cigarette smoke. Our previous efforts on cured tobacco leaf extracts have led to the characterization of 2,3,6-trimethyl-1,4-naphthoquinone, a non-selective monoamine oxidase inhibitor, and farnesylacetone, a selective monoamine oxidase B inhibitor. We now have extended these studies to tobacco smoke constituents. Fractionation of the smoke extracts has confirmed and extended the qualitative results of an earlier report [J. Korean Soc. Tob. Sci.1997, 19, 136] demonstrating the inhibitory activity of the terpene trans,trans-farnesol on rat brain MAO-B. In the present study, K(i) values for the inhibition of human, baboon, monkey, dog, rat, and mouse liver MAO-B have been determined. Noteworthy is the absence of inhibitory effects on human placental MAO-A and beef liver MAO-B. A limited structure-activity relationship study of analogs of trans,trans-farnesol is reported. Although the health hazards associated with the use of tobacco products preclude any therapeutic opportunities linked to smoking, these results suggest the possibility of identifying novel structures of compounds that could lead to the development of neuroprotective agents.

Recovery of choline oxidase activity by in vitro recombination of individual segments.

PubMed

Heinze, Birgit; Hoven, Nina; O'Connell, Timothy; Maurer, Karl-Heinz; Bartsch, Sebastian; Bornscheuer, Uwe T

2008-11-01

Initial attempts to express a choline oxidase from Arthrobacter pascens (APChO-syn) in Escherichia coli starting from a synthetic gene only led to inactive protein. However, activity was regained by the systematic exchange of individual segments of the gene with segments from a choline oxidase-encoding gene from Arthrobacter globiformis yielding a functional chimeric enzyme. Next, a sequence alignment of the exchanged segment with other choline oxidases revealed a mutation in the APChO-syn, showing that residue 200 was a threonine instead of an asparagine, which is, thus, crucial for confering enzyme activity and, hence, provides an explanation for the initial lack of activity. The active recombinant APChO-syn-T200N variant was biochemically characterized showing an optimum at pH 8.0 and at 37 degrees C. Furthermore, the substrate specificity was examined using N,N-dimethylethanolamine, N-methylethanolamine and 3,3-dimethyl-1-butanol.

Layer-by-Layer Assembly of Glucose Oxidase on Carbon Nanotube Modified Electrodes.

PubMed

Suroviec, Alice H

2017-01-01

The use of enzymatically modified electrodes for the detection of glucose or other non-electrochemically active analytes is becoming increasingly common. Direct heterogeneous electron transfer to glucose oxidase has been shown to be kinetically difficult, which is why electron transfer mediators or indirect detection is usually used for monitoring glucose with electrochemical sensors. It has been found, however, that electrodes modified with single or multi-walled carbon nanotubes (CNTs) demonstrate fast heterogeneous electron transfer kinetics as compared to that found for traditional electrodes. Incorporating CNTs into the assembly of electrochemical glucose sensors, therefore, affords the possibility of facile electron transfer to glucose oxidase, and a more direct determination of glucose. This chapter describes the methods used to use CNTs in a layer-by-layer structure along with glucose oxidase to produce an enzymatically modified electrode with high turnover rates, increased stability and shelf-life.

Regulation of the nitric oxide oxidase activity of myeloperoxidase by pharmacological agents.

PubMed

Maiocchi, Sophie L; Morris, Jonathan C; Rees, Martin D; Thomas, Shane R

2017-07-01

The leukocyte-derived heme enzyme myeloperoxidase (MPO) is released extracellularly during inflammation and impairs nitric oxide (NO) bioavailability by directly oxidizing NO or producing NO-consuming substrate radicals. Here, structurally diverse pharmacological agents with activities as MPO substrates/inhibitors or antioxidants were screened for their effects on MPO NO oxidase activity in human plasma and physiological model systems containing endogenous MPO substrates/antioxidants (tyrosine, urate, ascorbate). Hydrazide-based irreversible/reversible MPO inhibitors (4-ABAH, isoniazid) or the sickle cell anaemia drug, hydroxyurea, all promoted MPO NO oxidase activity. This involved the capacity of NO to antagonize MPO inhibition by hydrazide-derived radicals and/or the ability of drug-derived radicals to stimulate MPO turnover thereby increasing NO consumption by MPO redox intermediates or NO-consuming radicals. In contrast, the mechanism-based irreversible MPO inhibitor 2-thioxanthine, potently inhibited MPO turnover and NO consumption. Although the phenolics acetaminophen and resveratrol initially increased MPO turnover and NO consumption, they limited the overall extent of NO loss by rapidly depleting H 2 O 2 and promoting the formation of ascorbyl radicals, which inefficiently consume NO. The vitamin E analogue trolox inhibited MPO NO oxidase activity in ascorbate-depleted fluids by scavenging NO-consuming tyrosyl and urate radicals. Tempol and related nitroxides decreased NO consumption in ascorbate-replete fluids by scavenging MPO-derived ascorbyl radicals. Indoles or apocynin yielded marginal effects. Kinetic analyses rationalized differences in drug activities and identified criteria for the improved inhibition of MPO NO oxidase activity. This study reveals that widely used agents have important implications for MPO NO oxidase activity under physiological conditions, highlighting new pharmacological strategies for preserving NO bioavailability during

Enzymatic browning and biochemical alterations in black spots of pineapple [Ananas comosus (L.) Merr.].

PubMed

Avallone, Sylvie; Guiraud, Joseph-Pierre; Brillouet, Jean-Marc; Teisson, Claude

2003-08-01

Penicillium funiculosum Thom. was consistently isolated from pineapple-infected fruitlet (black spots). Polyphenol oxidase, peroxidase, and laccase activities were determined in extracts from contiguous and infected fruitlets. Healthy fruitlets showed a rather high level of polyphenol oxidase (optimum pH 7.0), and this activity was tremendously increased (X 10) in contiguous infected fruitlets. Furthermore, infected fruitlets also exhibited laccase activity (optimum pH 4.0), while peroxidase was rather constant in both fruitlets. Browning reactions were attributed to qualitative and quantitative modifications of the enzymatic equipment (polyphenol oxidase and laccase) (p < 0.0001). In infected fruiltets, sucrose and L-malic acid were present at significantly lower amounts than in healthy ones, likely owing to fungal metabolism (p < 0.0001), whereas cell wall material was three times higher, which could be viewed as a defense mechanism to limit expansion of the mycelium.

Spectrophotometric determination of H2O2-generating oxidases using oxyhemoglobin as oxygen donor and indicator.

PubMed

Bârzu, O; Dânşoreanu, M

1980-01-01

1. Spectrophotometric determination of oxygen uptake using oxyhemoglobin as oxygen donor and indicator was used for assay of H2O2-generating oxidases like monoamine oxidase and glucose oxidase. 2. In order to decompose H2O2 formed during the oxygen uptake, catalase and methanol (or ethanol) was added to the respiratory system. At pH values higher than 7.5 the oxydation of deoxygenated hemoglobin to methemoglobin was less than 3%. 2. Oxidases with low Km for oxygen can be assayed using the spectrophotometric method if suitable correction factors are introduced into the calculation of oxygen uptake. The correction factor represents the ratio of the rate of formation (or disappearance) of one of the reactants and the rate of oxyhemoglobin deoxygenation, measured under identical experimental conditions.

Hydroxychavicol: a potent xanthine oxidase inhibitor obtained from the leaves of betel, Piper betle.

PubMed

Murata, Kazuya; Nakao, Kikuyo; Hirata, Noriko; Namba, Kensuke; Nomi, Takao; Kitamura, Yoshihisa; Moriyama, Kenzo; Shintani, Takahiro; Iinuma, Munekazu; Matsuda, Hideaki

2009-07-01

The screening of Piperaceous plants for xanthine oxidase inhibitory activity revealed that the extract of the leaves of Piper betle possesses potent activity. Activity-guided purification led us to obtain hydroxychavicol as an active principle. Hydroxychavicol is a more potent xanthine oxidase inhibitor than allopurinol, which is clinically used for the treatment of hyperuricemia.

Cloning of a phenol oxidase gene from Acremonium murorum and its expression in Aspergillus awamori.

PubMed

Gouka, R J; van der Heiden, M; Swarthoff, T; Verrips, C T

2001-06-01

Fungal multicopper oxidases have many potential industrial applications, since they perform reactions under mild conditions. We isolated a phenol oxidase from the fungus Acremonium murorum var. murorum that was capable of decolorizing plant chromophores (such as anthocyanins). This enzyme is of interest in laundry-cleaning products because of its broad specificity for chromophores. We expressed an A. murorum cDNA library in Saccharomyces cerevisiae and subsequently identified enzyme-producing yeast colonies based on their ability to decolor a plant chromophore. The cDNA sequence contained an open reading frame of 1,806 bp encoding an enzyme of 602 amino acids. The phenol oxidase was overproduced by Aspergillus awamori as a fusion protein with glucoamylase, cleaved in vivo, and purified from the culture broth by hydrophobic-interaction chromatography. The phenol oxidase is active at alkaline pH (the optimum for syringaldazine is pH 9) and high temperature (optimum, 60 degrees C) and is fully stable for at least 1 h at 60 degrees C under alkaline conditions. These characteristics and the high production level of 0.6 g of phenol oxidase per liter in shake flasks, which is equimolar with the glucoamylase protein levels, make this enzyme suitable for use in processes that occur under alkaline conditions, such as laundry cleaning.

Cloning of a Phenol Oxidase Gene from Acremonium murorum and Its Expression in Aspergillus awamori

PubMed Central

Gouka, Robin J.; van der Heiden, Monique; Swarthoff, Ton; Verrips, C. Theo

2001-01-01

Fungal multicopper oxidases have many potential industrial applications, since they perform reactions under mild conditions. We isolated a phenol oxidase from the fungus Acremonium murorum var. murorum that was capable of decolorizing plant chromophores (such as anthocyanins). This enzyme is of interest in laundry-cleaning products because of its broad specificity for chromophores. We expressed an A. murorum cDNA library in Saccharomyces cerevisiae and subsequently identified enzyme-producing yeast colonies based on their ability to decolor a plant chromophore. The cDNA sequence contained an open reading frame of 1,806 bp encoding an enzyme of 602 amino acids. The phenol oxidase was overproduced by Aspergillus awamori as a fusion protein with glucoamylase, cleaved in vivo, and purified from the culture broth by hydrophobic-interaction chromatography. The phenol oxidase is active at alkaline pH (the optimum for syringaldazine is pH 9) and high temperature (optimum, 60°C) and is fully stable for at least 1 h at 60°C under alkaline conditions. These characteristics and the high production level of 0.6 g of phenol oxidase per liter in shake flasks, which is equimolar with the glucoamylase protein levels, make this enzyme suitable for use in processes that occur under alkaline conditions, such as laundry cleaning. PMID:11375170

Knockdown of Polyphenol Oxidase Gene Expression in Potato (Solanum tuberosum L.) with Artificial MicroRNAs.

PubMed

Chi, Ming; Bhagwat, Basdeo; Tang, Guiliang; Xiang, Yu

2016-01-01

It is of great importance and interest to develop crop varieties with low polyphenol oxidase (PPO) activity for the food industry because PPO-mediated oxidative browning is a main cause of post-harvest deterioration and quality loss of fresh produce and processed foods. We recently demonstrated that potato tubers with reduced browning phenotypes can be produced by inhibition of the expression of several PPO gene isoforms using artificial microRNA (amiRNA) technology. The approach introduces a single type of 21-nucleotide RNA population to guide silencing of the PPO gene transcripts in potato tissues. Some advantages of the technology are: small RNA molecules are genetically transformed, off-target gene silencing can be avoided or minimized at the stage of amiRNA designs, and accuracy and efficiency of the processes can be detected at every step using molecular biological techniques. Here we describe the methods for transformation and regeneration of potatoes with amiRNA vectors, detection of the expression of amiRNAs, identification of the cleaved product of the target gene transcripts, and assay of the expression level of PPO gene isoforms in potatoes.

An orphan cbb3-type cytochrome oxidase subunit supports Pseudomonas aeruginosa biofilm growth and virulence

PubMed Central

Jo, Jeanyoung; Cortez, Krista L; Cornell, William Cole; Price-Whelan, Alexa

2017-01-01

Hypoxia is a common challenge faced by bacteria during associations with hosts due in part to the formation of densely packed communities (biofilms). cbb3-type cytochrome c oxidases, which catalyze the terminal step in respiration and have a high affinity for oxygen, have been linked to bacterial pathogenesis. The pseudomonads are unusual in that they often contain multiple full and partial (i.e. ‘orphan’) operons for cbb3-type oxidases and oxidase subunits. Here, we describe a unique role for the orphan catalytic subunit CcoN4 in colony biofilm development and respiration in the opportunistic pathogen Pseudomonas aeruginosa PA14. We also show that CcoN4 contributes to the reduction of phenazines, antibiotics that support redox balancing for cells in biofilms, and to virulence in a Caenorhabditis elegans model of infection. These results highlight the relevance of the colony biofilm model to pathogenicity and underscore the potential of cbb3-type oxidases as therapeutic targets. PMID:29160206

Evolution of NADPH Oxidase Inhibitors: Selectivity and Mechanisms for Target Engagement.

PubMed

Altenhöfer, Sebastian; Radermacher, Kim A; Kleikers, Pamela W M; Wingler, Kirstin; Schmidt, Harald H H W

2015-08-10

Oxidative stress, an excess of reactive oxygen species (ROS) production versus consumption, may be involved in the pathogenesis of different diseases. The only known enzymes solely dedicated to ROS generation are nicotinamide adenine dinucleotide phosphate (NADPH) oxidases with their catalytic subunits (NOX). After the clinical failure of most antioxidant trials, NOX inhibitors are the most promising therapeutic option for diseases associated with oxidative stress. Historical NADPH oxidase inhibitors, apocynin and diphenylene iodonium, are un-specific and not isoform selective. Novel NOX inhibitors stemming from rational drug discovery approaches, for example, GKT137831, ML171, and VAS2870, show improved specificity for NADPH oxidases and moderate NOX isoform selectivity. Along with NOX2 docking sequence (NOX2ds)-tat, a peptide-based inhibitor, the use of these novel small molecules in animal models has provided preliminary in vivo evidence for a pathophysiological role of specific NOX isoforms. Here, we discuss whether novel NOX inhibitors enable reliable validation of NOX isoforms' pathological roles and whether this knowledge supports translation into pharmacological applications. Modern NOX inhibitors have increased the evidence for pathophysiological roles of NADPH oxidases. However, in comparison to knockout mouse models, NOX inhibitors have limited isoform selectivity. Thus, their use does not enable clear statements on the involvement of individual NOX isoforms in a given disease. The development of isoform-selective NOX inhibitors and biologicals will enable reliable validation of specific NOX isoforms in disease models other than the mouse. Finally, GKT137831, the first NOX inhibitor in clinical development, is poised to provide proof of principle for the clinical potential of NOX inhibition.

Rationally engineered flavin-dependent oxidase reveals steric control of dioxygen reduction.

PubMed

Zafred, Domen; Steiner, Barbara; Teufelberger, Andrea R; Hromic, Altijana; Karplus, P Andrew; Schofield, Christopher J; Wallner, Silvia; Macheroux, Peter

2015-08-01

The ability of flavoenzymes to reduce dioxygen varies greatly, and is controlled by the protein environment, which may cause either a rapid reaction (oxidases) or a sluggish reaction (dehydrogenases). Previously, a 'gatekeeper' amino acid residue was identified that controls the reactivity to dioxygen in proteins from the vanillyl alcohol oxidase superfamily of flavoenzymes. We have identified an alternative gatekeeper residue that similarly controls dioxygen reactivity in the grass pollen allergen Phl p 4, a member of this superfamily that has glucose dehydrogenase activity and the highest redox potential measured in a flavoenzyme. A substitution at the alternative gatekeeper site (I153V) transformed the enzyme into an efficient oxidase by increasing dioxygen reactivity by a factor of 60,000. An inverse exchange (V169I) in the structurally related berberine bridge enzyme (BBE) decreased its dioxygen reactivity by a factor of 500. Structural and biochemical characterization of these and additional variants showed that our model enzymes possess a cavity that binds an anion and resembles the 'oxyanion hole' in the proximity of the flavin ring. We showed also that steric control of access to this site is the most important parameter affecting dioxygen reactivity in BBE-like enzymes. Analysis of flavin-dependent oxidases from other superfamilies revealed similar structural features, suggesting that dioxygen reactivity may be governed by a common mechanistic principle. Structural data are available in PDB database under the accession numbers 4PVE, 4PVH, 4PVJ, 4PVK, 4PWB, 4PWC and 4PZF. © 2015 FEBS.

Characterization of two brassinosteroid C-6 oxidase genes in pea.

PubMed

Jager, Corinne E; Symons, Gregory M; Nomura, Takahito; Yamada, Yumiko; Smith, Jennifer J; Yamaguchi, Shinjiro; Kamiya, Yuji; Weller, James L; Yokota, Takao; Reid, James B

2007-04-01

C-6 oxidation genes play a key role in the regulation of biologically active brassinosteroid (BR) levels in the plant. They control BR activation, which involves the C-6 oxidation of 6-deoxocastasterone (6-DeoxoCS) to castasterone (CS) and in some cases the further conversion of CS to brassinolide (BL). C-6 oxidation is controlled by the CYP85A family of cytochrome P450s, and to date, two CYP85As have been isolated in tomato (Solanum lycopersicum), two in Arabidopsis (Arabidopsis thaliana), one in rice (Oryza sativa), and one in grape (Vitis vinifera). We have now isolated two CYP85As (CYP85A1 and CYP85A6) from pea (Pisum sativum). However, unlike Arabidopsis and tomato, which both contain one BR C-6 oxidase that converts 6-DeoxoCS to CS and one BR C-6 Baeyer-Villiger oxidase that converts 6-DeoxoCS right through to BL, the two BR C-6 oxidases in pea both act principally to convert 6-DeoxoCS to CS. The isolation of these two BR C-6 oxidation genes in pea highlights the species-specific differences associated with C-6 oxidation. In addition, we have isolated a novel BR-deficient mutant, lke, which blocks the function of one of these two BR C-6 oxidases (CYP85A6). The lke mutant exhibits a phenotype intermediate between wild-type plants and previously characterized pea BR mutants (lk, lka, and lkb) and contains reduced levels of CS and increased levels of 6-DeoxoCS. To date, lke is the only mutant identified in pea that blocks the latter steps of BR biosynthesis and it will therefore provide an excellent tool to further examine the regulation of BR biosynthesis and the relative biological activities of CS and BL in pea.

Purification and properties of two terminal oxidase complexes of Escherichia coli aerobic respiratory chain.

PubMed

Kita, K; Konishi, K; Anraku, Y

1986-01-01

Two terminal oxidase complexes, cytochrome b-562-o complex and cytochrome b-558-d complex, are isolated in highly purified forms which show ubiquinol oxidase activities. From the result of steady-state kinetics of cytochromes in the membrane and E'm values of purified cytochromes, we propose a branched arrangement of the late exponential phase of aerobic growth, as shown in Fig. 10. Cytochrome b-556 is reduced by several dehydrogenases and the gene for this cytochrome (cybA) is located in the sdh gene cluster. Recently, we found another low-potential b-type cytochrome, cytochrome b-561 (Em' = 20 mV), which is also reduced by dehydrogenases. The position of this new cytochrome in the aerobic respiratory chain is under investigation. Two terminal oxidase complexes branch at the site of ubiquinone-8, and the Km value for oxygen of the purified cytochrome b-558-d complex is about 8-fold lower than that of the purified cytochrome b-562-o complex when ubiquinol-1 is used as substrate. This result is consistent with the idea that the cytochrome b-558-d complex is synthesized as an alternative oxidase for more efficient utilization of oxygen at low oxygen concentration. Thus, E. coli cells can maintain efficient oxidative energy conservation over a wide range of oxygen pressures by simply changing the contents of the two terminal oxidases, each of which functions as a coupling site.

