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Sample records for acid oxidase l-amino

  1. Snake Venom L-Amino Acid Oxidases: Trends in Pharmacology and Biochemistry

    PubMed Central

    Izidoro, Luiz Fernando M.; Sobrinho, Juliana C.; Mendes, Mirian M.; Costa, Tássia R.; Grabner, Amy N.; Rodrigues, Veridiana M.; da Silva, Saulo L.; Zanchi, Fernando B.; Zuliani, Juliana P.; Fernandes, Carla F. C.; Calderon, Leonardo A.; Stábeli, Rodrigo G.; Soares, Andreimar M.

    2014-01-01

    L-amino acid oxidases are enzymes found in several organisms, including venoms of snakes, where they contribute to the toxicity of ophidian envenomation. Their toxicity is primarily due to enzymatic activity, but other mechanisms have been proposed recently which require further investigation. L-amino acid oxidases exert biological and pharmacological effects, including actions on platelet aggregation and the induction of apoptosis, hemorrhage, and cytotoxicity. These proteins present a high biotechnological potential for the development of antimicrobial, antitumor, and antiprotozoan agents. This review provides an overview of the biochemical properties and pharmacological effects of snake venom L-amino acid oxidases, their structure/activity relationship, and supposed mechanisms of action described so far. PMID:24738050

  2. Screening of Bothrops snake venoms for L-amino acid oxidase activity

    SciTech Connect

    Pessati, M.L.; Fontana, J.D.; Guimaraes, M.F.

    1995-12-31

    Toxins, enzymes, and biologically active peptides are the main components of snake venoms from the genus Bothrops. Following the venom inoculation, the local effects are hemorrhage, edema, and myonecrosis. Nineteen different species of Brazilian Bothrops were screened for protein content and L-amino acid oxidase activity. B. cotiara, formerly found in the South of Brazil, is now threatened with extinction. Its venom contains a highly hemorrhagic fraction and, as expected from the deep yellow color of the corresponding lyophilized powder, a high L-amino acid oxidase (LAO) activity was also characterized. Flavin adenine dinucleotide (FAD) is its associate coenzyme. B. cotiara venom LAO catalyzed the oxidative deamination of several L-amino acids, and the best substrates were methionine, leucine, tryptophan, and phenylalanine, hence, its potential application for the use in biosensors for aspartame determination and for the removal of amino acids from plasma. High levels for LAO were also found in other species than B. cotiara. In addition, the technique of isoelectric focusing (IEF) was employed as a powerful tool to study the iso- or multi-enzyme distribution for LAO activity in the B. cotiara snake venom.

  3. Screening of Bothrops snake venoms for L-amino acid oxidase activity.

    PubMed

    Pessatti, M; Fontana, J D; Furtado, M F; Guimãraes, M F; Zanette, L R; Costa, W T; Baron, M

    1995-01-01

    Toxins, enzymes, and biologically active peptides are the main components of snake venoms from the genus Bothrops. Following the venom inoculation, the local effects are hemorrhage, edema, and myonecrosis. Nineteen different species of Brazilian Bothrops were screened for protein content and L-amino acid oxidase activity. B. cotiara, formerly found in the South of Brazil, is now threatened with extinction. Its venom contains a highly hemorrhagic fraction and, as expected from the deep yellow color of the corresponding lyophilized powder, a high L-amino acid oxidase (LAO) activity was also characterized. Flavin adenine dinucleotide (FAD) is its associate coenzyme. B. cotiara venom LAO catalyzed the oxidative deamination of several L-amino acids, and the best substrates were methionine, leucine, tryptophan, and phenylalanine, hence, its potential application for the use of biosensors for aspartame determination and for the removal of amino acids from plasma. High levels for LAO were also found in other species than B. cotiara. In addition, the technique of isoelectric focusing (IEF) was employed as a powerful tool to study the iso- or multi-enzyme distribution for LAO activity in the B. cotiara snake venom.

  4. Substrate specificity of king cobra (Ophiophagus hannah) venom L-amino acid oxidase.

    PubMed

    Tan, N H; Saifuddin, M N

    1991-01-01

    1. Substrate specificity of purified king cobra (Ophiophagus hannah) venom L-amino acid oxidase was investigated. 2. The enzyme was highly specific for the L-enantiomer of amino acid. Effective oxidation of L-amino acid by the enzyme requires the presence of a free primary alpha-amino group but the alpha-carboxylate group is not as critical for the catalysis. 3. The enzyme was very active against L-Lys, L-Phe, L-Leu, L-Tyr, L-Tryp, L-Arg, L-Met, L-ornithine, L-norleucine and L-norvaline and moderately active against L-His, L-cystine and L-Ileu. Other L-amino acids were oxidized slowly or not oxidized. 4. The data suggest the presence of a side chain binding site in the enzyme, and that the binding site comprises at least five 'subsites': the hydrophobic subsites a, b and c; and the two 'amino' binding subsites d and e. Subsite b appears to be able to accommodate two methylene/methyl carbons.

  5. L-amino acid biosensor based on L-amino acid oxidase immobilized onto NiHCNFe/c-MWCNT/PPy/GC electrode.

    PubMed

    Lata, Suman; Pundir, C S

    2013-03-01

    A method is described for construction of a highly sensitive amperometric L-amino acid biosensor based on covalent immobilization of goat kidney L-amino acid oxidase (LAAO) onto carboxylated multiwalled carbon nanotubes/nickel hexacyanoferrate/polypyrrole hybrid film electrodeposited on surface of glassy carbon electrode. The biosensor showed optimum response within 5s at pH 7.5 and 35 °C, when polarized at 0.15 V vs. Ag/AgCl. There was a linear relationship between biosensor response (μA) and L-phenylalanine concentration in the range, 0.5 μM to 100 mM. The sensitivity of the biosensor was 79.31 nA cm(-2) μM(-1) with a detection limit of 0.5 μM (S/N=3). The enzyme electrode was used 160 times over a period of 140 days, when stored dry at 4 °C. The biosensor was evaluated and employed for determination of L-amino acid level in fruit juices and alcoholic beverages. The biosensor showed excellent analytical characteristics along with short response time, good shelf life, and negligible interference of commonly present interferents. The film could be used for improvement of other biosensors also.

  6. Antibacterial Properties of the Mammalian L-Amino Acid Oxidase IL4I1

    PubMed Central

    Puiffe, Marie-Line; Lachaise, Isabelle

    2013-01-01

    L-amino acid oxidases (LAAO) are flavoproteins that catalyze the oxidative deamination of L-amino acids to a keto-acid along with the production of H2O2 and ammonia. Interleukin 4 induced gene 1 (IL4I1) is a secreted LAAO expressed by macrophages and dendritic cells stimulated by microbial derived products or interferons, which is endowed with immunoregulatory properties. It is the first LAAO described in mammalian innate immune cells. In this work, we show that this enzyme blocks the in vitro and in vivo growth of Gram negative and Gram positive bacteria. This antibiotic effect is primarily mediated by H2O2 production but is amplified by basification of the medium due to the accumulation of ammonia. The depletion of phenylalanine (the primary amino acid catabolized by IL4I1) may also participate in the in vivo inhibition of staphylococci growth. Thus, IL4I1 plays a distinct role compared to other antibacterial enzymes produced by mononuclear phagocytes. PMID:23355881

  7. Snake venom L-amino acid oxidases: an overview on their antitumor effects

    PubMed Central

    2014-01-01

    The L-amino acid oxidases (LAAOs) constitute a major component of snake venoms and have been widely studied due to their widespread presence and various effects, such as apoptosis induction, cytotoxicity, induction and/or inhibition of platelet aggregation, hemorrhage, hemolysis, edema, as well as antimicrobial, antiparasitic and anti-HIV activities. The isolated and characterized snake venom LAAOs have become important research targets due to their potential biotechnological applications in pursuit for new drugs of interest in the scientific and medical fields. The current study discusses the antitumor effects of snake venom LAAOs described in the literature to date, highlighting the mechanisms of apoptosis induction proposed for this class of proteins. PMID:24940304