[Effects of nitrogen additions on soil hydrolase and oxidase activities in Pinus elliottii plantations.

PubMed

Zhang, Chuang; Zou, Hong Tao; Zhang, Xin Yu; Kou, Liang; Yang, Yang; Sun, Xiao Min; Li, Sheng Gong; Wang, Hui Min

2016-11-18

We evaluated responses of hydrolase and oxidase activities in a subtropical Pinus elliottii plantation through a nitrogen (N) addition field experiment (dosage level: 0, 40, 120 kg N·hm -2 ·a -1 ). The results showed that N additions significantly decreased the carbon, nitrogen and phosphorus related hydrolase and oxidase activities. The activities of β-1,4-glucosidase (BG), cellobiohydrolase (CBH), β-1,4-N-acetylglucosaminidase (NAG) and peroxidase (PER) activities were decreased by 16.5%-51.1% due to N additions, and the decrease was more remarkable in the higher N addition treatment. The activities of α-1,4-glucosidase (aG), β-1,4-xylosidase (BX), acid phosphatase (AP) and phenol oxidase (PPO) were decreased by 14.5%-38.6% by N additions, however, there was no significant difference among the different N addition treatments. Soil enzyme activities varied obviously in different seasons. The activities of BG, NAG, BX, CBH, AP and PPO were in the order of March > June > October, and aG and PER activities were in the order of October > March > June. Most of the soil hydrolase and oxidase activities were positively correlated with soil pH, but negatively with NO 3 - -N content. It indicated that N additions inhibited soil hydrolase and oxidase activities by reducing soil pH and increasing soil nitrification. N additions inhibited the soil organic matter mineralization and turnover in the subtropical area, and the effects were obvious with the increasing dosage of N additions.

A Xylenol Orange-Based Screening Assay for the Substrate Specificity of Flavin-Dependent para-Phenol Oxidases.

PubMed

Ewing, Tom A; van Noord, Aster; Paul, Caroline E; van Berkel, Willem J H

2018-01-14

Vanillyl alcohol oxidase (VAO) and eugenol oxidase (EUGO) are flavin-dependent enzymes that catalyse the oxidation of para -substituted phenols. This makes them potentially interesting biocatalysts for the conversion of lignin-derived aromatic monomers to value-added compounds. To facilitate their biocatalytic exploitation, it is important to develop methods by which variants of the enzymes can be rapidly screened for increased activity towards substrates of interest. Here, we present the development of a screening assay for the substrate specificity of para -phenol oxidases based on the detection of hydrogen peroxide using the ferric-xylenol orange complex method. The assay was used to screen the activity of VAO and EUGO towards a set of twenty-four potential substrates. This led to the identification of 4-cyclopentylphenol as a new substrate of VAO and EUGO and 4-cyclohexylphenol as a new substrate of VAO. Screening of a small library of VAO and EUGO active-site variants for alterations in their substrate specificity led to the identification of a VAO variant (T457Q) with increased activity towards vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) and a EUGO variant (V436I) with increased activity towards chavicol (4-allylphenol) and 4-cyclopentylphenol. This assay provides a quick and efficient method to screen the substrate specificity of para -phenol oxidases, facilitating the enzyme engineering of known para- phenol oxidases and the evaluation of the substrate specificity of novel para -phenol oxidases.

The cytochrome bd oxidase of Escherichia coli prevents respiratory inhibition by endogenous and exogenous hydrogen sulfide

PubMed Central

Korshunov, Sergey; Imlay, Karin R. C.; Imlay, James A.

2016-01-01

Summary When sulfur compounds are scarce or difficult to process, E. coli adapts by inducing the high-level expression of sulfur-compound importers. If cystine then becomes available, the cystine is rapidly overimported and reduced, leading to a burgeoning pool of intracellular cysteine. Most of the excess cysteine is exported, but some is adventitiously degraded, with the consequent release of sulfide. Sulfide is a potent ligand of copper and heme moieties, raising the prospect that it interferes with enzymes. We observed that when cystine was provided and sulfide levels rose, E. coli became strictly dependent upon cytochrome bd oxidase for continued respiration. Inspection revealed that low-micromolar levels of sulfide inhibited the proton-pumping cytochrome bo oxidase that is regarded as the primary respiratory oxidase. In the absence of the back-up cytochrome bd oxidase, growth failed. Exogenous sulfide elicited the same effect. The potency of sulfide was enhanced when oxygen concentrations were low. Natural oxic-anoxic interfaces are often sulfidic, including the intestinal environment where E. coli dwells. We propose that the sulfide resistance of the cytochrome bd oxidase is a key trait that permits respiration in such habitats. PMID:26991114

A Plastid Terminal Oxidase Associated with Carotenoid Desaturation during Chromoplast Differentiation1

PubMed Central

Josse, Eve-Marie; Simkin, Andrew J.; Gaffé, Joël; Labouré, Anne-Marie; Kuntz, Marcel; Carol, Pierre

2000-01-01

The Arabidopsis IMMUTANS gene encodes a plastid homolog of the mitochondrial alternative oxidase, which is associated with phytoene desaturation. Upon expression in Escherichia coli, this protein confers a detectable cyanide-resistant electron transport to isolated membranes. In this assay this activity is sensitive to n-propyl-gallate, an inhibitor of the alternative oxidase. This protein appears to be a plastid terminal oxidase (PTOX) that is functionally equivalent to a quinol:oxygen oxidoreductase. This protein was immunodetected in achlorophyllous pepper (Capsicum annuum) chromoplast membranes, and a corresponding cDNA was cloned from pepper and tomato (Lycopersicum esculentum) fruits. Genomic analysis suggests the presence of a single gene in these organisms, the expression of which parallels phytoene desaturase and ζ-carotene desaturase gene expression during fruit ripening. Furthermore, this PTOX gene is impaired in the tomato ghost mutant, which accumulates phytoene in leaves and fruits. These data show that PTOX also participates in carotenoid desaturation in chromoplasts in addition to its role during early chloroplast development. PMID:10938359

Experimental Evidence for a Hydride Transfer Mechanism in Plant Glycolate Oxidase Catalysis*

PubMed Central

Dellero, Younès; Mauve, Caroline; Boex-Fontvieille, Edouard; Flesch, Valérie; Jossier, Mathieu; Tcherkez, Guillaume; Hodges, Michael

2015-01-01

In plants, glycolate oxidase is involved in the photorespiratory cycle, one of the major fluxes at the global scale. To clarify both the nature of the mechanism and possible differences in glycolate oxidase enzyme chemistry from C3 and C4 plant species, we analyzed kinetic parameters of purified recombinant C3 (Arabidopsis thaliana) and C4 (Zea mays) plant enzymes and compared isotope effects using natural and deuterated glycolate in either natural or deuterated solvent. The 12C/13C isotope effect was also investigated for each plant glycolate oxidase protein by measuring the 13C natural abundance in glycolate using natural or deuterated glycolate as a substrate. Our results suggest that several elemental steps were associated with an hydrogen/deuterium isotope effect and that glycolate α-deprotonation itself was only partially rate-limiting. Calculations of commitment factors from observed kinetic isotope effect values support a hydride transfer mechanism. No significant differences were seen between C3 and C4 enzymes. PMID:25416784

Dual inhibitors of cholinesterases and monoamine oxidases for Alzheimer's disease.

PubMed

Knez, Damijan; Sova, Matej; Košak, Urban; Gobec, Stanislav

2017-05-01

Accumulating evidence indicates a solid relationship between several enzymes and Alzheimer's disease. Cholinesterases and monoamine oxidases are closely associated with the disease symptomatology and progression and have been tackled simultaneously using several multifunctional ligands. This design strategy offers great chances to alter the course of Alzheimer's disease, in addition to alleviation of the symptoms. More than 15 years of research has led to the identification of various dual cholinesterase/monoamine oxidase inhibitors, while some showing positive outcomes in clinical trials, thus giving rise to additional research efforts in the field. The aim of this review is to provide an update on the novel dual inhibitors identified recently and to shed light on their therapeutic potential.

Enzymatic properties of the membrane-bound NADH oxidase system in the aerobic respiratory chain of Bacillus cereus.

PubMed

Kim, Man Suk; Kim, Young Jae

2004-11-30

Membranes prepared from Bacillus cereus KCTC 3674, grown aerobically on a complex medium, oxidized NADH exclusively, whereas deamino-NADH was little oxidized. The respiratory chain-linked NADH oxidase exhibited an apparent K(m) value of approximately 65 microM for NADH. The maximum activity of the NADH oxidase was obtained at about pH 8.5 in the presence of 0.1 M KCl (or NaCl). Respiratory chain inhibitor 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) inhibited the activity of the NADH oxidase by about 90% at a concentration of 40 microM. Interestingly, rotenone and capsaicin inhibited the activity of the NADH oxidase by about 60% at a concentration of 40 microM and the activity was also highly sensitive to Ag(+).

Bisphenol A 3,4-quinone induces the conversion of xanthine dehydrogenase into oxidase in vitro.

PubMed

Sakuma, Satoru; Nakanishi, Masahiko; Morinaga, Kazuhiro; Fujitake, Mihoyo; Wada, Shun-ichi; Fujimoto, Yohko

2010-01-01

In the present study, we assessed the influence of bisphenol A (BPA) and bisphenol A 3,4-quinone (BPAQ) on the conversion of xanthine dehydrogenase (XD) into xanthine oxidase (XO) in the rat liver in vitro. BPA up to 100 micromol/L did not affect the XO and XD activities in the partially purified cytosolic fraction from rat liver, whereas BPAQ (2-10 micromol/L) dose-dependently enhanced the XO activity concomitant with a decrease in the XD activity, implying that BPAQ, but not BPA, can convert XD into the reactive oxygen species (ROS) producing the form XO. Furthermore, it was found that BPAQ could increase the generation of ROS and oxidize the guanine moiety of deoxyguanosine in the DNA of primary rat hepatocyte cultures. These results suggest that BPAQ has the potential to convert XD into XO in the liver, which in turn may lead to ROS generation and oxidative DNA damage in this region. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Mapping the primary structure of copper/topaquinone-containing methylamine oxidase from Aspergillus niger.

PubMed

Lenobel, R; Sebela, M; Frébort, I

2005-01-01

The amino acid sequence of methylamine oxidase (MeAO) from the fungus Aspergillus niger was analyzed using mass spectrometry (MS). First, MeAO was characterized by an accurate molar mass of 72.4 kDa of the monomer measured using MALDI-TOF-MS and by a pI value of 5.8 determined by isoelectric focusing. MALDI-TOF-MS revealed a clear peptide mass fingerprint after tryptic digestion, which did not provide any relevant hit when searched against a nonredundant protein database and was different from that of A. niger amine oxidase AO-I. Tandem mass spectrometry with electrospray ionization coupled to liquid chromatography allowed unambiguous reading of six peptide sequences (11-19 amino acids) and seven sequence tags (4-15 amino acids), which were used for MS BLAST homology searching. MeAO was found to be largely homologous to a hypothetical protein AN7641.2 (EMBL/GenBank protein-accession code EAA61827) from Aspergillus nidulans FGSC A4 with a theoretical molar mass of 76.46 kDa and pI 6.14, which belongs to the superfamily of copper amine oxidases. The protein AN7641.2 is only little homologous to the amine oxidase AO-I (32% identity, 49 % similarity).

Monoamine oxidase inhibitors from Gentiana lutea.

PubMed

Haraguchi, Hiroyuki; Tanaka, Yasumasa; Kabbash, Amal; Fujioka, Toshihiro; Ishizu, Takashi; Yagi, Akira

2004-08-01

Three monoamine oxidase (MAO) inhibitors were isolated from Gentiana lutea. Their structures were elucidated to be 3-3''linked-(2'-hydroxy-4-O-isoprenylchalcone)-(2'''-hydroxy-4''-O-isoprenyldihydrochalcone) (1), 2-methoxy-3-(1,1'-dimethylallyl)-6a,10a-dihydrobenzo(1,2-c)chroman-6-one and 5-hydroxyflavanone. These compounds, and the hydrolysis product of 1, displayed competitive inhibitory properties against MAO-B which was more effective than MAO-A.

Oxidase catalysis via aerobically generated hypervalent iodine intermediates

NASA Astrophysics Data System (ADS)

Maity, Asim; Hyun, Sung-Min; Powers, David C.

2018-02-01

The development of sustainable oxidation chemistry demands strategies to harness O2 as a terminal oxidant. Oxidase catalysis, in which O2 serves as a chemical oxidant without necessitating incorporation of oxygen into reaction products, would allow diverse substrate functionalization chemistry to be coupled to O2 reduction. Direct O2 utilization suffers from intrinsic challenges imposed by the triplet ground state of O2 and the disparate electron inventories of four-electron O2 reduction and two-electron substrate oxidation. Here, we generate hypervalent iodine reagents—a broadly useful class of selective two-electron oxidants—from O2. This is achieved by intercepting reactive intermediates of aldehyde autoxidation to aerobically generate hypervalent iodine reagents for a broad array of substrate oxidation reactions. The use of aryl iodides as mediators of aerobic oxidation underpins an oxidase catalysis platform that couples substrate oxidation directly to O2 reduction. We anticipate that aerobically generated hypervalent iodine reagents will expand the scope of aerobic oxidation chemistry in chemical synthesis.

Diamine oxidase is involved in H(2)O(2) production in the chalazal cells during barley grain filling.

PubMed

Asthir, Bavita; Duffus, Carol M; Smith, Rachel C; Spoor, William

2002-04-01

The localization and activities of diamine oxidase (DAO, EC 1.4.3.6) and polyamine oxidase (PAO, EC 1.4.3.4) together with polyamine levels have been investigated in developing grains of barley (Hordeum vulgare L.). DAO (pH 7.5) is present mainly in vascular tissue and its neighbouring cells, namely chalazal cells and nucellar projection, while PAO (pH 6.0) is mainly localized in the chlorenchymatous cells of the crease and at the base of the vascular tissue. Activities of both these enzymes appear to be independently-regulated, as DAO activity increased steadily throughout grain development while PAO activity was higher during the early stages of grain filling, declined thereafter and again increased towards maturity. The maximum activities of DAO coincided with the maximum content of putrescine while the levels of PAO did not seem to be directly correlated with spermidine or spermine contents. Isoelectric focusing (IEF) of DAO and PAO activities revealed the presence of bands at 30 and 45 DPA. The possible involvement of DAO and PAO in the supply of H(2)O(2) to peroxidase-catalysed reactions in the chalazal cells during grain filling is discussed.

A point mutation of valine-311 to methionine in Bacillus subtilis protoporphyrinogen oxidase does not greatly increase resistance to the diphenyl ether herbicide oxyfluorfen.

PubMed

Jeong, Eunjoo; Houn, Thavrak; Kuk, Yongin; Kim, Eun-Seon; Chandru, Hema Kumar; Baik, Myunggi; Back, Kyoungwhan; Guh, Ja-Ock; Han, Oksoo

2003-10-01

In an effort to asses the effect of Val311Met point mutation of Bacillus subtilis protoporphyrinogen oxidase on the resistance to diphenyl ether herbicides, a Val311Met point mutant of B. subtilis protoporphyrinogen oxidase was prepared, heterologously expressed in Escherichia coli, and the purified recombinant Val311Met mutant protoporphyrinogen oxidase was kinetically characterized. The mutant protoporphyrinogen oxidase showed very similar kinetic patterns to wild type protoporphyrinogen oxidase, with slightly decreased activity dependent on pH and the concentrations of NaCl, Tween 20, and imidazole. When oxyfluorfen was used as a competitive inhibitor, the Val311Met mutant protoporphyrinogen oxidase showed an increased inhibition constant about 1.5 times that of wild type protoporphyrinogen oxidase. The marginal increase of the inhibition constant indicates that the Val311Met point mutation in B. subtilis protoporphyrinogen oxidase may not be an important determinant in the mechanism that protects protoporphyrinogen oxidase against diphenyl ether herbicides.

The Regulation of Aldehyde Oxidase in Imaginal Wing Discs of Drosophila Hybrids: Evidence for cis- and trans-Acting Control Elements

PubMed Central

Sprey, Th. E.; Kuhn, David T.

1987-01-01

The aldehyde oxidase (Aldox) distribution pattern was determined for wing discs of partial hybrids between D. melanogaster and D. simulans. In these animals the regulation of Aldox activity is not uniform over the disc epithelium as both cis-dominant and trans -acting control were evident in different regions of the disc. The Aldox expression was shown to be regulated by loci on the X chromosome, 2L and 3R of D. melanogaster and 2R and 3R of D. simulans. PMID:17246366

Purification and characterization of polyphenol oxidase from rape flower.

PubMed

Sun, Han-Ju; Wang, Jing; Tao, Xue-Ming; Shi, Juan; Huang, Mei-Ying; Chen, Zhe

2012-01-25

The purification and partial enzymology characteristics of polyphenol oxidase (PPO) from rape flower were studied. After preliminary treatments, the crude enzyme solution was in turn purified with ammonium sulfate, dialysis, and Sephadex G-75 gel chromatography. The optimal conditions and stability of PPO were examined at different pH values and temperatures. Subsequently, PPO was also characterized by substrate (catechol) concentrations, inhibitors, kinetic parameters, and molecular weight. Results showed that the optimal pH for PPO activity was 5.5 in the presence of catechol and that PPO was relatively stable at pH 3.5-5.5. PPO was moderately stable at temperatures from 60 to 70 °C, whereas it was easily denatured at 80-90 °C. Ethylenediaminetetraacetic acid, sodium chloride, and calcium chloride had little inhibitive effects on PPO, whereas citric acid, sodium sulfite, and ascorbic acid had strongly inhibitive effects. The Michaelis-Menten constant (K(m)) and maximal reaction velocity (V(max)) of PPO were 0.767 mol/L and 0.519 Ab/min/mL of the crude PPO solution, respectively. PPO was finally purified to homogeneity with a purification factor of 4.41-fold and a recovery of 12.41%. Its molecular weight was 60.4 kDa, indicating that the PPO is a dimer. The data obtained in this research may help to prevent the enzymatic browning of rape flower during its storage and processing.

Activation of NADPH oxidase mediates increased endoplasmic reticulum stress and left ventricular remodeling after myocardial infarction in rabbits.

PubMed

Li, Bao; Tian, Jing; Sun, Yi; Xu, Tao-Rui; Chi, Rui-Fang; Zhang, Xiao-Li; Hu, Xin-Ling; Zhang, Yue-An; Qin, Fu-Zhong; Zhang, Wei-Fang

2015-05-01

Nicotinamide adenine dinucleotide 3-phosphate (NADPH) oxidase activity and endoplasmic reticulum (ER) stress are increased after myocardial infarction (MI). In this study, we proposed to test whether activation of the NADPH oxidase in the remote non-infarcted myocardium mediates ER stress and left ventricular (LV) remodeling after MI. Rabbits with MI or sham operation were randomly assigned to orally receive an NADPH oxidase inhibitor apocynin or placebo for 30 days. The agents were administered beginning at 1 week after surgery. MI rabbits exhibited decreases in LV fractional shortening, LV ejection fraction and the first derivative of the LV pressure rise, which were abolished by apocynin treatment. NADPH oxidase Nox2 protein and mRNA expressions were increased in the remote non-infarcted myocardium after MI. Immunolabeling further revealed that Nox2 was increased in cardiac myocytes in the remote myocardium. The apocynin treatment prevented increases in the Nox2 expression, NADPH oxidase activity, oxidative stress, myocyte apoptosis and GRP78, CHOP and cleaved caspase 12 protein expression in the remote myocardium. The apocynin treatment also attenuated increases in myocyte diameter and cardiac fibrosis. In cultured H9C2 cardiomyocytes exposed to angiotensin II, an important stimulus for post-MI remodeling, Nox2 knockdown with siRNA significantly inhibited angiotensin II-induced NADPH oxidase activation, reactive oxygen species and GRP78 and CHOP protein expression. We conclude that NADPH oxidase inhibition attenuates increased ER stress in the remote non-infarcted myocardium and LV remodeling late after MI in rabbits. These findings suggest that the activation of NADPH oxidase in the remote non-infarcted myocardium mediates increased ER stress, contributing to myocyte apoptosis and LV remodeling after MI. Copyright © 2015 Elsevier B.V. All rights reserved.