  8. Isolation and characterization of an unusual form of L-amino acid oxidase from King cobra (Ophiophagus hannah) venom.

    PubMed

    Tan, N H; Saifuddin, M N

    1989-10-01

    The L-amino acid oxidase (EC 1. 4. 3. 2) from King cobra (Ophiophagus hannah) venom was purified to electrophoretic homogeneity. The molecular weight of the enzyme was determined to be 140000 when examined by gel filtration and 68000 by SDS-polyacrylamide gel electrophoresis. The enzyme had an isoelectric point of 4.5 and an intravenous LD50 of 5 micrograms/g in mice. It is a glycoprotein and contains two moles of FAD per mole of enzyme. The enzyme exhibited unusual thermal stability and unlike most other venom L-amino acid oxidases, it was stable in alkaline solution and was not inactivated by freezing.

  9. Purification and biological effects of L-amino acid oxidase isolated from Bothrops insularis venom.

    PubMed

    Braga, Marcus Davis Machado; Martins, Alice Maria Costa; Amora, Daniela Nascimento; de Menezes, Dalgimar Beserra; Toyama, Marcos Hikari; Toyama, Daniela Oliveira; Marangoni, Sergio; Alves, Claudênio Diógenes; Barbosa, Paulo Sérgio Ferreira; de Sousa Alves, Renata; Fonteles, Manassés Claudino; Monteiro, Helena Serra Azul

    2008-02-01

    Bothrops insularis is a snake from Ilha da Queimada Grande, an island located about 20 miles away from the Southeastern coast of Brazil. Compared with other Brazilian species of Bothrops, the toxinology of B. insularis is still poorly understood, and so far, no fraction from this venom with amino acid oxidase activity had been isolated or its biological activity tested. We investigated the biochemical and biological effects of one l-amino acid oxidase enzyme isolated from B. insularis snake venom (BiLAO), which was purified using HPLC and sequence grade. We also evaluated the renal effects induced by BiLAO. Chromatographic profile of B. insularis whole venom disclosed seven main fractions (I, II, III, IV, V, VI and VII) and the main LAO enzymatic activity was detected in fraction II. The group treated with BiLAO showed a decrease in perfusion pressure (C(120)=110.28+/-3.69; BiLAO(120)=82.2+/-5.6 mmHg*); renal vascular resistance (C(120)=5.48+/-0.53; BiLAO(120)=4.12+/-0.42 mmHg/mL/g/min*), urinary flow (C(120)=0.160+/-0.020; BiLAO(120)=0.064+/-0.012 mL/g/min*), glomerular filtration rate (C(120)=0.697+/-0.084; BiLAO(120)=0.176+/-0.017 mL/g/min*), sodium (C(120)=79.76+/-0.56; BiLAO(120)=65.39+/-6.19%*), potassium (C(120)=69.94+/-6.86; BiLAO(120)=60.26+/-2.24%*) and chloride tubular reabsortion (C(120)=78.53+/-2.33; BiLAO(120)=64.58+/-6.68%*). Acute tubular necrosis foci were observed in the group treated with the LAO fraction of the B. insularis snake venom. Some findings have the same morphological aspect of apoptosis, more evident cortically; otherwise, reversible degenerative phenomena represented by hydropic ballooning with extensive cytoplasmic vacuolization and discontinuity of the cell brush borders in the proximal tubular epithelium were observed; furthermore, necrotic detachment of these cells into the tubular lumina, and increased amount of protein deposits in the distal and proximal tubules were observed. In conclusion, the slowness of blood flow and of