Molecular Insights of p47phox Phosphorylation Dynamics in the Regulation of NADPH Oxidase Activation and Superoxide Production*

PubMed Central

Meijles, Daniel N.; Fan, Lampson M.; Howlin, Brendan J.; Li, Jian-Mei

2014-01-01

Phagocyte superoxide production by a multicomponent NADPH oxidase is important in host defense against microbial invasion. However inappropriate NADPH oxidase activation causes inflammation. Endothelial cells express NADPH oxidase and endothelial oxidative stress due to prolonged NADPH oxidase activation predisposes many diseases. Discovering the mechanism of NADPH oxidase activation is essential for developing novel treatment of these diseases. The p47phox is a key regulatory subunit of NADPH oxidase; however, due to the lack of full protein structural information, the mechanistic insight of p47phox phosphorylation in NADPH oxidase activation remains incomplete. Based on crystal structures of three functional domains, we generated a computational structural model of the full p47phox protein. Using a combination of in silico phosphorylation, molecular dynamics simulation and protein/protein docking, we discovered that the C-terminal tail of p47phox is critical for stabilizing its autoinhibited structure. Ser-379 phosphorylation disrupts H-bonds that link the C-terminal tail to the autoinhibitory region (AIR) and the tandem Src homology 3 (SH3) domains, allowing the AIR to undergo phosphorylation to expose the SH3 pocket for p22phox binding. These findings were confirmed by site-directed mutagenesis and gene transfection of p47phox−/− coronary microvascular cells. Compared with wild-type p47phox cDNA transfected cells, the single mutation of S379A completely blocked p47phox membrane translocation, binding to p22phox and endothelial O2⨪ production in response to acute stimulation of PKC. p47phox C-terminal tail plays a key role in stabilizing intramolecular interactions at rest. Ser-379 phosphorylation is a molecular switch which initiates p47phox conformational changes and NADPH oxidase-dependent superoxide production by cells. PMID:24970888

Effects of co-administration of dietary sodium arsenite and an NADPH oxidase inhibitor on the rat bladder epithelium.

PubMed

Suzuki, Shugo; Arnold, Lora L; Pennington, Karen L; Kakiuchi-Kiyota, Satoko; Cohen, Samuel M

2009-06-30

Arsenite (As(III)), an inorganic arsenical, is a known human carcinogen, inducing tumors of the skin, urinary bladder and lung. It is metabolized to organic methylated arsenicals. Oxidative stress has been suggested as a mechanism for arsenic-induced carcinogenesis. Reactive oxygen species (ROS) can be important factors for carcinogenesis and tumor progression. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is known to produce intracellular ROS, therefore, we investigated the ability of apocynin (acetovanillone), an NADPH oxidase inhibitor, to inhibit the cytotoxicity and regenerative cell proliferation of arsenic in vitro and in vivo. Apocynin had similar effects in reducing the cytotoxicity of As(III) and dimethylarsinous acid (DMA(III)) in rat urothelial cells in vitro. When tested at the same concentrations as apocynin, other antioxidants, such as l-ascorbate and N-acetylcysteine, did not inhibit As(III)-induced cytotoxicity but they were more effective at inhibiting DMA(III)-induced cytotoxicity compared with apocynin. In vivo, female rats were treated for 3 weeks with 100ppm As(III). Immunohistochemical staining for 8-hydroxy-2'-deoxyguanosine (8-OHdG) showed that apocynin reduced oxidative stress partially induced by As(III) treatment on rat urothelium, and significantly reduced the cytotoxicity of superficial cells detected by scanning electron microscopy (SEM). However, based on the incidence of simple hyperplasia and the bromodeoxyuridine (BrdU) labeling index, apocynin did not inhibit As(III)-induced urothelial cell proliferation. These data suggest that the NADPH oxidase inhibitor, apocynin, may have the ability to partially inhibit arsenic-induced oxidative stress and cytotoxicity of the rat bladder epithelium in vitro and in vivo. However, apocynin did not inhibit the regenerative cell proliferation induced by arsenite in a short-term study.

Heterologous Expression of Phanerochaete chrysoporium Glyoxal Oxidase and its Application for the Coupled Reaction with Manganese Peroxidase to Decolorize Malachite Green

PubMed Central

Son, Yu-Lim; Kim, Hyoun-Young; Thiyagarajan, Saravanakumar; Xu, Jing Jing

2012-01-01

cDNA of the glx1 gene encoding glyoxal oxidase (GLX) from Phanerochaete chrysosporium was isolated and expressed in Pichia pastoris. The recombinant GLX (rGLX) produces H2O2 over 7.0 nmol/min/mL using methyl glyoxal as a substrate. Use of rGLX as a generator of H2O2 improved the coupled reaction with recombinant manganese peroxidase resulting in decolorization of malachite green up to 150 µM within 90 min. PMID:23323052

Molecular evolution of the polyamine oxidase gene family in Metazoa

PubMed Central

2012-01-01

Background Polyamine oxidase enzymes catalyze the oxidation of polyamines and acetylpolyamines. Since polyamines are basic regulators of cell growth and proliferation, their homeostasis is crucial for cell life. Members of the polyamine oxidase gene family have been identified in a wide variety of animals, including vertebrates, arthropodes, nematodes, placozoa, as well as in plants and fungi. Polyamine oxidases (PAOs) from yeast can oxidize spermine, N1-acetylspermine, and N1-acetylspermidine, however, in vertebrates two different enzymes, namely spermine oxidase (SMO) and acetylpolyamine oxidase (APAO), specifically catalyze the oxidation of spermine, and N1-acetylspermine/N1-acetylspermidine, respectively. Little is known about the molecular evolutionary history of these enzymes. However, since the yeast PAO is able to catalyze the oxidation of both acetylated and non acetylated polyamines, and in vertebrates these functions are addressed by two specialized polyamine oxidase subfamilies (APAO and SMO), it can be hypothesized an ancestral reference for the former enzyme from which the latter would have been derived. Results We analysed 36 SMO, 26 APAO, and 14 PAO homologue protein sequences from 54 taxa including various vertebrates and invertebrates. The analysis of the full-length sequences and the principal domains of vertebrate and invertebrate PAOs yielded consensus primary protein sequences for vertebrate SMOs and APAOs, and invertebrate PAOs. This analysis, coupled to molecular modeling techniques, also unveiled sequence regions that confer specific structural and functional properties, including substrate specificity, by the different PAO subfamilies. Molecular phylogenetic trees revealed a basal position of all the invertebrates PAO enzymes relative to vertebrate SMOs and APAOs. PAOs from insects constitute a monophyletic clade. Two PAO variants sampled in the amphioxus are basal to the dichotomy between two well supported monophyletic clades including

Molecular evolution of the polyamine oxidase gene family in Metazoa.

PubMed

Polticelli, Fabio; Salvi, Daniele; Mariottini, Paolo; Amendola, Roberto; Cervelli, Manuela

2012-06-20

Polyamine oxidase enzymes catalyze the oxidation of polyamines and acetylpolyamines. Since polyamines are basic regulators of cell growth and proliferation, their homeostasis is crucial for cell life. Members of the polyamine oxidase gene family have been identified in a wide variety of animals, including vertebrates, arthropodes, nematodes, placozoa, as well as in plants and fungi. Polyamine oxidases (PAOs) from yeast can oxidize spermine, N1-acetylspermine, and N1-acetylspermidine, however, in vertebrates two different enzymes, namely spermine oxidase (SMO) and acetylpolyamine oxidase (APAO), specifically catalyze the oxidation of spermine, and N1-acetylspermine/N1-acetylspermidine, respectively. Little is known about the molecular evolutionary history of these enzymes. However, since the yeast PAO is able to catalyze the oxidation of both acetylated and non acetylated polyamines, and in vertebrates these functions are addressed by two specialized polyamine oxidase subfamilies (APAO and SMO), it can be hypothesized an ancestral reference for the former enzyme from which the latter would have been derived. We analysed 36 SMO, 26 APAO, and 14 PAO homologue protein sequences from 54 taxa including various vertebrates and invertebrates. The analysis of the full-length sequences and the principal domains of vertebrate and invertebrate PAOs yielded consensus primary protein sequences for vertebrate SMOs and APAOs, and invertebrate PAOs. This analysis, coupled to molecular modeling techniques, also unveiled sequence regions that confer specific structural and functional properties, including substrate specificity, by the different PAO subfamilies. Molecular phylogenetic trees revealed a basal position of all the invertebrates PAO enzymes relative to vertebrate SMOs and APAOs. PAOs from insects constitute a monophyletic clade. Two PAO variants sampled in the amphioxus are basal to the dichotomy between two well supported monophyletic clades including, respectively, all

Ectopic Expression of Pumpkin Gibberellin Oxidases Alters Gibberellin Biosynthesis and Development of Transgenic Arabidopsis Plants1

PubMed Central

Radi, Abeer; Lange, Theo; Niki, Tomoya; Koshioka, Masaji; Lange, Maria João Pimenta

2006-01-01

Immature pumpkin (Cucurbita maxima) seeds contain gibberellin (GA) oxidases with unique catalytic properties resulting in GAs of unknown function for plant growth and development. Overexpression of pumpkin GA 7-oxidase (CmGA7ox) in Arabidopsis (Arabidopsis thaliana) resulted in seedlings with elongated roots, taller plants that flower earlier with only a little increase in bioactive GA4 levels compared to control plants. In the same way, overexpression of the pumpkin GA 3-oxidase1 (CmGA3ox1) resulted in a GA overdose phenotype with increased levels of endogenous GA4. This indicates that, in Arabidopsis, 7-oxidation and 3-oxidation are rate-limiting steps in GA plant hormone biosynthesis that control plant development. With an opposite effect, overexpression of pumpkin seed-specific GA 20-oxidase1 (CmGA20ox1) in Arabidopsis resulted in dwarfed plants that flower late with reduced levels of GA4 and increased levels of physiological inactive GA17 and GA25 and unexpected GA34 levels. Severe dwarfed plants were obtained by overexpression of the pumpkin GA 2-oxidase1 (CmGA2ox1) in Arabidopsis. This dramatic change in phenotype was accompanied by a considerable decrease in the levels of bioactive GA4 and an increase in the corresponding inactivation product GA34 in comparison to control plants. In this study, we demonstrate the potential of four pumpkin GA oxidase-encoding genes to modulate the GA plant hormone pool and alter plant stature and development. PMID:16384902

A peroxidase/dual oxidase system modulates midgut epithelial immunity in Anopheles gambiae.

PubMed

Kumar, Sanjeev; Molina-Cruz, Alvaro; Gupta, Lalita; Rodrigues, Janneth; Barillas-Mury, Carolina

2010-03-26

Extracellular matrices in diverse biological systems are cross-linked by dityrosine covalent bonds catalyzed by the peroxidase/oxidase system. We show that a peroxidase, secreted by the Anopheles gambiae midgut, and dual oxidase form a dityrosine network that decreases gut permeability to immune elicitors. This network protects the microbiota by preventing activation of epithelial immunity. It also provides a suitable environment for malaria parasites to develop within the midgut lumen without inducing nitric oxide synthase expression. Disruption of this barrier results in strong and effective pathogen-specific immune responses.

Timing of electron and proton transfer in the ba(3) cytochrome c oxidase from Thermus thermophilus.

PubMed

von Ballmoos, Christoph; Lachmann, Peter; Gennis, Robert B; Ädelroth, Pia; Brzezinski, Peter

2012-06-05

Heme-copper oxidases are membrane-bound proteins that catalyze the reduction of O(2) to H(2)O, a highly exergonic reaction. Part of the free energy of this reaction is used for pumping of protons across the membrane. The ba(3) oxidase from Thermus thermophilus presumably uses a single proton pathway for the transfer of substrate protons used during O(2) reduction as well as for the transfer of the protons that are pumped across the membrane. The pumping stoichiometry (0.5 H(+)/electron) is lower than that of most other (mitochondrial-like) oxidases characterized to date (1 H(+)/electron). We studied the pH dependence and deuterium isotope effect of the kinetics of electron and proton transfer reactions in the ba(3) oxidase. The results from these studies suggest that the movement of protons to the catalytic site and movement to a site located some distance from the catalytic site [proposed to be a "proton-loading site" (PLS) for pumped protons] are separated in time, which allows individual investigation of these reactions. A scenario in which the uptake and release of a pumped proton occurs upon every second transfer of an electron to the catalytic site would explain the decreased proton pumping stoichiometry compared to that of mitochondrial-like oxidases.

Role of F357 as an Oxygen Gate in the Oxidative Half-Reaction of Choline Oxidase.

PubMed

Salvi, Francesca; Rodriguez, Isela; Hamelberg, Donald; Gadda, Giovanni

2016-03-15

Choline oxidase from Arthrobacter globiformis catalyzes the oxidation of choline to glycine betaine by using oxygen as an electron acceptor. A partially rate limiting isomerization of the reduced wild-type enzyme during the reaction with oxygen was previously detected using solvent viscosity effects. In this study, we hypothesized that the side chains of M62 and F357, located at the entrance to the active site of choline oxidase, may be related to the slow isomerization detected. We engineered a double-variant enzyme M62A/F357A. The kinetic characterization of the double-variant enzyme showed a lack of the isomerization detected in wild-type choline oxidase, and a lack of saturation with an oxygen concentration as high as 1 mM, while most other kinetic parameters were similar to those of wild-type choline oxidase. The kinetic characterization of the single-variant enzymes established that only the side chain of F357 plays a role in the isomerization of choline oxidase in the oxidative half-reaction. Molecular dynamics studies suggest that the slow isomerization related to F357 is possibly due to the participation of the phenyl ring in a newly proposed gating mechanism for a narrow tunnel, assumed to regulate the access of oxygen to the reduced cofactor.

Glitazones inhibit human monoamine oxidase but their anti-inflammatory actions are not mediated by VAP-1/semicarbazide-sensitive amine oxidase inhibition.

PubMed

Carpéné, Christian; Bizou, Mathilde; Tréguer, Karine; Hasnaoui, Mounia; Grès, Sandra

2015-09-01

Glitazones are peroxisome proliferator-activated receptor gamma (PPARγ) agonists widely used as antidiabetic drugs also known as thiazolidinediones. Most of them exert other effects such as anti-inflammatory actions via mechanisms supposed to be independent from PPARγ activation (e.g., decreased plasma monocyte chemoattractant protein-1 (MCP-1) levels). Recently, pioglitazone has been shown to inhibit the B form of monoamine oxidase (MAO) in mouse, while rosiglitazone and troglitazone were described as non-covalent inhibitors of both human MAO A and MAO B. Since molecules interacting with MAO might also inhibit semicarbazide-sensitive amine oxidase (SSAO), known as vascular adhesion protein-1 (VAP-1), and since VAP-1/SSAO inhibitors exhibit anti-inflammatory activity, our aim was to elucidate whether VAP-1/SSAO inhibition could be a mechanism involved in the anti-inflammatory behaviour of glitazones. To this aim, MAO and SSAO activities were measured in human subcutaneous adipose tissue biopsies obtained from overweight women undergoing plastic surgery. The production of hydrogen peroxide, an end-product of amine oxidase activity, was determined in tissue homogenates using a fluorometric method. The oxidation of 1 mM tyramine was inhibited by pargyline and almost resistant to semicarbazide, therefore predominantly MAO-dependent. Rosiglitazone was more potent than pioglitazone in inhibiting tyramine oxidation. By contrast, benzylamine oxidation was only abolished by semicarbazide: hence SSAO-mediated. Pioglitazone hampered SSAO activity only when tested at 1 mM while rosiglitazone was inefficient. However, rosiglitazone exhibited anti-inflammatory activity in human adipocytes by limiting MCP-1 expression. Our observations rule out any involvement of VAP-1/SSAO inhibition and subsequent limitation of leukocyte extravasation in the anti-inflammatory action of glitazones.

Attenuation of NADPH oxidase activation and glomerular filtration barrier remodeling with statin treatment.

PubMed

Whaley-Connell, Adam; Habibi, Javad; Nistala, Ravi; Cooper, Shawna A; Karuparthi, Poorna R; Hayden, Melvin R; Rehmer, Nathan; DeMarco, Vincent G; Andresen, Bradley T; Wei, Yongzhong; Ferrario, Carlos; Sowers, James R

2008-02-01

Activation of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase by angiotensin II is integral to the formation of oxidative stress in the vasculature and the kidney. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibition is associated with reductions of oxidative stress in the vasculature and kidney and associated decreases in albuminuria. Effects of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibition on oxidative stress in the kidney and filtration barrier integrity are poorly understood. To investigate, we used transgenic TG(mRen2)27 (Ren2) rats, which harbor the mouse renin transgene and renin-angiotensin system activation, and an immortalized murine podocyte cell line. We treated young, male Ren2 and Sprague-Dawley rats with rosuvastatin (20 mg/kg IP) or placebo for 21 days. Compared with controls, we observed increases in systolic blood pressure, albuminuria, renal NADPH oxidase activity, and 3-nitrotryosine staining, with reductions in the rosuvastatin-treated Ren2. Structural changes on light and transmission electron microscopy, consistent with periarteriolar fibrosis and podocyte foot-process effacement, were attenuated with statin treatment. Nephrin expression was diminished in the Ren2 kidney and trended to normalize with statin treatment. Angiotensin II-dependent increases in podocyte NADPH oxidase activity and subunit expression (NOX2, NOX4, Rac, and p22(phox)) and reactive oxygen species generation were decreased after in vitro statin treatment. These data support a role for increased NADPH oxidase activity and subunit expression with resultant reactive oxygen species formation in the kidney and podocyte. Furthermore, statin attenuation of NADPH oxidase activation and reactive oxygen species formation in the kidney/podocyte seems to play roles in the abrogation of oxidative stress-induced filtration barrier injury and consequent albuminuria.

Development of a glucose oxidase-based biocatalyst adopting both physical entrapment and crosslinking, and its use in biofuel cells

NASA Astrophysics Data System (ADS)

Chung, Yongjin; Ahn, Yeonjoo; Christwardana, Marcelinus; Kim, Hansung; Kwon, Yongchai

2016-04-01

New enzymatic catalysts prepared using physical entrapment and chemical bonding were used as anodic catalysts to enhance the performance of enzymatic biofuel cells (EBCs). For estimating the physical entrapment effect, the best glucose oxidase (GOx) concentration immobilized on polyethyleneimine (PEI) and carbon nanotube (CNT) (GOx/PEI/CNT) was determined, while for inspecting the chemical bonding effect, terephthalaldehyde (TPA) and glutaraldehyde (GA) crosslinkers were employed. According to the enzyme activity and XPS measurements, when the GOx concentration is 4 mg mL-1, they are most effectively immobilized (via the physical entrapment effect) and TPA-crosslinked GOx/PEI/CNT(TPA/[GOx/PEI/CNT]) forms π conjugated bonds via chemical bonding, inducing the promotion of electron transfer by delocalization of electrons. Due to the optimized GOx concentration and π conjugated bonds, TPA/[GOx/PEI/CNT], including 4 mg mL-1 GOx displays a high electron transfer rate, followed by excellent catalytic activity and EBC performance.New enzymatic catalysts prepared using physical entrapment and chemical bonding were used as anodic catalysts to enhance the performance of enzymatic biofuel cells (EBCs). For estimating the physical entrapment effect, the best glucose oxidase (GOx) concentration immobilized on polyethyleneimine (PEI) and carbon nanotube (CNT) (GOx/PEI/CNT) was determined, while for inspecting the chemical bonding effect, terephthalaldehyde (TPA) and glutaraldehyde (GA) crosslinkers were employed. According to the enzyme activity and XPS measurements, when the GOx concentration is 4 mg mL-1, they are most effectively immobilized (via the physical entrapment effect) and TPA-crosslinked GOx/PEI/CNT(TPA/[GOx/PEI/CNT]) forms π conjugated bonds via chemical bonding, inducing the promotion of electron transfer by delocalization of electrons. Due to the optimized GOx concentration and π conjugated bonds, TPA/[GOx/PEI/CNT], including 4 mg mL-1 GOx displays a high

Phosphatidylinositol 3-Kinase Plays a Vital Role in Regulation of Rice Seed Vigor via Altering NADPH Oxidase Activity

PubMed Central

Liu, Jian; Zhou, Jun; Xing, Da

2012-01-01

Phosphatidylinositol 3-kinase (PI3K) has been reported to be important in normal plant growth and stress responses. In this study, it was verified that PI3K played a vital role in rice seed germination through regulating NADPH oxidase activity. Suppression of PI3K activity by inhibitors wortmannin or LY294002 could abate the reactive oxygen species (ROS) formation, which resulted in disturbance to the seed germination. And then, the signal cascades that PI3K promoted the ROS liberation was also evaluated. Diphenylene iodonium (DPI), an NADPH oxidase inhibitor, suppressed most of ROS generation in rice seed germination, which suggested that NADPH oxidase was the main source of ROS in this process. Pharmacological experiment and RT-PCR demonstrated that PI3K promoted the expression of Os rboh9. Moreover, functional analysis by native PAGE and the measurement of the 2, 3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazo-lium-5- carboxanilide (XTT) formazan concentration both showed that PI3K promoted the activity of NADPH oxidase. Furthermore, the western blot analysis of OsRac-1 demonstrated that the translocation of Rac-1 from cytoplasm to plasma membrane, which was known as a key factor in the assembly of NADPH oxidase, was suppressed by treatment with PI3K inhibitors, resulting in the decreased activity of NADPH oxidase. Taken together, these data favored the novel conclusion that PI3K regulated NADPH oxidase activity through modulating the recruitment of Rac-1 to plasma membrane and accelerated the process of rice seed germination. PMID:22448275

Selective Rac1 inhibition protects renal tubular epithelial cells from oxalate-induced NADPH oxidase-mediated oxidative cell injury

PubMed Central

Thamilselvan, Vijayalakshmi; Menon, Mani

2013-01-01

Oxalate-induced oxidative cell injury is one of the major mechanisms implicated in calcium oxalate nucleation, aggregation and growth of kidney stones. We previously demonstrated that oxalate-induced NADPH oxidase-derived free radicals play a significant role in renal injury. Since NADPH oxidase activation requires several regulatory proteins, the primary goal of this study was to characterize the role of Rac GTPase in oxalate-induced NADPH oxidase-mediated oxidative injury in renal epithelial cells. Our results show that oxalate significantly increased membrane translocation of Rac1 and NADPH oxidase activity of renal epithelial cells in a time-dependent manner. We found that NSC23766, a selective inhibitor of Rac1, blocked oxalate-induced membrane translocation of Rac1 and NADPH oxidase activity. In the absence of Rac1 inhibitor, oxalate exposure significantly increased hydrogen peroxide formation and LDH release in renal epithelial cells. In contrast, Rac1 inhibitor pretreatment, significantly decreased oxalate-induced hydrogen peroxide production and LDH release. Furthermore, PKC α and δ inhibitor, oxalate exposure did not increase Rac1 protein translocation, suggesting that PKC resides upstream from Rac1 in the pathway that regulates NADPH oxidase. In conclusion, our data demonstrate for the first time that Rac1-dependent activation of NADPH oxidase might be a crucial mechanism responsible for oxalate-induced oxidative renal cell injury. These findings suggest that Rac1 signaling plays a key role in oxalate-induced renal injury, and may serve as a potential therapeutic target to prevent calcium oxalate crystal deposition in stone formers and reduce recurrence. PMID:21814770

Advanced oxidation protein products induce inflammatory response in fibroblast-like synoviocytes through NADPH oxidase -dependent activation of NF-κB.

PubMed

Zheng, Shuai; Zhong, Zhao-Ming; Qin, Shuai; Chen, Guo-Xian; Wu, Qian; Zeng, Ji-Huan; Ye, Wen-Bin; Li, Wei; Yuan, Kai; Yao, Ling; Chen, Jian-Ting

2013-01-01

Advanced oxidation protein products (AOPPs), a marker of oxidative stress, are prevalent in many kinds of disorders. Rheumatoid arthritis (RA), mainly resulting from the dysfunction of fibroblast-like synoviocytes (FLSs), is related to oxidative stress. Although the increased levels of AOPPs in RA patients were reported, the effect of AOPPs on FLSs function still remains unclear. Therefore, our study aims to investigate whether AOPPs have an effect on the inflammatory response of FLSs in vitro. FLSs obtained from both knees of rats were treated with or without AOPPs-modified rat serum albumin (AOPPs-RSA) in vitro. The mRNA and protein expression of tumor necrosis factor (TNF)-α, interleukin(IL)-1β, matrix metalloproteinases(MMP)-3, MMP-13 and vascular endothelial growth factor (VEGF) were measured by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA), respectively. Reactive oxygen species (ROS) generation was detected by fluorescent microscope and fluorescence microplate reader. Immunoprecipitation, Co-Immunoprecipitation and western blot were performed to examine the activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and nuclear factor kappa B (NF-κB). Exposure of FLSs to AOPPs upregulated the mRNA and protein expression of TNF-α, IL-1β, MMP-3, MMP-13 and VEGF in a concentration dependent manner. AOPPs treatment triggered ROS production in FLSs, which was significantly abolished by ROS scavenger N-acetyl-L-cysteine (NAC), superoxide dismutase (SOD), NADPH oxidase inhibitors diphenyleneiodonium (DPI) and apocynin. Challenged AOPPs induced phosphorylation of p47(phox), triggered an interaction between p47(phox), p22(phox) and gp91(phox), and significantly upregulated expression of NADPH oxidase subunits p47(phox), p22(phox) and gp91(phox). IκB degradation and nuclear translocation of NF-κB p65 induced by AOPPs were significantly blocked by SOD, NAC, DPI and apocynin. These data indicate that

Exercise training decreases NADPH oxidase activity and restores skeletal muscle mass in heart failure rats.

PubMed

Cunha, Telma F; Bechara, Luiz R G; Bacurau, Aline V N; Jannig, Paulo R; Voltarelli, Vanessa A; Dourado, Paulo M; Vasconcelos, Andrea R; Scavone, Cristóforo; Ferreira, Júlio C B; Brum, Patricia C

2017-04-01

We have recently demonstrated that NADPH oxidase hyperactivity, NF-κB activation, and increased p38 phosphorylation lead to atrophy of glycolytic muscle in heart failure (HF). Aerobic exercise training (AET) is an efficient strategy to counteract skeletal muscle atrophy in this syndrome. Therefore, we tested whether AET would regulate muscle redox balance and protein degradation by decreasing NADPH oxidase hyperactivity and reestablishing NF-κB signaling, p38 phosphorylation, and proteasome activity in plantaris muscle of myocardial infarcted-induced HF (MI) rats. Thirty-two male Wistar rats underwent MI or fictitious surgery (SHAM) and were randomly assigned into untrained (UNT) and trained (T; 8 wk of AET on treadmill) groups. AET prevented HF signals and skeletal muscle atrophy in MI-T, which showed an improved exercise tolerance, attenuated cardiac dysfunction and increased plantaris fiber cross-sectional area. To verify the role of inflammation and redox imbalance in triggering protein degradation, circulating TNF-α levels, NADPH oxidase profile, NF-κB signaling, p38 protein levels, and proteasome activity were assessed. MI-T showed a reduced TNF-α levels, NADPH oxidase activity, and Nox2 mRNA expression toward SHAM-UNT levels. The rescue of NADPH oxidase activity induced by AET in MI rats was paralleled by reducing nuclear binding activity of the NF-κB, p38 phosphorylation, atrogin-1, mRNA levels, and 26S chymotrypsin-like proteasome activity. Taken together our data provide evidence for AET improving plantaris redox homeostasis in HF associated with a decreased NADPH oxidase, redox-sensitive proteins activation, and proteasome hyperactivity further preventing atrophy. These data reinforce the role of AET as an efficient therapy for muscle wasting in HF. NEW & NOTEWORTHY This study demonstrates, for the first time, the contribution of aerobic exercise training (AET) in decreasing muscle NADPH oxidase activity associated with reduced reactive oxygen

Probing the ubiquinol-binding site of recombinant Sauromatum guttatum alternative oxidase expressed in E. coli membranes through site-directed mutagenesis.

PubMed

Young, Luke; May, Benjamin; Pendlebury-Watt, Alice; Shearman, Julia; Elliott, Catherine; Albury, Mary S; Shiba, Tomoo; Inaoka, Daniel Ken; Harada, Shigeharu; Kita, Kiyoshi; Moore, Anthony L

2014-07-01

In the present paper we have investigated the effect of mutagenesis of a number of highly conserved residues (R159, D163, L177 and L267) which we have recently shown to line the hydrophobic inhibitor/substrate cavity in the alternative oxidases (AOXs). Measurements of respiratory activity in rSgAOX expressed in Escherichia coli FN102 membranes indicate that all mutants result in a decrease in maximum activity of AOX and in some cases (D163 and L177) a decrease in the apparent Km (O2). Of particular importance was the finding that when the L177 and L267 residues, which appear to cause a bottleneck in the hydrophobic cavity, are mutated to alanine the sensitivity to AOX antagonists is reduced. When non-AOX anti-malarial inhibitors were also tested against these mutants widening the bottleneck through removal of isobutyl side chain allowed access of these bulkier inhibitors to the active-site and resulted in inhibition. Results are discussed in terms of how these mutations have altered the way in which the AOX's catalytic cycle is controlled and since maximum activity is decreased we predict that such mutations result in an increase in the steady state level of at least one O2-derived AOX intermediate. Such mutations should therefore prove to be useful in future stopped-flow and electron paramagnetic resonance experiments in attempts to understand the catalytic cycle of the alternative oxidase which may prove to be important in future rational drug design to treat diseases such as trypanosomiasis. Furthermore since single amino acid mutations in inhibitor/substrate pockets have been found to be the cause of multi-drug resistant strains of malaria, the decrease in sensitivity to main AOX antagonists observed in the L-mutants studied in this report suggests that an emergence of drug resistance to trypanosomiasis may also be possible. Therefore we suggest that the design of future AOX inhibitors should have structures that are less reliant on the orientation by the two

Phosphatidic acid as a second messenger in human polymorphonuclear leukocytes. Effects on activation of NADPH oxidase.

PubMed Central

Agwu, D E; McPhail, L C; Sozzani, S; Bass, D A; McCall, C E

1991-01-01

Receptor-mediated agonists, such as FMLP, induce an early, phospholipase D (PLD)-mediated accumulation of phosphatidic acid (PA) which may play a role in the activation of NADPH oxidase in human PMN. We have determined the effect of changes in PA production on O2 consumption in intact PMN and the level of NADPH oxidase activity measured in a cell-free assay. Pretreatment of cells with various concentrations of propranolol enhanced (less than or equal to 200 microM) or inhibited (greater than 300 microM) PLD-induced production of PA (mass and radiolabel) in a manner that correlated with enhancement or inhibition of O2 consumption in PMN stimulated with 1 microM FMLP in the absence of cytochalasin B. The concentration-dependent effects of propranolol on FMLP-induced NADPH oxidase activation was confirmed by direct assay of the enzyme in subcellular fractions. In PA extracted from cells pretreated with 200 microM propranolol before stimulation with 1 microM FMLP, phospholipase A1 (PLA1)-digestion for 90 min, followed by quantitation of residual PA, showed that a minimum of 44% of PA in control (undigested) sample was diacyl-PA; alkylacyl-PA remained undigested by PLA1. Propranolol was also observed to have a concentration-dependent enhancement of mass of 1,2-DG formed in PMN stimulated with FMLP. DG levels reached a maximum at 300 microM propranolol and remained unchanged up to 500 microM propranolol. However, in contrast to PA levels, the level of DG produced did not correlate with NADPH oxidase activation. Exogenously added didecanoyl-PA activated NADPH oxidase in a concentration-dependent manner (1-300 microM) in a reconstitution assay using membrane and cytosolic fractions from unstimulated PMN. In addition, PA synergized with SDS for oxidase activation. Taken together, these results indicate that PA plays a second messenger role in the activation of NADPH oxidase in human PMN and that regulation of phospholipase D is a key step in the activation pathway. Images

NADPH oxidase-mediated redox signaling promotes oxidative stress resistance and longevity through memo-1 in C. elegans

PubMed Central

Ewald, Collin Yvès; Hourihan, John M; Bland, Monet S; Obieglo, Carolin; Katic, Iskra; Moronetti Mazzeo, Lorenza E; Alcedo, Joy; Blackwell, T Keith; Hynes, Nancy E

2017-01-01

Transient increases in mitochondrially-derived reactive oxygen species (ROS) activate an adaptive stress response to promote longevity. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases produce ROS locally in response to various stimuli, and thereby regulate many cellular processes, but their role in aging remains unexplored. Here, we identified the C. elegans orthologue of mammalian mediator of ErbB2-driven cell motility, MEMO-1, as a protein that inhibits BLI-3/NADPH oxidase. MEMO-1 is complexed with RHO-1/RhoA/GTPase and loss of memo-1 results in an enhanced interaction of RHO-1 with BLI-3/NADPH oxidase, thereby stimulating ROS production that signal via p38 MAP kinase to the transcription factor SKN-1/NRF1,2,3 to promote stress resistance and longevity. Either loss of memo-1 or increasing BLI-3/NADPH oxidase activity by overexpression is sufficient to increase lifespan. Together, these findings demonstrate that NADPH oxidase-induced redox signaling initiates a transcriptional response that protects the cell and organism, and can promote both stress resistance and longevity. DOI: http://dx.doi.org/10.7554/eLife.19493.001 PMID:28085666

SIRPα controls the activity of the phagocyte NADPH oxidase by restricting the expression of gp91(phox).

PubMed

van Beek, Ellen M; Zarate, Julian Alvarez; van Bruggen, Robin; Schornagel, Karin; Tool, Anton T J; Matozaki, Takashi; Kraal, Georg; Roos, Dirk; van den Berg, Timo K

2012-10-25

The phagocyte NADPH oxidase mediates oxidative microbial killing in granulocytes and macrophages. However, because the reactive oxygen species produced by the NADPH oxidase can also be toxic to the host, it is essential to control its activity. Little is known about the endogenous mechanism(s) that limits NADPH oxidase activity. Here, we demonstrate that the myeloid-inhibitory receptor SIRPα acts as a negative regulator of the phagocyte NADPH oxidase. Phagocytes isolated from SIRPα mutant mice were shown to have an enhanced respiratory burst. Furthermore, overexpression of SIRPα in human myeloid cells prevented respiratory burst activation. The inhibitory effect required interactions between SIRPα and its natural ligand, CD47, as well as signaling through the SIRPα cytoplasmic immunoreceptor tyrosine-based inhibitory motifs. Suppression of the respiratory burst by SIRPα was caused by a selective repression of gp91(phox) expression, the catalytic component of the phagocyte NADPH oxidase complex. Thus, SIRPα can limit gp91(phox) expression during myeloid development, thereby controlling the magnitude of the respiratory burst in phagocytes. Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.

Flow-injection amperometric determination of glucose using a biosensor based on immobilization of glucose oxidase onto Au seeds decorated on core Fe₃O₄ nanoparticles.

PubMed

Samphao, Anchalee; Butmee, Preeyanut; Jitcharoen, Juthamas; Švorc, Ľubomír; Raber, Georg; Kalcher, Kurt

2015-09-01

An amperometric biosensor based on chemisorption of glucose oxidase (GOx) on Au seeds decorated on magnetic core Fe3O4 nanoparticles (Fe3O4@Au) and their immobilization on screen-printed carbon electrode bulk-modified with manganese oxide (SPCE{MnO2}) was designed for the determination of glucose. The Fe3O4@Au/GOx modified SPCE{MnO2} was used in a flow-injection analysis (FIA) arrangement. The experimental conditions were investigated in amperometric mode with the following optimized parameters: flow rate 1.7 mL min(-1), applied potential +0.38 V, phosphate buffer solution (PBS; 0.1 mol L(-1), pH 7.0) as carrier and 3.89 unit mm(-2) enzyme glucose oxidase loading on the active surface of the SPCE. The designed biosensor in FIA arrangement yielded a linear dynamic range for glucose from 0.2 to 9.0 mmol L(-1) with a sensitivity of 2.52 µA mM(-1) cm(-2), a detection limit of 0.1 mmol L(-1) and a quantification limit of 0.3 mmol L(-1). Moreover, a good repeatability of 2.8% (number of measurements n=10) and a sufficient reproducibility of 4.0% (number of sensors n=3) were achieved. It was found that the studied system Fe3O4@Au facilitated not only a simpler enzyme immobilization but also provided wider linear range. The practical application of the proposed biosensor for FIA quantification of glucose was tested in glucose sirup samples, honeys and energy drinks with the results in good accordance with those obtained by an optical glucose meter and with the contents declared by the producers. Copyright © 2015. Published by Elsevier B.V.

Rationale and design of a multicenter randomized study for evaluating vascular function under uric acid control using the xanthine oxidase inhibitor, febuxostat: the PRIZE study.

PubMed

Oyama, Jun-Ichi; Tanaka, Atsushi; Sato, Yasunori; Tomiyama, Hirofumi; Sata, Masataka; Ishizu, Tomoko; Taguchi, Isao; Kuroyanagi, Takanori; Teragawa, Hiroki; Ishizaka, Nobukazu; Kanzaki, Yumiko; Ohishi, Mitsuru; Eguchi, Kazuo; Higashi, Yukihito; Yamada, Hirotsugu; Maemura, Koji; Ako, Junya; Bando, Yasuko K; Ueda, Shinichiro; Inoue, Teruo; Murohara, Toyoaki; Node, Koichi

2016-06-18

Xanthine oxidase inhibitors are anti-hyperuricemic drugs that decrease serum uric acid levels by inhibiting its synthesis. Xanthine oxidase is also recognized as a pivotal enzyme in the production of oxidative stress. Excess oxidative stress induces endothelial dysfunction and inflammatory reactions in vascular systems, leading to atherosclerosis. Many experimental studies have suggested that xanthine oxidase inhibitors have anti-atherosclerotic effects by decreasing in vitro and in vivo oxidative stress. However, there is only limited evidence on the clinical implications of xanthine oxidase inhibitors on atherosclerotic cardiovascular disease in patients with hyperuricemia. We designed the PRIZE study to evaluate the effects of febuxostat on a surrogate marker of cardiovascular disease risk, ultrasonography-based intima-media thickness of the carotid artery in patients with hyperuricemia. The study is a multicenter, prospective, randomized, open-label and blinded-endpoint evaluation (PROBE) design. A total of 500 patients with asymptomatic hyperuricemia (uric acid >7.0 mg/dL) and carotid intima-media thickness ≥1.1 mm will be randomized centrally to receive either febuxostat (10-60 mg/day) or non-pharmacological treatment. Randomization is carried out using the dynamic allocation method stratified according to age (<65, ≥65 year), gender, presence or absence of diabetes mellitus, serum uric acid (<8.0, ≥8.0 mg/dL), and carotid intima-media thickness (<1.3, ≥1.3 mm). In addition to administering the study drug, we will also direct lifestyle modification in all participants, including advice on control of body weight, sleep, exercise and healthy diet. Carotid intima-media thickness will be evaluated using ultrasonography performed by skilled technicians at a central laboratory. Follow-up will be continued for 24 months. The primary endpoint is percentage change in mean intima-media thickness of the common carotid artery 24 months after baseline, measured by

Cell-free NADPH oxidase activation assays: "in vitro veritas".