  10. Molecular characterization of L-amino acid oxidase from king cobra venom.

    PubMed

    Jin, Yang; Lee, Wen-Hui; Zeng, Lin; Zhang, Yun

    2007-09-15

    An L-amino acid oxidase from Ophiophagus hannah snake venom (Oh-LAAO) was purified by successive gel filtration, ion-exchange and heparin chromatography. Oh-LAAO did not induce platelet aggregation; however, it had potent inhibitory activity on platelet aggregation induced by ADP and U46619, but showed no effect on platelet aggregation induced by thrombin, mucetin, ristocetin and stejnulxin. By RT-PCR and 5'-RACE methods, the complete Oh-LAAO cDNA was cloned from the venom gland total RNA preparations. The cDNA sequence contains an open-reading frame (ORF) of 1476-bp, which encodes a protein of 491 amino acids comprising a signal peptide of 25 amino acids and 466-residue mature protein. The predicted protein sequence of Oh-LAAO was confirmed by N-terminal and trypsin-digested internal peptides sequencing together with peptide mass fingerprinting. cDNAs encoding for ORF of LAAOs from Bungarus fasciatus and B. multicinctus were cloned and reported in this study. In addition, partial cDNA encoding for Naja atra LAAO was also reported. Oh-LAAO shared approximately 50% protein sequence identity with other known snake venom LAAOs. Phylogenetic analysis indicated that Oh-LAAO is evolutionary distant to other snake venom LAAOs. PMID:17543361

  11. Molecular characterization of L-amino acid oxidase from king cobra venom.

    PubMed

    Jin, Yang; Lee, Wen-Hui; Zeng, Lin; Zhang, Yun

    2007-09-15

    An L-amino acid oxidase from Ophiophagus hannah snake venom (Oh-LAAO) was purified by successive gel filtration, ion-exchange and heparin chromatography. Oh-LAAO did not induce platelet aggregation; however, it had potent inhibitory activity on platelet aggregation induced by ADP and U46619, but showed no effect on platelet aggregation induced by thrombin, mucetin, ristocetin and stejnulxin. By RT-PCR and 5'-RACE methods, the complete Oh-LAAO cDNA was cloned from the venom gland total RNA preparations. The cDNA sequence contains an open-reading frame (ORF) of 1476-bp, which encodes a protein of 491 amino acids comprising a signal peptide of 25 amino acids and 466-residue mature protein. The predicted protein sequence of Oh-LAAO was confirmed by N-terminal and trypsin-digested internal peptides sequencing together with peptide mass fingerprinting. cDNAs encoding for ORF of LAAOs from Bungarus fasciatus and B. multicinctus were cloned and reported in this study. In addition, partial cDNA encoding for Naja atra LAAO was also reported. Oh-LAAO shared approximately 50% protein sequence identity with other known snake venom LAAOs. Phylogenetic analysis indicated that Oh-LAAO is evolutionary distant to other snake venom LAAOs.

  12. Crystallization and preliminary X-ray analysis of a bacterial l-amino-acid oxidase from Rhodococcus opacus

    SciTech Connect

    Faust, Annette; Geueke, Birgit; Niefind, Karsten; Hummel, Werner; Schomburg, Dietmar

    2006-03-01

    The crystallization and preliminary X-ray analysis of a bacterial l-amino acid oxidase from R. opacus is described. The homodimeric protein contains one molecule of non-covalently bound FAD per monomer. Crystals with good diffraction properties were grown in two different orthorhombic space groups (P2{sub 1}2{sub 1}2{sub 1} and C222{sub 1}). l-Amino-acid oxidases (EC 1.4.3.2) catalyse the stereospecific oxidative deamination of an l-amino-acid substrate to an α-keto acid with the production of ammonia and hydrogen peroxide. In this study, the crystallization and preliminary X-ray analysis of a bacterial l-amino-acid oxidase from Rhodococcus opacus (RoLAAO) is described. RoLAAO is a dimeric protein consisting of two identical subunits of 489 amino acids with a calculated molecular weight of 54.2 kDa and a non-covalently bound FAD molecule. RoLAAO was crystallized by the vapour-diffusion method in two different space groups: P2{sub 1}2{sub 1}2{sub 1} (unit-cell parameters a = 65.7, b = 109.7, c = 134.4 Å) and C222{sub 1} (unit-cell parameters a = 68.3, b = 88.4, c = 186.6 Å). Both crystal forms diffracted X-rays to a resolution of at least 1.6 Å.