PubMed

Pick, Edgar

2014-01-01

The superoxide (O2 (∙-))-generating NADPH oxidase complex of phagocytes comprises a membrane-imbedded heterodimeric flavocytochrome, known as cytochrome b 558 (consisting of Nox2 and p22 (phox) ) and four cytosolic regulatory proteins, p47 (phox) , p67 (phox) , p40 (phox) , and the small GTPase Rac. Under physiological conditions, in the resting phagocyte, O2 (∙-) generation is initiated by engagement of membrane receptors by a variety of stimuli, followed by specific signal transduction sequences leading to the translocation of the cytosolic components to the membrane and their association with the cytochrome. A consequent conformational change in Nox2 initiates the electron "flow" along a redox gradient, from NADPH to oxygen, leading to the one-electron reduction of molecular oxygen to O2 (∙-). Methodological difficulties in the dissection of this complex mechanism led to the design "cell-free" systems (also known as "broken cells" or in vitro systems). In these, membrane receptor stimulation and all or part of the signal transduction sequence are missing, the accent being placed on the actual process of "NADPH oxidase assembly," thus on the formation of the complex between cytochrome b 558 and the cytosolic components and the resulting O2 (∙-) generation. Cell-free assays consist of a mixture of the individual components of the NADPH oxidase complex, derived from resting phagocytes or in the form of purified recombinant proteins, exposed in vitro to an activating agent (distinct from and unrelated to whole cell stimulants), in the presence of NADPH and oxygen. Activation is commonly quantified by measuring the primary product of the reaction, O2 (∙-), trapped immediately after its generation by an appropriate acceptor in a kinetic assay, permitting the calculation of the linear rate of O2 (∙-) production, but numerous variations exist, based on the assessment of reaction products or the consumption of substrates. Cell-free assays played a paramount

Coordination chemistry controls the thiol oxidase activity of the B12-trafficking protein CblC

PubMed Central

Li, Zhu; Shanmuganathan, Aranganathan; Ruetz, Markus; Yamada, Kazuhiro; Lesniak, Nicholas A.; Kräutler, Bernhard; Brunold, Thomas C.; Koutmos, Markos; Banerjee, Ruma

2017-01-01

The cobalamin or B12 cofactor supports sulfur and one-carbon metabolism and the catabolism of odd-chain fatty acids, branched-chain amino acids, and cholesterol. CblC is a B12-processing enzyme involved in an early cytoplasmic step in the cofactor-trafficking pathway. It catalyzes the glutathione (GSH)-dependent dealkylation of alkylcobalamins and the reductive decyanation of cyanocobalamin. CblC from Caenorhabditis elegans (ceCblC) also exhibits a robust thiol oxidase activity, converting reduced GSH to oxidized GSSG with concomitant scrubbing of ambient dissolved O2. The mechanism of thiol oxidation catalyzed by ceCblC is not known. In this study, we demonstrate that novel coordination chemistry accessible to ceCblC-bound cobalamin supports its thiol oxidase activity via a glutathionyl-cobalamin intermediate. Deglutathionylation of glutathionyl-cobalamin by a second molecule of GSH yields GSSG. The crystal structure of ceCblC provides insights into how architectural differences at the α- and β-faces of cobalamin promote the thiol oxidase activity of ceCblC but mute it in wild-type human CblC. The R161G and R161Q mutations in human CblC unmask its latent thiol oxidase activity and are correlated with increased cellular oxidative stress disease. In summary, we have uncovered key architectural features in the cobalamin-binding pocket that support unusual cob(II)alamin coordination chemistry and enable the thiol oxidase activity of ceCblC. PMID:28442570

Single mutations that redirect internal proton transfer in the ba3 oxidase from Thermus thermophilus

PubMed Central

Smirnova, Irina; Chang, Hsin-Yang; von Ballmoos, Christoph; Ädelroth, Pia; Gennis, Robert B.; Brzezinski, Peter

2014-01-01

The ba3-type cytochrome c oxidase from Thermus thermophilus is a membrane-bound proton pump. Results from earlier studies have shown that with the aa3-type oxidases proton uptake to the catalytic site and “pump site” occur simultaneously. However, with the ba3 oxidase the pump site is loaded before proton transfer to the catalytic site because the proton transfer to the latter is slower than with the aa3 oxidases. In addition, the timing of formation and decay of catalytic intermediates is different in the two types of oxidases. In the present study, we have investigated two mutant ba3 CytcOs in which residues of the proton pathway leading to the catalytic site as well as the pump site were exchanged, Thr312Val and Tyr244Phe. Even though the ba3 CytcO uses only a single proton pathway for transfer of the substrate and “pumped” protons, the amino-acid residue substitutions had distinctly different effects on the kinetics of proton transfer to the catalytic site and the pump site, respectively. The results indicate that the rates of these reactions can be modified independently by replacement of single residues within the proton pathway. Furthermore, the data suggest that the Thr312Val and Tyr244Phe mutations interfere with a structural rearrangement in the proton pathway that is rate limiting for proton transfer to the catalytic site. PMID:24004023

Switching an O2 sensitive glucose oxidase bioelectrode into an almost insensitive one by cofactor redesign.

PubMed

Tremey, Emilie; Suraniti, Emmanuel; Courjean, Olivier; Gounel, Sébastien; Stines-Chaumeil, Claire; Louerat, Frédéric; Mano, Nicolas

2014-06-04

In the 5-8 mM glucose concentration range, of particular interest for diabetes management, glucose oxidase bioelectrodes are O2 dependent, which decrease their efficiencies. By replacing the natural cofactor of glucose oxidase, we succeeded in turning an O2 sensitive bioelectrode into an almost insensitive one.

Use of the Yeast Pichia pastoris as an Expression Host for Secretion of Enterocin L50, a Leaderless Two-Peptide (L50A and L50B) Bacteriocin from Enterococcus faecium L50▿

PubMed Central

Basanta, Antonio; Gómez-Sala, Beatriz; Sánchez, Jorge; Diep, Dzung B.; Herranz, Carmen; Hernández, Pablo E.; Cintas, Luis M.

2010-01-01

In this work, we report the expression and secretion of the leaderless two-peptide (EntL50A and EntL50B) bacteriocin enterocin L50 from Enterococcus faecium L50 by the methylotrophic yeast Pichia pastoris X-33. The bacteriocin structural genes entL50A and entL50B were fused to the Saccharomyces cerevisiae gene region encoding the mating pheromone α-factor 1 secretion signal (MFα1s) and cloned, separately and together (entL50AB), into the P. pastoris expression and secretion vector pPICZαA, which contains the methanol-inducible alcohol oxidase promoter (PAOX1) to express the fusion genes. After transfer into the yeast, the recombinant plasmids were integrated into the genome, resulting in three bacteriocinogenic yeast strains able to produce and secrete the individual bacteriocin peptides EntL50A and EntL50B separately and together. The secretion was efficiently directed by MFα1s through the Sec system, and the precursor peptides were found to be correctly processed to form mature and active bacteriocin peptides. The present work describes for the first time the heterologous expression and secretion of a two-peptide non-pediocin-like bacteriocin by a yeast. PMID:20348300

Involvement of alternative oxidase in the regulation of sensitivity of Sclerotinia sclerotiorum to the fungicides azoxystrobin and procymidone.

PubMed

Xu, Ting; Wang, Ya-Ting; Liang, Wu-Sheng; Yao, Fei; Li, Yong-Hong; Li, Dian-Rong; Wang, Hao; Wang, Zheng-Yi

2013-06-01

Sclerotinia sclerotiorum is a filamentous fungal pathogen that can infect many economically important crops and vegetables. Alternative oxidase is the terminal oxidase of the alternative respiratory pathway in fungal mitochondria. The function of alternative oxidase was investigated in the regulation of sensitivity of S. sclerotiorum to two commercial fungicides, azoxystrobin and procymidone which have different fungitoxic mechanisms. Two isolates of S. sclerotiorum were sensitive to both fungicides. Application of salicylhydroxamic acid, a specific inhibitor of alternative oxidase, significantly increased the values of effective concentration causing 50% mycelial growth inhibition (EC50) of azoxystrobin to both S. sclerotiorum isolates, whereas notably decreased the EC50 values of procymidone. In mycelial respiration assay azoxystrobin displayed immediate inhibitory effect on cytochrome pathway capacity, but had no immediate effect on alternative pathway capacity. In contrast, procymidone showed no immediate impact on capacities of both cytochrome and alternative pathways in the mycelia. However, alternative oxidase encoding gene (aox) transcript and protein levels, alternative respiration pathway capacity of the mycelia were obviously increased by pre-treatment for 24 h with both azoxystrobin and procymidone. These results indicate that alternative oxidase was involved in the regulation of sensitivity of S. sclerotiorum to the fungicides azoxystrobin and procymidone, and that both fungicides could affect aox gene expression and the alternative respiration pathway capacity development in mycelia of this fungal pathogen.

Regulation of the NADPH Oxidase RBOHD During Plant Immunity.

PubMed

Kadota, Yasuhiro; Shirasu, Ken; Zipfel, Cyril

2015-08-01

Pathogen recognition induces the production of reactive oxygen species (ROS) by NADPH oxidases in both plants and animals. ROS have direct antimicrobial properties, but also serve as signaling molecules to activate further immune outputs. However, ROS production has to be tightly controlled to avoid detrimental effects on host cells, but yet must be produced in the right amount, at the right place and at the right time upon pathogen perception. Plant NADPH oxidases belong to the respiratory burst oxidase homolog (RBOH) family, which contains 10 members in the model plant Arabidopsis thaliana. The perception of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) leads to a rapid, specific and strong production of ROS, which is dependent on RBOHD. RBOHD is mainly controlled by Ca(2+) via direct binding to EF-hand motifs and phosphorylation by Ca(2+)-dependent protein kinases. Recent studies have, however, revealed a critical role for a Ca(2+)-independent regulation of RBOHD. The plasma membrane-associated cytoplasmic kinase BIK1 (BOTRYTIS-INDUCED KINASE1), which is a direct substrate of the PRR complex, directly interacts with and phosphorylates RBOHD upon PAMP perception. Impairment of these phosphorylation events completely abolishes the function of RBOHD in immunity. These results suggest that RBOHD activity is tightly controlled by multilayered regulations. In this review, we summarize recent advances in our understanding of the regulatory mechanisms controlling RBOHD activation. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Ciproxifan, a histamine H3 receptor antagonist, reversibly inhibits monoamine oxidase A and B

PubMed Central

Hagenow, S.; Stasiak, A.; Ramsay, R. R.; Stark, H.

2017-01-01

Ciproxifan is a well-investigated histamine H3 receptor (H3R) inverse agonist/antagonist, showing an exclusively high species-specific affinity at rodent compared to human H3R. It is well studied as reference compound for H3R in rodent models for neurological diseases connected with neurotransmitter dysregulation, e.g. attention deficit hyperactivity disorder or Alzheimer’s disease. In a screening for potential monoamine oxidase A and B inhibition ciproxifan showed efficacy on both enzyme isoforms. Further characterization of ciproxifan revealed IC50 values in a micromolar concentration range for human and rat monoamine oxidases with slight preference for monoamine oxidase B in both species. The inhibition by ciproxifan was reversible for both human isoforms. Regarding inhibitory potency of ciproxifan on rat brain MAO, these findings should be considered, when using high doses in rat models for neurological diseases. As the H3R and monoamine oxidases are all capable of affecting neurotransmitter modulation in brain, we consider dual targeting ligands as interesting approach for treatment of neurological disorders. Since ciproxifan shows only moderate activity at human targets, further investigations in animals are not of primary interest. On the other hand, it may serve as starting point for the development of dual targeting ligands. PMID:28084411

In vivo oxalate degradation by liposome encapsulated oxalate oxidase in rat model of hyperoxaluria

PubMed Central

Dahiya, Tulika; Pundir, C.S.

2013-01-01

Background & objectives: High level of urinary oxalate substantially increases the risk of hyperoxaluria, a significant risk factor for urolithiasis. The primary goal of this study was to reduce urinary oxalate excretion employing liposome encapsulated oxalate oxidase in animal model. Methods: A membrane bound oxalate oxidase was purified from Bougainvillea leaves. The enzyme in its native form was less effective at the physiological pH of the recipient animal. To increase its functional viability, the enzyme was immobilized on to ethylene maleic anhydride (EMA). Rats were injected with liposome encapsulated EMA- oxalate oxidase and the effect was observed on degradation of oxalic acid. Results: The enzyme was purified to apparent homogeneity with 60-fold purification and 31 per cent yield. The optimum pH of EMA-derivative enzyme was 6.0 and it showed 70 per cent of its optimal activity at pH 7.0. The EMA-bound enzyme encapsulated into liposome showed greater oxalate degradation in 15 per cent casein vitamin B6 deficient fed rats as compared with 30 per cent casein vitamin B6 deficient fed rats and control rats. Interpretation & conclusions: EMA-oxalate oxidase encapsulated liposome caused oxalate degradation in experimental hyperoxaluria indicating that the enzyme could be used as a therapeutic agent in hyperoxaluria leading to urinary stones. PMID:23481063

Immobilization techniques to avoid enzyme loss from oxidase-based biosensors: a one-year study.

PubMed

House, Jody L; Anderson, Ellen M; Ward, W Kenneth

2007-01-01

Continuous amperometric sensors that measure glucose or lactate require a stable sensitivity, and glutaraldehyde crosslinking has been used widely to avoid enzyme loss. Nonetheless, little data is published on the effectiveness of enzyme immobilization with glutaraldehyde. A combination of electrochemical testing and spectrophotometric assays was used to study the relationship between enzyme shedding and the fabrication procedure. In addition, we studied the relationship between the glutaraldehyde concentration and sensor performance over a period of one year. The enzyme immobilization process by glutaraldehyde crosslinking to glucose oxidase appears to require at least 24-hours at room temperature to reach completion. In addition, excess free glucose oxidase can be removed by soaking sensors in purified water for 20 minutes. Even with the addition of these steps, however, it appears that there is some free glucose oxidase entrapped within the enzyme layer which contributes to a decline in sensitivity over time. Although it reduces the ultimate sensitivity (probably via a change in the enzyme's natural conformation), glutaraldehyde concentration in the enzyme layer can be increased in order to minimize this instability. After exposure of oxidase enzymes to glutaraldehyde, effective crosslinking requires a rinse step and a 24-hour incubation step. In order to minimize the loss of sensor sensitivity over time, the glutaraldehyde concentration can be increased.

The dual actions of morin (3,5,7,2',4'-pentahydroxyflavone) as a hypouricemic agent: uricosuric effect and xanthine oxidase inhibitory activity.

PubMed

Yu, Zhifeng; Fong, Wing Ping; Cheng, Christopher H K

2006-01-01

Hyperuricemia is associated with a number of pathological conditions such as gout. Lowering of elevated uric acid level in the blood could be achieved by xanthine oxidase inhibitors and inhibitors of renal urate reabsorption. Some natural compounds isolated from herbs used in traditional Chinese medicine have been previously demonstrated to possess xanthine oxidase inhibitory activities. In the present investigation, morin (3,5,7,2',4'-pentahydroxyflavone), which occurs in the twigs of Morus alba L. documented in traditional Chinese medicinal literature to treat conditions akin to gout, was demonstrated to exert potent inhibitory action on urate uptake in rat renal brush-border membrane vesicles, indicating that this compound acts on the kidney to inhibit urate reabsorption. Lineweaver-Burk transformation of the inhibition kinetics data demonstrated that the inhibition of urate uptake was of a competitive type, with a K(i) value of 17.4 microM. In addition, morin was also demonstrated to be an inhibitor of xanthine oxidase. Lineweaver-Burk analysis of the enzyme kinetics indicated that the mode of inhibition was of a mixed type, with K(i) and K(ies) values being 7.9 and 35.1 microM, respectively. Using an oxonate-induced hyperuricemic rat model, morin was indeed shown to exhibit an in vivo uricosuric action, which could explain, in part at least, the observed hypouricemic effect of morin in these rats. The potential application of this compound in the treatment of conditions associated with hyperuricemia was discussed.

A New Transgenic Mouse Model for Studying the Neurotoxicity of Spermine Oxidase Dosage in the Response to Excitotoxic Injury

PubMed Central

Cervelli, Manuela; Bellavia, Gabriella; D'Amelio, Marcello; Cavallucci, Virve; Moreno, Sandra; Berger, Joachim; Nardacci, Roberta; Marcoli, Manuela; Maura, Guido; Piacentini, Mauro; Amendola, Roberto; Cecconi, Francesco; Mariottini, Paolo

2013-01-01

Spermine oxidase is a FAD-containing enzyme involved in polyamines catabolism, selectively oxidizing spermine to produce H2O2, spermidine, and 3-aminopropanal. Spermine oxidase is highly expressed in the mouse brain and plays a key role in regulating the levels of spermine, which is involved in protein synthesis, cell division and cell growth. Spermine is normally released by neurons at synaptic sites where it exerts a neuromodulatory function, by specifically interacting with different types of ion channels, and with ionotropic glutamate receptors. In order to get an insight into the neurobiological roles of spermine oxidase and spermine, we have deregulated spermine oxidase gene expression producing and characterizing the transgenic mouse model JoSMOrec, conditionally overexpressing the enzyme in the neocortex. We have investigated the effects of spermine oxidase overexpression in the mouse neocortex by transcript accumulation, immunohistochemical analysis, enzymatic assays and polyamine content in young and aged animals. Transgenic JoSMOrec mice showed in the neocortex a higher H2O2 production in respect to Wild-Type controls, indicating an increase of oxidative stress due to SMO overexpression. Moreover, the response of transgenic mice to excitotoxic brain injury, induced by kainic acid injection, was evaluated by analysing the behavioural phenotype, the immunodistribution of neural cell populations, and the ultrastructural features of neocortical neurons. Spermine oxidase overexpression and the consequently altered polyamine levels in the neocortex affects the cytoarchitecture in the adult and aging brain, as well as after neurotoxic insult. It resulted that the transgenic JoSMOrec mouse line is more sensitive to KA than Wild-Type mice, indicating an important role of spermine oxidase during excitotoxicity. These results provide novel evidences of the complex and critical functions carried out by spermine oxidase and spermine in the mammalian brain. PMID

The proteolytic processing site of the precursor of lysyl oxidase.

PubMed Central

Cronshaw, A D; Fothergill-Gilmore, L A; Hulmes, D J

1995-01-01

The precise cleavage site of the N-terminal propeptide region of the precursor of lysyl oxidase has not yet been established, due to N-terminal blocking of the mature protein. Using a combination of peptide fragmentation, amino acid sequencing, time-of-flight m.s. and partial chemical unblocking procedures, it is shown that the mature form of lysyl oxidase begins at residue Asp-169 of the precursor protein (numbered according to the human sequence). The cleavage site is 28 residues to the C-terminal side of the site previously suggested on the basis of apparant molecular mass by SDS/PAGE, with the consequence that the two putative, N-linked glycosylation sites and the position of the Arg/Gln sequence polymorphism are now all in the precursor region. PMID:7864821

Acupuncture elicits neuroprotective effect by inhibiting NAPDH oxidase-mediated reactive oxygen species production in cerebral ischaemia.

PubMed

Shi, Guang-Xia; Wang, Xue-Rui; Yan, Chao-Qun; He, Tian; Yang, Jing-Wen; Zeng, Xiang-Hong; Xu, Qian; Zhu, Wen; Du, Si-Qi; Liu, Cun-Zhi

2015-12-10

In the current study, we aimed to investigate whether NADPH oxidase, a major ROS-producing enzyme, was involved in the antioxidant effect of acupuncture on cognitive impairment after cerebral ischaemia. The cognitive function, infract size, neuron cell loss, level of superoxide anion and expression of NADPH oxidase subunit in hippocampus of two-vessel occlusion (2VO) rats were determined after 2-week acupuncture. Furthermore, the cognitive function and production of O2(-) were determined in the presence and absence of NADPH oxidase agonist (TBCA) and antagonist (Apocynin). The effect of acupuncture on cognitive function after cerebral ischaemia in gp91phox-KO mice was evaluated by Morris water maze. Acupuncture reduced infarct size, attenuated overproduction of O2(-), and reversed consequential cognitive impairment and neuron cell loss in 2VO rats. The elevations of gp91phox and p47phox after 2VO were significantly decreased after acupuncture treatment. However, no differences of gp91phox mRNA were found among any experimental groups. Furthermore, these beneficial effects were reversed by TBCA, whereas apocynin mimicked the effect of acupuncture by improving cognitive function and decreasing O2(-) generation. Acupuncture failed to improve the memory impairment in gp91phox KO mice. Full function of the NADPH oxidase enzyme plays an important role in neuroprotective effects against cognitive impairment via inhibition of NAPDH oxidase-mediated oxidative stress.

Screening, separation, and evaluation of xanthine oxidase inhibitors from Paeonia lactiflora using chromatography combined with a multi-mode microplate reader.

PubMed

Wang, Jing; Shi, Dongfang; Zheng, Meizhu; Ma, Bing; Cui, Jing; Liu, Chunming; Liu, Chengyu

2017-11-01

Natural products have become one of the most important resources for discovering novel xanthine oxidase inhibitors, which are commonly employed in the treatment of hyperuricemia and gout. However, to date, few reports exist regarding the use of monoterpene glycosides as xanthine oxidase inhibitors. Thus, we herein report the use of ultrafiltration coupled with liquid chromatography in the screening of monoterpene glycoside xanthine oxidase inhibitors from the extract of Paeonia lactiflora (P. lactiflora), and both high-performance counter-current chromatography and medium-pressure liquid chromatography were employed to separate the main constituents. Furthermore, the xanthine oxidase inhibitory activities and the mechanisms of inhibition of the isolated compounds were evaluated using a multi-mode microplate reader by Molecular Devices. As a result, three monoterpene glycosides were separated by combined high-performance counter-current chromatography and medium-pressure liquid chromatography in purities of 90.4, 98.0, and 86.3%, as determined by liquid chromatography. These three compounds were identified as albiflorin, paeoniflorin, and 1-O-β-ᴅ-glucopyranosyl-8-O-benzoylpaeonisuffrone by electrospray ionization tandem mass spectrometry, and albiflorin and paeoniflorin were screened as potential xanthine oxidase inhibitors by ultrafiltration with liquid chromatography. The evaluation results of xanthine oxidase inhibitory activity corresponded with the screening results, as only albiflorin and paeoniflorin exhibited xanthine oxidase inhibitory activity. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Expression analysis of polyphenol oxidase isozymes by active staining method and tissue browning of head lettuce (Lactuca sativa L.).

PubMed

Noda, Takahiro; Iimure, Kazuhiko; Okamoto, Shunsuke; Saito, Akira

2017-08-01

Browning of plant tissue is generally considered attributable to enzymatic oxidation by polyphenol oxidase (PPO). Electrophoresis followed by activity staining has been used as an effective procedure to visually detect and isolate isozymes; however, it has not been applied for examination of various PPO isozymes in lettuce. Our study demonstrated that different lettuce PPO isozymes could be detected at different pH in active staining, and multiple isozymes were detected only under alkaline conditions. As a result, we concluded that activity staining with approximately pH 8 enabled to detect various PPO isozymes in lettuce. By expression analysis of the PPO isozymes after wounding, PPO isozymes that correlated with time-course of tissue browning were detected. The wound-induced PPO may play a key role in enzymatic browning.

Comparison of brain mitochondrial cytochrome c oxidase activity with cyanide LD(50) yields insight into the efficacy of prophylactics.

PubMed

Marziaz, Mandy L; Frazier, Kathryn; Guidry, Paul B; Ruiz, Robyn A; Petrikovics, Ilona; Haines, Donovan C

2013-01-01

Cyanide inhibits cytochrome c oxidase, the terminal oxidase of the mitochondrial respiratory pathway, therefore inhibiting the cell oxygen utilization and resulting in the condition of histotoxic anoxia. The enzyme rhodanese detoxifies cyanide by utilizing sulfur donors to convert cyanide to thiocyanate, and new and improved sulfur donors are actively sought as researchers seek to improve cyanide prophylactics. We have determined brain cytochrome c oxidase activity as a marker for cyanide exposure for mice pre-treated with various cyanide poisoning prophylactics, including sulfur donors thiosulfate (TS) and thiotaurine (TT3). Brain mitochondria were isolated by differential centrifugation, the outer mitochondrial membrane was disrupted by a maltoside detergent, and the decrease in absorbance at 550 nm as horse heart ferrocytochrome c (generated by the dithiothreitol reduction of ferricytochrome c) was oxidized was monitored. Overall, the TS control prophylactic treatment provided significant protection of the cytochrome c oxidase activity. The TT3-treated mice showed reduced cytochrome c oxidase activity even in the absence of cyanide. In both treatment series, addition of exogenous Rh did not significantly enhance the prevention of cytochrome c oxidase inhibition, but the addition of sodium nitrite did. These findings can lead to a better understanding of the protection mechanism by various cyanide antidotal systems. Copyright © 2011 John Wiley & Sons, Ltd.

NADH Oxidase of Streptococcus thermophilus 1131 is Required for the Effective Yogurt Fermentation with Lactobacillus delbrueckii subsp. bulgaricus 2038.

PubMed

Sasaki, Yasuko; Horiuchi, Hiroshi; Kawashima, Hiroko; Mukai, Takao; Yamamoto, Yuji

2014-01-01

We previously reported that dissolved oxygen (DO) suppresses yogurt fermentation with an industrial starter culture composed of Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) 2038 and Streptococcus thermophilus 1131, and also found that reducing the DO in the medium prior to fermentation (deoxygenated fermentation) shortens the fermentation time. In this study, we found that deoxygenated fermentation primarily increased the cell number of S. thermophilus 1131 rather than that of L. bulgaricus 2038, resulting in earlier l-lactate and formate accumulation. Measurement of the DO concentration and hydrogen peroxide generation in the milk medium suggested that DO is mainly removed by S. thermophilus 1131. The results using an H2O-forming NADH oxidase (Nox)-defective mutant of S. thermophilus 1131 revealed that Nox is the major oxygen-consuming enzyme of the bacterium. Yogurt fermentation with the S. thermophilus Δnox mutant and L. bulgaricus 2038 was significantly slower than with S. thermophilus 1131 and L. bulgaricus 2038, and the DO concentrations of the mixed culture did not decrease to less than 2 mg/kg within 3 hr. These observations suggest that Nox of S. thermophilus 1131 contributes greatly to yogurt fermentation, presumably by removing the DO in milk.

Human protoporphyrinogen oxidase: expression, purification, and characterization of the cloned enzyme.

PubMed Central

Dailey, T. A.; Dailey, H. A.

1996-01-01

Protoporphyrinogen oxidase (E.C.1.3.3.4) catalyzes the oxygen-dependent oxidation of protoporphyrinogen IX to protoporphyrin IX. The enzyme from human placenta has been cloned, sequenced, expressed in Escherichia coli, purified to homogeneity, and characterized. Northern blot analysis of eight different human tissues show evidence for only a single transcript in all tissue types and the size of this transcript is approximately 1.8 kb. The human cDNA has been inserted into an expression vector for E. coli and the protein produced at high levels in these cells. The protein is found in both membrane and cytoplasmic fractions. The enzyme was purified to homogeneity in the presence of detergents using a metal chelate affinity column. The purified protein is a homodimer composed of subunits of molecular weight of 51,000. The enzyme contains one noncovalently bound FAD per dimer, has a monomer extinction coefficient of 48,000 at 270 nm and contains no detectable redox active metals. The apparent K(m) and Kcat for protoporphyrinogen IX are 1.7 microM and 10.5 min-1, respectively. The enzyme does not use coproporphyrinogen III as a substrate and is inhibited by micromolar concentrations of the herbicide acifluorfen. Protein database searches reveal significant homology between protoporphyrinogen oxidase and monoamine oxidase. PMID:8771201

Spermine oxidase maintains basal skeletal muscle gene expression and fiber size and is strongly repressed by conditions that cause skeletal muscle atrophy

PubMed Central

Bongers, Kale S.; Fox, Daniel K.; Kunkel, Steven D.; Stebounova, Larissa V.; Murry, Daryl J.; Pufall, Miles A.; Ebert, Scott M.; Dyle, Michael C.; Bullard, Steven A.; Dierdorff, Jason M.

2014-01-01

Skeletal muscle atrophy is a common and debilitating condition that remains poorly understood at the molecular level. To better understand the mechanisms of muscle atrophy, we used mouse models to search for a skeletal muscle protein that helps to maintain muscle mass and is specifically lost during muscle atrophy. We discovered that diverse causes of muscle atrophy (limb immobilization, fasting, muscle denervation, and aging) strongly reduced expression of the enzyme spermine oxidase. Importantly, a reduction in spermine oxidase was sufficient to induce muscle fiber atrophy. Conversely, forced expression of spermine oxidase increased muscle fiber size in multiple models of muscle atrophy (immobilization, fasting, and denervation). Interestingly, the reduction of spermine oxidase during muscle atrophy was mediated by p21, a protein that is highly induced during muscle atrophy and actively promotes muscle atrophy. In addition, we found that spermine oxidase decreased skeletal muscle mRNAs that promote muscle atrophy (e.g., myogenin) and increased mRNAs that help to maintain muscle mass (e.g., mitofusin-2). Thus, in healthy skeletal muscle, a relatively low level of p21 permits expression of spermine oxidase, which helps to maintain basal muscle gene expression and fiber size; conversely, during conditions that cause muscle atrophy, p21 expression rises, leading to reduced spermine oxidase expression, disruption of basal muscle gene expression, and muscle fiber atrophy. Collectively, these results identify spermine oxidase as an important positive regulator of muscle gene expression and fiber size, and elucidate p21-mediated repression of spermine oxidase as a key step in the pathogenesis of skeletal muscle atrophy. PMID:25406264

Heterologous Production and Characterization of Two Glyoxal Oxidases from Pycnoporus cinnabarinus

PubMed Central

Daou, Marianne; Piumi, François; Cullen, Daniel; Record, Eric

2016-01-01

ABSTRACT The genome of the white rot fungus Pycnoporus cinnabarinus includes a large number of genes encoding enzymes implicated in lignin degradation. Among these, three genes are predicted to encode glyoxal oxidase, an enzyme previously isolated from Phanerochaete chrysosporium. The glyoxal oxidase of P. chrysosporium is physiologically coupled to lignin-oxidizing peroxidases via generation of extracellular H2O2 and utilizes an array of aldehydes and α-hydroxycarbonyls as the substrates. Two of the predicted glyoxal oxidases of P. cinnabarinus, GLOX1 (PciGLOX1) and GLOX2 (PciGLOX2), were heterologously produced in Aspergillus niger strain D15#26 (pyrG negative) and purified using immobilized metal ion affinity chromatography, yielding 59 and 5 mg of protein for PciGLOX1 and PciGLOX2, respectively. Both proteins were approximately 60 kDa in size and N-glycosylated. The optimum temperature for the activity of these enzymes was 50°C, and the optimum pH was 6. The enzymes retained most of their activity after incubation at 50°C for 4 h. The highest relative activity and the highest catalytic efficiency of both enzymes occurred with glyoxylic acid as the substrate. The two P. cinnabarinus enzymes generally exhibited similar substrate preferences, but PciGLOX2 showed a broader substrate specificity and was significantly more active on 3-phenylpropionaldehyde. IMPORTANCE This study addresses the poorly understood role of how fungal peroxidases obtain an in situ supply of hydrogen peroxide to enable them to oxidize a variety of organic and inorganic compounds. This cooperative activity is intrinsic in the living organism to control the amount of toxic H2O2 in its environment, thus providing a feed-on-demand scenario, and can be used biotechnologically to supply a cheap source of peroxide for the peroxidase reaction. The secretion of multiple glyoxal oxidases by filamentous fungi as part of a lignocellulolytic mechanism suggests a controlled system, especially as these

Characterisation of phenol oxidase and peroxidase from maize silk.

PubMed

Sukalović, V Hadzi-Tasković; Veljović-Jovanović, S; Maksimović, J Dragisić; Maksimović, V; Pajić, Z

2010-05-01

Silk of some maize genotypes contains a high level of phenolics that undergo enzymatic oxidation to form quinones, which condense among themselves or with proteins to form brown pigments. Two phenolic oxidizing enzymes, peroxidase (POD; EC 1.11.1.7) and polyphenol oxidase (PPO; EC 1.10.3.1), from maize (Zea mays L.) silk were characterised with respect to their preferred substrate, different isoforms and specific effectors. One browning silk sample with high, and two non-browning samples with low phenolic content were investigated. Although POD oxidizes a wide range of phenolic substrates in vitro, its activity rate was independent of silk phenolic content. PPO activity, detected with o-diphenolic substrates, was abundant only in browning silk, and low or absent in non-browning silk. Pollination increased POD but not PPO activity. Isoelectric-focusing (IEF) and specific staining for POD and PPO showed a high degree of polymorphism that varied with silk origin. The IEF pattern of POD revealed a number of anionic and several cationic isoenzymes, with the most pronounced having neutral pI 7 and a basic isoform with pI 10. Detected isoforms of PPO were anionic, except for one neutral form found only in browning silk, and occupied positions different from those of POD. Different inhibitory effects of NaN(3), EDTA, KCN, and L-cysteine, as well as different impacts of a variety of cations on the oxidation of chlorogenic acid, mediated by PPO or POD, were detected. The findings are discussed in terms of a possible roles of these enzymes in defence and pollination.

Reusable and Mediator-Free Cholesterol Biosensor Based on Cholesterol Oxidase Immobilized onto TGA-SAM Modified Smart Bio-Chips

PubMed Central

Rahman, Mohammed M.

2014-01-01

A reusable and mediator-free cholesterol biosensor based on cholesterol oxidase (ChOx) was fabricated based on self-assembled monolayer (SAM) of thioglycolic acid (TGA) (covalent enzyme immobilization by dropping method) using bio-chips. Cholesterol was detected with modified bio-chip (Gold/Thioglycolic-acid/Cholesterol-oxidase i.e., Au/TGA/ChOx) by reliable cyclic voltammetric (CV) technique at room conditions. The Au/TGA/ChOx modified bio-chip sensor demonstrates good linearity (1.0 nM to 1.0 mM; R = 0.9935), low-detection limit (∼0.42 nM, SNR∼3), and higher sensitivity (∼74.3 µAµM−1cm−2), lowest-small sample volume (50.0 μL), good stability, and reproducibility. To the best of our knowledge, this is the first statement with a very high sensitivity, low-detection limit, and low-sample volumes are required for cholesterol biosensor using Au/TGA/ChOx-chips assembly. The result of this facile approach was investigated for the biomedical applications for real samples at room conditions with significant assembly (Au/TGA/ChOx) towards the development of selected cholesterol biosensors, which can offer analytical access to a large group of enzymes for wide range of biomedical applications in health-care fields. PMID:24949733

Identification of a xanthine oxidase-inhibitory component from Sophora flavescens using NMR-based metabolomics.

PubMed

Suzuki, Ryuichiro; Hasuike, Yuka; Hirabayashi, Moeka; Fukuda, Tatsuo; Okada, Yoshihito; Shirataki, Yoshiaki

2013-10-01

We demonstrate that NMR-based metabolomics studies can be used to identify xanthine oxidase-inhibitory compounds in the diethyl ether soluble fraction prepared from a methanolic extract of Sophora flavescens. Loading plot analysis, accompanied by direct comparison of 1H NMR spectraexhibiting characteristic signals, identified compounds exhibiting inhibitory activity. NMR analysis indicated that these characteristic signals were attributed to flavanones such as sophoraflavanone G and kurarinone. Sophoraflavanone G showed inhibitory activity towards xanthine oxidase in an in vitro assay.

Molecular Interface of S100A8 with Cytochrome b558 and NADPH Oxidase Activation

PubMed Central

Berthier, Sylvie; Hograindleur, Marc-André; Paclet, Marie-Hélène; Polack, Benoît; Morel, Françoise

2012-01-01

S100A8 and S100A9 are two calcium binding Myeloid Related Proteins, and important mediators of inflammatory diseases. They were recently introduced as partners for phagocyte NADPH oxidase regulation. However, the precise mechanism of their interaction remains elusive. We had for aim (i) to evaluate the impact of S100 proteins on NADPH oxidase activity; (ii) to characterize molecular interaction of either S100A8, S100A9, or S100A8/S100A9 heterocomplex with cytochrome b 558; and (iii) to determine the S100A8 consensus site involved in cytochrome b 558/S100 interface. Recombinant full length or S100A9-A8 truncated chimera proteins and ExoS-S100 fusion proteins were expressed in E. coli and in P. aeruginosa respectively. Our results showed that S100A8 is the functional partner for NADPH oxidase activation contrary to S100A9, however, the loading with calcium and a combination with phosphorylated S100A9 are essential in vivo. Endogenous S100A9 and S100A8 colocalize in differentiated and PMA stimulated PLB985 cells, with Nox2/gp91phox and p22phox. Recombinant S100A8, loaded with calcium and fused with the first 129 or 54 N-terminal amino acid residues of the P. aeruginosa ExoS toxin, induced a similar oxidase activation in vitro, to the one observed with S100A8 in the presence of S100A9 in vivo. This suggests that S100A8 is the essential component of the S100A9/S100A8 heterocomplex for oxidase activation. In this context, recombinant full-length rS100A9-A8 and rS100A9-A8 truncated 90 chimera proteins as opposed to rS100A9-A8 truncated 86 and rS100A9-A8 truncated 57 chimeras, activate the NADPH oxidase function of purified cytochrome b 558 suggesting that the C-terminal region of S100A8 is directly involved in the molecular interface with the hemoprotein. The data point to four strategic 87HEES90 amino acid residues of the S100A8 C-terminal sequence that are involved directly in the molecular interaction with cytochrome b558 and then in the phagocyte NADPH oxidase

Molecular Characterization of the Oxalate Oxidase Involved in the Response of Barley to the Powdery Mildew Fungus1

PubMed Central

Zhou, Fasong; Zhang, Ziguo; Gregersen, Per L.; Mikkelsen, Jørn D.; de Neergaard, Eigil; Collinge, David B.; Thordal-Christensen, Hans

1998-01-01

Previously we reported that oxalate oxidase activity increases in extracts of barley (Hordeum vulgare) leaves in response to the powdery mildew fungus (Blumeria [syn. Erysiphe] graminis f.sp. hordei) and proposed this as a source of H2O2 during plant-pathogen interactions. In this paper we show that the N terminus of the major pathogen-response oxalate oxidase has a high degree of sequence identity to previously characterized germin-like oxalate oxidases. Two cDNAs were isolated, pHvOxOa, which represents this major enzyme, and pHvOxOb', representing a closely related enzyme. Our data suggest the presence of only two oxalate oxidase genes in the barley genome, i.e. a gene encoding HvOxOa, which possibly exists in several copies, and a single-copy gene encoding HvOxOb. The use of 3′ end gene-specific probes has allowed us to demonstrate that the HvOxOa transcript accumulates to 6 times the level of the HvOxOb transcript in response to the powdery mildew fungus. The transcripts were detected in both compatible and incompatible interactions with a similar accumulation pattern. The oxalate oxidase is found exclusively in the leaf mesophyll, where it is cell wall located. A model for a signal transduction pathway in which oxalate oxidase plays a central role is proposed for the regulation of the hypersensitive response. PMID:9576772

Characterization of the cydAB-Encoded Cytochrome bd Oxidase from Mycobacterium smegmatis

PubMed Central

Kana, Bavesh D.; Weinstein, Edward A.; Avarbock, David; Dawes, Stephanie S.; Rubin, Harvey; Mizrahi, Valerie

2001-01-01

The cydAB genes from Mycobacterium smegmatis have been cloned and characterized. The cydA and cydB genes encode the two subunits of a cytochrome bd oxidase belonging to the widely distributed family of quinol oxidases found in prokaryotes. The cydD and cydC genes located immediately downstream of cydB encode a putative ATP-binding cassette-type transporter. At room temperature, reduced minus oxidized difference spectra of membranes purified from wild-type M. smegmatis displayed spectral features that are characteristic of the γ-proteobacterial type cytochrome bd oxidase. Inactivation of cydA or cydB by insertion of a kanamycin resistance marker resulted in loss of d-heme absorbance at 631 nm. The d-heme could be restored by transformation of the M. smegmatis cyd mutants with a replicating plasmid carrying the highly homologous cydABDC gene cluster from Mycobacterium tuberculosis. Inactivation of cydA had no effect on the ability of M. smegmatis to exit from stationary phase at 37 or 42°C. The growth rate of the cydA mutant was tested under oxystatic conditions. Although no discernible growth defect was observed under moderately aerobic conditions (9.2 to 37.5 × 102 Pa of pO2 or 5 to 21% air saturation), the mutant displayed a significant growth disadvantage when cocultured with the wild type under extreme microaerophilia (0.8 to 1.7 × 102 Pa of pO2 or 0.5 to 1% air saturation). These observations were in accordance with the two- to threefold increase in cydAB gene expression observed upon reduction of the pO2 of the growth medium from 21 to 0.5% air saturation and with the concomitant increase in d-heme absorbance in spectra of membranes isolated from wild-type M. smegmatis cultured at 1% air saturation. Finally, the cydA mutant displayed a competitive growth disadvantage in the presence of the terminal oxidase inhibitor, cyanide, when cocultured with wild type at 21% air saturation in an oxystat. In conjunction with these findings, our results suggest that

A multicopper oxidase-related protein is essential for insect viability, longevity and ovary development.

PubMed

Peng, Zeyu; Green, Peter G; Arakane, Yasuyuki; Kanost, Michael R; Gorman, Maureen J

2014-01-01

Typical multicopper oxidases (MCOs) have ten conserved histidines and one conserved cysteine that coordinate four copper atoms. These copper ions are required for oxidase activity. During our studies of insect MCOs, we discovered a gene that we named multicopper oxidase-related protein (MCORP). MCORPs share sequence similarity with MCOs, but lack many of the copper-coordinating residues. We identified MCORP orthologs in many insect species, but not in other invertebrates or vertebrates. We predicted that MCORPs would lack oxidase activity due to the absence of copper-coordinating residues. To test this prediction, we purified recombinant Tribolium castaneum (red flour beetle) MCORP and analyzed its enzymatic activity using a variety of substrates. As expected, no oxidase activity was detected. To study MCORP function in vivo, we analyzed expression profiles of TcMCORP and Anopheles gambiae (African malaria mosquito) MCORP, and assessed RNAi-mediated knockdown phenotypes. We found that both MCORPs are constitutively expressed at a low level in all of the tissues we analyzed. Injection of TcMCORP dsRNA into larvae resulted in 100% mortality prior to adult eclosion, with death occurring mainly during the pharate pupal stage or late pharate adult stage. Injection of TcMCORP dsRNA into pharate pupae resulted in the death of approximately 20% of the treated insects during the pupal to adult transition and a greatly shortened life span for the remaining insects. In addition, knockdown of TcMCORP in females prevented oocyte maturation and, thus, greatly decreased the number of eggs laid. These results indicate that TcMCORP is an essential gene and that its function is required for reproduction. An understanding of the role MCORP plays in insect physiology may help to develop new strategies for controlling insect pests.

Inhibition of monoamine oxidase B (MAO-B) by Chinese herbal medicines.

PubMed

Lin, R D; Hou, W C; Yen, K Y; Lee, M H

2003-11-01

Monoamine oxidase (MAO) catalyzes the oxidative deamination of biogenic amines accompaned by the release of H2O2. Two subtypes, MAO-A and MAO-B, exist on the basis of their specificities to substrates and inhibitors. The regulation of MAO-B activity is important in the treatment of neurodegenerative diseases. Twenty-seven species of plants used in traditional Chinese medicines, selected from an enthnobotanical survey, were used in an investigation of their inhibitory effect on MAO-B in rat brain homogenates. The 50% aqueous methanol extracts of four active extracts, Arisaema amurense, Lilium brownii var. colchesteri, Lycium chinense, and Uncaria rhynchophylla, exhibited the best activity and selectivity towards MAO-B with IC50 values of 0.44, 0.29, 0.40, and 0.03 mg/ml, respectively. A kinetic study of MAO-B inhibition by the four extracts using the Lineweaver-Burk plot for each active extract revealed the IC50 concentrations, and results show that: Ki = 0.59 mg/ml for A. amurense for the mixed-type mode, Ki = 0.58 mg/ml for L. brownii var. colchesteri for the mixed-type mode, Ki = 5.01 mg/ml for L. chinense for the uncompetitive mode, and Ki = 0.02 mg/ml for U. rhynchophylla for the uncompetitive mode. These may therefore be candidates for use in delaying the progressive degeneration caused by neurological diseases.

Pseudo-bi-enzyme glucose sensor: ZnS hollow spheres and glucose oxidase concerted catalysis glucose.

PubMed

Shuai, Ying; Liu, Changhua; Wang, Jia; Cui, Xiaoyan; Nie, Ling

2013-06-07

This work creatively uses peroxidase-like ZnS hollow spheres (ZnS HSs) to cooperate with glucose oxidase (GOx) for glucose determinations. This approach is that the ZnS HSs electrocatalytically oxidate the enzymatically generated H2O2 to O2, and then the O2 circularly participates in the previous glucose oxidation by glucose oxidase. Au nanoparticles (AuNPs) and carbon nanotubes (CNTs) are used as electron transfer and enzyme immobilization matrices, respectively. The biosensor of glucose oxidase-carbon nanotubes-Au nanoparticles-ZnS hollow spheres-gold electrode (GOx-CNT-AuNPs-ZnS HSs-GE) exhibits a rapid response, a low detection limit (10 μM), a wide linear range (20 μM to 7 mM) as well as good anti-interference, long-term longevity and reproducibility.

Time dependent inhibition of xanthine oxidase in irradiated solutions of folic acid, aminopterin and methotrexate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Robinson, K.; Pilot, T.F.; Meany, J.E.

1990-01-01

The xanthine oxidase catalyzed oxidation of hypoxanthine was followed by monitoring the formation of uric acid at 290 nm. Inhibition of xanthine oxidase occurs in aqueous solutions of folic acid methotrexate and aminopterin. These compounds are known to dissociate upon exposure to ultraviolet light resulting in the formation of their respective 6-formylpteridine derivatives. The relative rates of dissociation were monitored spectrophotometrically by determining the absorbance of their 2,4-dinitrophenylhydrazine derivatives at 500 nm. When aqueous solutions of folic acid, aminopterin and methotrexate were exposed to uv light, a direct correlation was observed between the concentrations of the 6-formylpteridine derivatives existing inmore » solution and the ability of these solutions to inhibit xanthine oxidase. The relative potency of the respective photolysis products were estimated.« less

4-Hydroxyanisole: the most suitable monophenolic substrate for determining spectrophotometrically the monophenolase activity of polyphenol oxidase from fruits and vegetables.

PubMed

Espín, J C; Tudela, J; García-Cánovas, F

1998-05-15

A continuous spectrophotometric method for determining the monophenolase activity of polyphenol oxidase from several plant sources is described. This assay method is based on the coupling reaction between 3-methyl-2-benzothiazolinone hydrazone and the quinone product of the oxidation of 4-hydroxyanisole in the presence of polyphenol oxidase. 4-Hydroxyanisole proved to be the best monophenol assayed to measure the monophenolase activity of polyphenol oxidase from apple, artichoke, avocado, medlar, pear, and strawberry. Kinetic constants of 4-hydroxyanisole were compared to those of p-hydroxyphenyl propionic acid, a very sensitive monophenol previously reported to assay the monophenolase activity of polyphenol oxidase from apple, pear, and mushroom. The high values of the maximum steady state rate obtained for 4-hydroxyanisole suggest the existence of high catalytic constant toward this monophenol. These kinetic values were supported by nuclear magnetic resonance assays which predicted the highest reactivity of 4-hydroxyanisole. Therefore nuclear magnetic resonance assays proved to be a valuable and useful tool to predict the best monophenolic substrate for plant polyphenol oxidases. The 3-methyl-2-benzothiazlolinone-adduct for 4-hydroxyanisole was stable, with high molar absorptivity at the optimum pHs of the polyphenol oxidases assayed. All this together makes the use of 4-hydroxyanisol as monophenolic substrate and 3-methyl-2-benzothiazolinone as coupling reagent the most sensitive and precise assay method up to date reported in the literature to determine the monophenolas activity of polyphenol oxidase from fruits and vegetables.

A Peroxidase-linked Spectrophotometric Assay for the Detection of Monoamine Oxidase Inhibitors.

PubMed

Zhi, Kangkang; Yang, Zhongduo; Sheng, Jie; Shu, Zongmei; Shi, Yin

2016-01-01

To develop a new more accurate spectrophotometric method for detecting monoamine oxidase inhibitors from plant extracts, a series of amine substrates were selected and their ability to be oxidized by monoamine oxidase was evaluated by the HPLC method and a new substrate was used to develop a peroxidase-linked spectrophotometric assay. 4-(Trifluoromethyl) benzylamine (11) was proved to be an excellent substrate for peroxidase-linked spectrophotometric assay. Therefore, a new peroxidase-linked spectrophotometric assay was set up. The principle of the method is that the MAO converts 11 into aldehyde, ammonia and hydrogen peroxide. In the presence of peroxidase, the hydrogen peroxide will oxidize 4-aminoantipyrine into oxidised 4-aminoantipyrine which can condense with vanillic acid to give a red quinoneimine dye. The production of the quinoneimine dye was detected at 490 nm by a microplate reader. The ⊿OD value between the blank group and blank negative control group in this new method is twice as much as that in Holt's method, which enables the procedure to be more accurate and avoids the produce of false positive results. The new method will be helpful for researchers to screening monoamine oxidase inhibitors from deep-color plant extracts.

Artificial Intelligence versus Statistical Modeling and Optimization of Cholesterol Oxidase Production by using Streptomyces Sp.

PubMed Central

Niwas, Ram; Osama, Khwaja; Khan, Saif; Haque, Shafiul; Tripathi, C. K. M.; Mishra, B. N.

2015-01-01

Cholesterol oxidase (COD) is a bi-functional FAD-containing oxidoreductase which catalyzes the oxidation of cholesterol into 4-cholesten-3-one. The wider biological functions and clinical applications of COD have urged the screening, isolation and characterization of newer microbes from diverse habitats as a source of COD and optimization and over-production of COD for various uses. The practicability of statistical/ artificial intelligence techniques, such as response surface methodology (RSM), artificial neural network (ANN) and genetic algorithm (GA) have been tested to optimize the medium composition for the production of COD from novel strain Streptomyces sp. NCIM 5500. All experiments were performed according to the five factor central composite design (CCD) and the generated data was analysed using RSM and ANN. GA was employed to optimize the models generated by RSM and ANN. Based upon the predicted COD concentration, the model developed with ANN was found to be superior to the model developed with RSM. The RSM-GA approach predicted maximum of 6.283 U/mL COD production, whereas the ANN-GA approach predicted a maximum of 9.93 U/mL COD concentration. The optimum concentrations of the medium variables predicted through ANN-GA approach were: 1.431 g/50 mL soybean, 1.389 g/50 mL maltose, 0.029 g/50 mL MgSO4, 0.45 g/50 mL NaCl and 2.235 ml/50 mL glycerol. The experimental COD concentration was concurrent with the GA predicted yield and led to 9.75 U/mL COD production, which was nearly two times higher than the yield (4.2 U/mL) obtained with the un-optimized medium. This is the very first time we are reporting the statistical versus artificial intelligence based modeling and optimization of COD production by Streptomyces sp. NCIM 5500. PMID:26368924

Artificial Intelligence versus Statistical Modeling and Optimization of Cholesterol Oxidase Production by using Streptomyces Sp.

PubMed

Pathak, Lakshmi; Singh, Vineeta; Niwas, Ram; Osama, Khwaja; Khan, Saif; Haque, Shafiul; Tripathi, C K M; Mishra, B N

2015-01-01

Cholesterol oxidase (COD) is a bi-functional FAD-containing oxidoreductase which catalyzes the oxidation of cholesterol into 4-cholesten-3-one. The wider biological functions and clinical applications of COD have urged the screening, isolation and characterization of newer microbes from diverse habitats as a source of COD and optimization and over-production of COD for various uses. The practicability of statistical/ artificial intelligence techniques, such as response surface methodology (RSM), artificial neural network (ANN) and genetic algorithm (GA) have been tested to optimize the medium composition for the production of COD from novel strain Streptomyces sp. NCIM 5500. All experiments were performed according to the five factor central composite design (CCD) and the generated data was analysed using RSM and ANN. GA was employed to optimize the models generated by RSM and ANN. Based upon the predicted COD concentration, the model developed with ANN was found to be superior to the model developed with RSM. The RSM-GA approach predicted maximum of 6.283 U/mL COD production, whereas the ANN-GA approach predicted a maximum of 9.93 U/mL COD concentration. The optimum concentrations of the medium variables predicted through ANN-GA approach were: 1.431 g/50 mL soybean, 1.389 g/50 mL maltose, 0.029 g/50 mL MgSO4, 0.45 g/50 mL NaCl and 2.235 ml/50 mL glycerol. The experimental COD concentration was concurrent with the GA predicted yield and led to 9.75 U/mL COD production, which was nearly two times higher than the yield (4.2 U/mL) obtained with the un-optimized medium. This is the very first time we are reporting the statistical versus artificial intelligence based modeling and optimization of COD production by Streptomyces sp. NCIM 5500.

Immobilization Techniques to Avoid Enzyme Loss from Oxidase-Based Biosensors: A One-Year Study

PubMed Central

House, Jody L.; Anderson, Ellen M.; Ward, W. Kenneth

2007-01-01

Background Continuous amperometric sensors that measure glucose or lactate require a stable sensitivity, and glutaraldehyde crosslinking has been used widely to avoid enzyme loss. Nonetheless, little data is published on the effectiveness of enzyme immobilization with glutaraldehyde. Methods A combination of electrochemical testing and spectrophotometric assays was used to study the relationship between enzyme shedding and the fabrication procedure. In addition, we studied the relationship between the glutaraldehyde concentration and sensor performance over a period of one year. Results The enzyme immobilization process by glutaraldehyde crosslinking to glucose oxidase appears to require at least 24-hours at room temperature to reach completion. In addition, excess free glucose oxidase can be removed by soaking sensors in purified water for 20 minutes. Even with the addition of these steps, however, it appears that there is some free glucose oxidase entrapped within the enzyme layer which contributes to a decline in sensitivity over time. Although it reduces the ultimate sensitivity (probably via a change in the enzyme's natural conformation), glutaraldehyde concentration in the enzyme layer can be increased in order to minimize this instability. Conclusions After exposure of oxidase enzymes to glutaraldehyde, effective crosslinking requires a rinse step and a 24-hour incubation step. In order to minimize the loss of sensor sensitivity over time, the glutaraldehyde concentration can be increased. PMID:19888375

Protein dynamics promote hydride tunnelling in substrate oxidation by aryl-alcohol oxidase.

PubMed

Carro, Juan; Martínez-Júlvez, Marta; Medina, Milagros; Martínez, Angel T; Ferreira, Patricia

2017-11-01

The temperature dependence of hydride transfer from the substrate to the N5 of the FAD cofactor during the reductive half-reaction of Pleurotus eryngii aryl-alcohol oxidase (AAO) is assessed here. Kinetic isotope effects on both the pre-steady state reduction of the enzyme and its steady-state kinetics, with differently deuterated substrates, suggest an environmentally-coupled quantum-mechanical tunnelling process. Moreover, those kinetic data, along with the crystallographic structure of the enzyme in complex with a substrate analogue, indicate that AAO shows a pre-organized active site that would only require the approaching of the hydride donor and acceptor for the tunnelled transfer to take place. Modification of the enzyme's active-site architecture by replacement of Tyr92, a residue establishing hydrophobic interactions with the substrate analogue in the crystal structure, in the Y92F, Y92L and Y92W variants resulted in different temperature dependence patterns that indicated a role of this residue in modulating the transfer reaction.

Identification of novel monoamine oxidase B inhibitors by structure-based virtual screening.

PubMed

Geldenhuys, Werner J; Darvesh, Altaf S; Funk, Max O; Van der Schyf, Cornelis J; Carroll, Richard T

2010-09-01

Parkinson's disease is a severe debilitating neurodegenerative disorder. Recently, it was shown that the peroxisome proliferating-activator receptor-gamma agonist pioglitazone protected mice from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity due to its ability to inhibit monoamine oxidase B (MAO-B). Docking studies were initiated to investigate pioglitazone's interactions within the substrate cavity of MAO-B. Modeling studies indicated that the thiazolidinedione (TZD) moiety was a likely candidate for its specificity to MAO-B. To explore this potential novel MAO-B scaffold, we performed a structure-based virtual screen to identify additional MAO-B inhibitors. Our search identified eight novel compounds containing the TZD-moiety that allowed for a limited study to identify structural requirements for binding to MAO-B. Inhibition assays identified two TZDs (A6355 and L136662) which were found to inhibit recombinant human MAO-B with IC(50) values of 82 and 195 nM, respectively. Copyright 2010 Elsevier Ltd. All rights reserved.

NADPH oxidase: an enzyme for multicellularity?

PubMed

Lalucque, Hervé; Silar, Philippe

2003-01-01

Multicellularity has evolved several times during the evolution of eukaryotes. One evolutionary pressure that permits multicellularity relates to the division of work, where one group of cells functions as nutrient providers and the other in specialized roles such as defence or reproduction. This requires signalling systems to ensure harmonious development of multicellular structures. Here, we show that NADPH oxidases are specifically present in organisms that differentiate multicellular structures during their life cycle and are absent from unicellular life forms. The biochemical properties of these enzymes make them ideal candidates for a role in intercellular signalling.

NADPH oxidase/ROS-dependent PYK2 activation is involved in TNF-α-induced matrix metalloproteinase-9 expression in rat heart-derived H9c2 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Chuen-Mao, E-mail: chuenmao@mail.cgu.edu.tw; Heart Failure Center, Division of Cardiology, Department of Internal Medicine, Chang Gung Memorial Hospital at Keelung, Keelung, Taiwan; Lee, I-Ta

TNF-α plays a mediator role in the pathogenesis of chronic heart failure contributing to cardiac remodeling and peripheral vascular disturbances. The implication of TNF-α in inflammatory responses has been shown to be mediated through up-regulation of matrix metalloproteinase-9 (MMP-9). However, the detailed mechanisms of TNF-α-induced MMP-9 expression in rat embryonic-heart derived H9c2 cells are largely not defined. We demonstrated that in H9c2 cells, TNF-α induced MMP-9 mRNA and protein expression associated with an increase in the secretion of pro-MMP-9. TNF-α-mediated responses were attenuated by pretreatment with the inhibitor of ROS (N-acetyl-L-cysteine, NAC), NADPH oxidase [apocynin (APO) or diphenyleneiodonium chloride (DPI)],more » MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), NF-κB (Bay11-7082), or PYK2 (PF-431396) and transfection with siRNA of TNFR1, p47{sup phox}, p42, p38, JNK1, p65, or PYK2. Moreover, TNF-α markedly induced NADPH oxidase-derived ROS generation in these cells. TNF-α-enhanced p42/p44 MAPK, p38 MAPK, JNK1/2, and NF-κB (p65) phosphorylation and in vivo binding of p65 to the MMP-9 promoter were inhibited by U0126, SB202190, SP600125, NAC, DPI, or APO. In addition, TNF-α-mediated PYK2 phosphorylation was inhibited by NAC, DPI, or APO. PYK2 inhibition could reduce TNF-α-stimulated MAPKs and NF-κB activation. Thus, in H9c2 cells, we are the first to show that TNF-α-induced MMP-9 expression is mediated through a TNFR1/NADPH oxidase/ROS/PYK2/MAPKs/NF-κB cascade. We demonstrated that NADPH oxidase-derived ROS generation is involved in TNF-α-induced PYK2 activation in these cells. Understanding the regulation of MMP-9 expression and NADPH oxidase activation by TNF-α on H9c2 cells may provide potential therapeutic targets of chronic heart failure. - Highlights: • TNF-α induces MMP-9 secretion and expression via a TNFR1-dependent pathway. • TNF-α induces ROS/PYK2-dependent MMP-9 expression in H9c2 cells. • TNF

Crystal structure of heterotetrameric sarcosine oxidase from Corynebacterium sp. U-96

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ida, Koh; E-mail: idakoh@sci.kitasato-u.ac.jp; Moriguchi, Tomotaka

2005-07-29

Sarcosine oxidase from Corynebacterium sp. U-96 is a heterotetrameric enzyme. Here we report the crystal structures of the enzyme in complex with dimethylglycine and folinic acid. The {alpha} subunit is composed of two domains, contains NAD{sup +}, and binds folinic acid. The {beta} subunit contains dimethylglycine, FAD, and FMN, and these flavins are approximately 10 A apart. The {gamma} subunit is in contact with two domains of {alpha} subunit and has possibly a folate-binding structure. The {delta} subunit contains a single atom of zinc and has a Cys{sub 3}His zinc finger structure. Based on the structures determined and on themore » previous works, the structure-function relationship on the heterotetrameric sarcosine oxidase is discussed.« less

Ag-doped CdO nanocatalysts: Preparation, characterization and catechol oxidase activity

NASA Astrophysics Data System (ADS)

El-Kemary, Maged; El-Mehasseb, Ibrahim; El-Shamy, Hany

2018-06-01

Silver doped cadmium oxide (Ag/CdO) nanoparticles with an average size of 41 nm have been successfully synthesized via thermal decomposition and liquid impregnation technique. The structural characterization has been performed by using several spectroscopic techniques, e.g., X-ray diffraction (XRD), scanning electron microscopy (SEM) and fourier-transform infrared (FT-IR). The catechol oxidase has been studied by UV-visible absorption spectroscopy and fourier-transform infrared as well as the mechanism has been assured by cyclic voltammetry and fluorescence spectroscopy. The results indicate that the oxidation does not occur in the presence of unsupported cadmium oxide particles by silver and in the same time, the catechol oxidase activity of silver doped CdO nanoparticles were improved by about three orders of magnitude than silver ions.

The arachidonic acid-binding protein S100A8/A9 promotes NADPH oxidase activation by interaction with p67phox and Rac-2.

PubMed

Kerkhoff, Claus; Nacken, Wolfgang; Benedyk, Malgorzata; Dagher, Marie Claire; Sopalla, Claudia; Doussiere, Jacques

2005-03-01

The Ca2+- and arachidonic acid-binding S100A8/A9 protein complex was recently identified by in vitro studies as a novel partner of the phagocyte NADPH oxidase. The present study demonstrated its functional relevance by the impaired oxidase activity in neutrophil-like NB4 cells, after specific blockage of S100A9 expression, and bone marrow polymorphonuclear neutrophils from S100A9-/- mice. The impaired oxidase activation could also be mimicked in a cell-free system by pretreatment of neutrophil cytosol with an S100A9-specific antibody. Further analyses gave insights into the molecular mechanisms by which S100A8/A9 promoted NADPH oxidase activation. In vitro analysis of oxidase activation as well as protein-protein interaction studies revealed that S100A8 is the privileged interaction partner for the NADPH oxidase complex since it bound to p67phox and Rac, whereas S100A9 did interact with neither p67phox nor p47phox. Moreover, S100A8/A9 transferred the cofactor arachidonic acid to NADPH oxidase as shown by the impotence of a mutant S100A8/A9 complex unable to bind arachidonic acid to enhance NADPH oxidase activity. It is concluded that S100A8/A9 plays an important role in phagocyte NADPH oxidase activation.

Xanthine oxidase inhibitory activities of extracts and flavonoids of the leaves of Blumea balsamifera.

PubMed

Nessa, Fazilatun; Ismail, Zhari; Mohamed, Nornisah

2010-12-01

Blumea balsamifera DC (Compositae) leaves have been recommended for use as a folk medicine in the treatment of various diseases related to urolithiasis in southeast Asia. Phytochemical studies of this plant revealed it contains four classes of flavonoids (e.g., flavonols, flavones, flavanones, and dihydroflavonol derivatives). In view of the broad pharmacological activity of flavonoids, this study was carried out to determine the xanthine oxidase (XO) inhibitory and enzymatically produced superoxide radical scavenging activity of different organic extracts and that of the isolated flavonoids from B. balsamifera leaves. The inhibitory activity of XO was assayed spectrophotometrically at 295 nm. The superoxide radicals scavenging activity was assessed by NBT reduction method, spectrophotometrically at 560 nm. A dose response curve was plotted for determining IC₅₀ values. The methanol extract (IC₅₀ = 0.111 mg/mL) showed higher XO inhibitory activity than the chloroform (0.138 mg/mL) and pet-ether extracts (0.516 mg/mL). IC₅₀ values of scavenging of superoxide radicals for extracts decreased in the order of: methanol (0.063 mg/mL) > chloroform (0.092 mg/mL) > pet-ether (0.321 mg/mL). The XO inhibitory activity of the isolated flavonoids and reference compounds tested decreased in the order of: allopurinol > luteolin > quercetin > tamarixetin > 5,7,3',5'-tetrahydroxyflavanone > rhamnetin > luteolin-7-methyl ether > blumeatin > dihydroquercetin-4'-methyl ether > dihydroquercetin-7,4'-dimethyl ether > L-ascorbic acid. The results indicated that the flavone derivatives were more active than the flavonol derivatives. The flavanone derivatives were moderately active and the dihydroflavonol derivatives were the least. The higher flavonoid content of extracts contributed to their higher XO inhibitory activity.

Osmolytic Effect of Sucrose on Thermal Denaturation of Pea Seedling Copper Amine Oxidase.

PubMed

Amani, Mojtaba; Barzegar, Aboozar; Mazani, Mohammad

2017-04-01

Protein stability is a subject of interest by many researchers. One of the common methods to increase the protein stability is using the osmolytes. Many studies and theories analyzed and explained osmolytic effect by equilibrium thermodynamic while most proteins undergo an irreversible denaturation. In current study we investigated the effect of sucrose as an osmolyte on the thermal denaturation of pea seedlings amine oxidase by the enzyme activity, fluorescence spectroscopy, circular dichroism, and differential scanning calorimetry. All experiments are in agreement that pea seedlings amine oxidase denaturation is controlled kinetically and its kinetic stability is increased in presence of sucrose. Differential scanning calorimetry experiments at different scanning rates showed that pea seedlings amine oxidase unfolding obeys two-state irreversible model. Fitting the differential scanning calorimetry data to two-state irreversible model showed that unfolding enthalpy and T * , temperature at which rate constant equals unit per minute, are increased while activation energy is not affected by increase in sucrose concentration. We concluded that osmolytes decrease the molecular oscillation of irreversible proteins which leads to decline in unfolding rate constant.

Understanding cerebral L-lysine metabolism: the role of L-pipecolate metabolism in Gcdh-deficient mice as a model for glutaric aciduria type I.

PubMed

Posset, Roland; Opp, Silvana; Struys, Eduard A; Völkl, Alfred; Mohr, Heribert; Hoffmann, Georg F; Kölker, Stefan; Sauer, Sven W; Okun, Jürgen G

2015-03-01

Inherited deficiencies of the L-lysine catabolic pathway cause glutaric aciduria type I and pyridoxine-dependent epilepsy. Dietary modulation of cerebral L-lysine metabolism is thought to be an important therapeutic intervention for these diseases. To better understand cerebral L-lysine degradation, we studied in mice the two known catabolic routes -- pipecolate and saccharopine pathways -- using labeled stable L-lysine and brain peroxisomes purified according to a newly established protocol. Experiments with labeled stable L-lysine show that cerebral L-pipecolate is generated along two pathways: i) a minor proportion retrograde after ε-deamination of L-lysine along the saccharopine pathway, and ii) a major proportion anterograde after α-deamination of L-lysine along the pipecolate pathway. In line with these findings, we observed only little production of saccharopine in the murine brain. L-pipecolate oxidation was only detectable in brain peroxisomes, but L-pipecolate oxidase activity was low (7 ± 2μU/mg protein). In conclusion, L-pipecolate is a major degradation product from L-lysine in murine brain generated by α-deamination of this amino acid.

The Role of Aldehyde Oxidase and Xanthine Oxidase in the Biotransformation of a Novel Negative Allosteric Modulator of Metabotropic Glutamate Receptor Subtype 5

PubMed Central

Morrison, Ryan D.; Blobaum, Anna L.; Byers, Frank W.; Santomango, Tammy S.; Bridges, Thomas M.; Stec, Donald; Brewer, Katrina A.; Sanchez-Ponce, Raymundo; Corlew, Melany M.; Rush, Roger; Felts, Andrew S.; Manka, Jason; Bates, Brittney S.; Venable, Daryl F.; Rodriguez, Alice L.; Jones, Carrie K.; Niswender, Colleen M.; Conn, P. Jeffrey; Lindsley, Craig W.; Emmitte, Kyle A.

2012-01-01

Negative allosteric modulation (NAM) of metabotropic glutamate receptor subtype 5 (mGlu5) represents a therapeutic strategy for the treatment of childhood developmental disorders, such as fragile X syndrome and autism. VU0409106 emerged as a lead compound within a biaryl ether series, displaying potent and selective inhibition of mGlu5. Despite its high clearance and short half-life, VU0409106 demonstrated efficacy in rodent models of anxiety after extravascular administration. However, lack of a consistent correlation in rat between in vitro hepatic clearance and in vivo plasma clearance for the biaryl ether series prompted an investigation into the biotransformation of VU0409106 using hepatic subcellular fractions. An in vitro appraisal in rat, monkey, and human liver S9 fractions indicated that the principal pathway was NADPH-independent oxidation to metabolite M1 (+16 Da). Both raloxifene (aldehyde oxidase inhibitor) and allopurinol (xanthine oxidase inhibitor) attenuated the formation of M1, thus implicating the contribution of both molybdenum hydroxylases in the biotransformation of VU0409106. The use of 18O-labeled water in the S9 experiments confirmed the hydroxylase mechanism proposed, because 18O was incorporated into M1 (+18 Da) as well as in a secondary metabolite (M2; +36 Da), the formation of which was exclusively xanthine oxidase-mediated. This unusual dual and sequential hydroxylase metabolism was confirmed in liver S9 and hepatocytes of multiple species and correlated with in vivo data because M1 and M2 were the principal metabolites detected in rats administered VU0409106. An in vitro-in vivo correlation of predicted hepatic and plasma clearance was subsequently established for VU0409106 in rats and nonhuman primates. PMID:22711749

Study on the activity of non-purine xanthine oxidase inhibitor by 3D-QSAR modeling and molecular docking

NASA Astrophysics Data System (ADS)

Li, Peizhen; Tian, Yueli; Zhai, Honglin; Deng, Fangfang; Xie, Meihong; Zhang, Xiaoyun

2013-11-01

Non-purine derivatives have been shown to be promising novel drug candidates as xanthine oxidase inhibitors. Based on three-dimensional quantitative structure-activity relationship (3D-QSAR) methods including comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA), two 3D-QSAR models for a series of non-purine xanthine oxidase (XO) inhibitors were established, and their reliability was supported by statistical parameters. Combined 3D-QSAR modeling and the results of molecular docking between non-purine xanthine oxidase inhibitors and XO, the main factors that influenced activity of inhibitors were investigated, and the obtained results could explain known experimental facts. Furthermore, several new potential inhibitors with higher activity predicted were designed, which based on our analyses, and were supported by the simulation of molecular docking. This study provided some useful information for the development of non-purine xanthine oxidase inhibitors with novel structures.

Season-controlled changes in biochemical constituents and oxidase enzyme activities in tomato (Lycopersicon esculentum Mill.).

PubMed

Sen, Supatra; Mukherji, S

2009-07-01

Season-controlled changes in biochemical constituents viz. carotenoids (carotene and xanthophyll) and pectic substances along with IAA-oxidase and polyphenol oxidase (PPO) enzyme activities were estimated/assayed in leaves of Lycopersicon esculentum Mill. (tomato) in two developmental stages--pre-flowering (35 days after sowing) and post-flowering (75 days after sowing) in three different seasons--summer rainy and winter Carotenoid content along with pectic substances were highest in winter and declined significantly in summer followed by rainy i.e. winter > summer > rainy. Carotenoid content was significantly higher in the pre-flowering as compared to post-flowering in all three seasons while pectic substances increased in the post-flowering as compared to pre-flowering throughout the annual cycle. IAA oxidase and PPO enzyme activities were enhanced in rainy and decreased sharply in summer and winter i.e. rainy > summer > winter. Both the enzymes exhibited higher activity in the post-flowering stage as compared to pre-flowering in all three seasons. These results indicate winter to be the most favourable season for tomato plants while rainy season environmental conditions prove to be unfavourable (stressful) with diminished content of carotenoid and pectic substances and low activities of IAA oxidase and PPO, ultimately leading to poor growth and productivity.

In vitro xanthine oxidase inhibitory and in vivo hypouricemic activity of herbal coded formulation (Gouticin).

PubMed

Akram, Muhammad; Usmanghani, Khan; Ahmed, Iqbal; Azhar, Iqbal; Hamid, Abdul

2014-05-01

Currently, natural products have been used in treating gouty arthritis and are recognized as xanthine oxidase inhibitors. Current study was designed to evaluate in vitro xanthine oxidase inhibitory potential of Gouticin and its ingredients extracts and in vivo hypouricemic activity of gouticin tablet 500 mg twice daily. Ethanol extracts of Gouticin and its ingredients were evaluated in vitro, at 200, 100, 50, 25 μ g/ml concentrations for xanthine oxidase inhibitory activity. IC(50) values of Gouticin and its ingredients were estimated. Further, in vivo therapeutic effect of Gouticin was investigated in comparison with allopathic medicine (Allopurinol) to treat gout. Total patients were 200 that were divided into test and control group. Herbal coded medicine (Gouticin) was given to test group and allopathic medicine allopurinol was administered to control group. In vitro, Gouticin has the highest percent inhibition at 96% followed by Allopurinol with 93% inhibition. In vivo study, mean serum uric acid level of patients was 4.62 mg/dl and 5.21mg/dl by use of Gouticin and Allopurinol at end of therapy. The study showed that herbal coded formulation gouticin and its ingredients are potential sources of natural xanthine oxidase inhibitors. Gouticin 500 mg twice daily is more effective than the allopurinol 300mg once daily in the management of gout.

Assessment of Antioxidant and Phenolic Compound Concentrations as well as Xanthine Oxidase and Tyrosinase Inhibitory Properties of Different Extracts of Pleurotus citrinopileatus Fruiting Bodies

PubMed Central

Alam, Nuhu; Yoon, Ki Nam; Lee, Kyung Rim; Kim, Hye Young; Shin, Pyung Gyun; Cheong, Jong Chun; Yoo, Young Bok; Shim, Mi Ja; Lee, Min Woong

2011-01-01

Cellular damage caused by reactive oxygen species has been implicated in several diseases, thus establishing a significant role for antioxidants in maintaining human health. Acetone, methanol, and hot water extracts of Pleurotus citrinopileatus were evaluated for their antioxidant activities against β-carotene-linoleic acid and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, reducing power, ferrous ion-chelating abilities, and xanthine oxidase inhibitory activities. In addition, the tyrosinase inhibitory effects and phenolic compound contents of the extracts were also analyzed. Methanol and acetone extracts of P. citrinopileatus showed stronger inhibition of β-carotene-linoleic acid compared to the hot water extract. Methanol extract (8 mg/mL) showed a significantly high reducing power of 2.92 compared to the other extracts. The hot water extract was more effective than the acetone and methanole extracts for scavenging DPPH radicals. The strongest chelating effect (92.72%) was obtained with 1.0 mg/mL of acetone extract. High performance liquid chromatography analysis detected eight phenolic compounds, including gallic acid, protocatechuic acid, chlorogenic acid, ferulic acid, naringenin, hesperetin, formononetin, and biochanin-A, in an acetonitrile and hydrochloric acid (5 : 1) solvent extract. Xanthine oxidase and tyrosinase inhibitory activities of the acetone, methanol, and hot water extracts increased with increasing concentration. This study suggests that fruiting bodies of P. citrinopileatus can potentially be used as a readily accessible source of natural antioxidants. PMID:22783067

Thermal Characterization of Purified Glucose Oxidase from A Newly Isolated Aspergillus Niger UAF-1

PubMed Central

Anjum Zia, Muhammad; Khalil-ur-Rahman; K. Saeed, Muhammad; Andaleeb, Fozia; I. Rajoka, Muhammad; A. Sheikh, Munir; A. Khan, Iftikhar; I. Khan, Azeem

2007-01-01

An intracellular glucose oxidase was isolated from the mycelium extract of a locally isolated strain of Aspergillus niger UAF-1. The enzyme was purified to a yield of 28.43% and specific activity of 135 U mg−1 through ammonium sulfate precipitation, anion exchange and gel filtration chromatography. The enzyme showed high affinity for D-glucose with a Km value of 2.56 mM. The enzyme exhibited optimum catalytic activity at pH 5.5. Temperature optimum for glucose oxidase, catalyzed D-glucose oxidation was 40°C. The enzyme showed a high thermostability having a half-life 30 min, enthalpy of denaturation 99.66 kJ mol−1 and free energy of denaturation 103.63 kJ mol−1. These characteristics suggest the use of glucose oxidase from Aspergillus niger UAF-1 as an analytical reagent and in the design of biosensors for clinical, biochemical and diagnostic assays. PMID:18193107

Involvement of alternative oxidase (AOX) in adventitious rooting of Olea europaea L. microshoots is linked to adaptive phenylpropanoid and lignin metabolism.

PubMed

Santos Macedo, E; Sircar, D; Cardoso, H G; Peixe, A; Arnholdt-Schmitt, B

2012-09-01

Alternative oxidase (AOX) has been proposed as a functional marker candidate in a number of events involving cell differentiation, including rooting efficiency in semi-hardwood shoot cuttings of olive (Olea europaea L.). To ascertain the general importance of AOX in olive rooting, the auxin-induced rooting process was studied in an in vitro system for microshoot propagation. Inhibition of AOX by salicylhydroxamic acid (SHAM) significantly reduced rooting efficiency. However, the inhibitor failed to exhibit any effect on the preceding calli stage. This makes the system appropriate for distinguishing dedifferentiation and de novo differentiation during root induction. Metabolite analyses of microshoots showed that total phenolics, total flavonoids and lignin contents were significantly reduced upon SHAM treatment. It was concluded that the influence of alternative respiration on root formation was associated to adaptive phenylpropanoid and lignin metabolism. Transcript profiles of two olive AOX genes (OeAOX1a and OeAOX2) were examined during the process of auxin-induced root induction. Both genes displayed stable transcript accumulation in semi-quantitative RT-PCR analysis during all experimental stages. In contrary, when the reverse primer for OeAOX2 was designed from the 3'-UTR instead of the ORF, differential transcript accumulation was observed suggesting posttranscriptional regulation of OeAOX2 during metabolic acclimation. This result confirms former observations in olive semi-hardwood shoot cuttings on differential OeAOX2 expression during root induction. It further points to the importance of future studies on the functional role of sequence and length polymorphisms in the 3'-UTR of this gene. The manuscript reports the general importance of AOX in olive adventitious rooting and the association of alternative respiration to adaptive phenylpropanoid and lignin metabolism.

Isolation and purification of pyranose 2-oxidase from Phanerochaete chrysosporium and characterization of gene structure and regulation

Treesearch

Theodorus H. de Koker; Michael D. Mozuch; Daniel Cullen; Jill Gaskell; Philip J. Kersten

2004-01-01

Pyranose 2-oxidase (POX) was recovered from Phanerochaete chrysosporium BKM-F-1767 solid substrate culture using mild extraction conditions and was purified. 13C-nuclear magnetic resonance confirmed production of D- arabino -hexos-2-ulose (glucosone) from D-glucose with the oxidase. Peptide fingerprints generated by liquid chromatography-tandem mass spectrometry of...