  13. Structure and characterization of the glycan moiety of L-amino-acid oxidase from the Malayan pit viper Calloselasma rhodostoma.

    PubMed

    Geyer, A; Fitzpatrick, T B; Pawelek, P D; Kitzing, K; Vrielink, A; Ghisla, S; Macheroux, P

    2001-07-01

    Ophidian L-amino-acid oxidase (L-amino-acid oxygen:oxidoreductase, deaminating, EC 1.4.3.2) is found in the venom of many poisonous snakes (crotalids, elapids and viperids). This FAD-dependent glycoprotein has been studied from several snake species (e.g. Crotalus adamanteus, Crotalus atrox and Calloselasma rhodostoma) in detail with regard to the biochemical and enzymatic properties. The nature of glycosylation, however, as well as the chemical structure(s) of the attached oligosaccharide(s) are unknown. In view of the putative involvement of the glycan moiety in the biological effects of ophidian L-amino-acid oxidase, notably the apoptotic activity of the enzyme, structural knowledge is needed to evaluate its exact function. In this study we report on the glycosylation of L-amino-acid oxidase from the venom of the Malayan pit viper (Calloselasma rhodostoma). Its glycosylation is remarkably homogeneous with the major oligosaccharide accounting for approximately 90% of the total sugar content. Based on detailed analysis of the isolated oligosaccharide by 2D NMR spectroscopies and MALDI-TOF mass spectrometry the glycan is identified as a bis-sialylated, biantennary, core-fucosylated dodecasaccharide. The biological significance of this finding is discussed in light of the biological activities of the enzyme. PMID:11453999

  14. A rational protocol for the successful crystallization of L-amino-acid oxidase from Bothrops atrox.

    PubMed

    Alves, Raquel Melo; Feliciano, Patricia Rosa; Sampaio, Suely Vilela; Nonato, Maria Cristina

    2011-04-01

    Despite the valuable contributions of robotics and high-throughput approaches to protein crystallization, the role of an experienced crystallographer in the evaluation and rationalization of a crystallization process is still crucial to obtaining crystals suitable for X-ray diffraction measurements. In this work, the difficult task of crystallizing the flavoenzyme L-amino-acid oxidase purified from Bothrops atrox snake venom was overcome by the development of a protocol that first required the identification of a non-amorphous precipitate as a promising crystallization condition followed by the implementation of a methodology that combined crystallization in the presence of oil and seeding techniques. Crystals were obtained and a complete data set was collected to 2.3 Å resolution. The crystals belonged to space group P2(1), with unit-cell parameters a = 73.64, b = 123.92, c = 105.08 Å, β = 96.03°. There were four protein subunits in the asymmetric unit, which gave a Matthews coefficient V(M) of 2.12 Å(3) Da(-1), corresponding to 42% solvent content. The structure has been solved by molecular-replacement techniques. PMID:21505245

  15. 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. PMID:26546307

  16. Crystallization and preliminary X-ray diffraction studies of an l-amino-acid oxidase from Lachesis muta venom

    PubMed Central

    Ullah, Anwar; Masood, Rehana; Spencer, Patrick Jack; Murakami, Mário Tyago; Arni, Raghuvir Krishnaswamy

    2014-01-01

    Snake-venom proteins form multi-component defence systems by the recruitment and rapid evolution of nonvenomous proteins and hence serve as model systems to understand the structural modifications that result in toxicity. l-Amino-acid oxidases (LAAOs) are encountered in a number of snake venoms and have been implicated in the inhibition of platelet aggregation, cytotoxicity, haemolysis, apoptosis and haemorrhage. An l-amino-acid oxidase from Lachesis muta venom has been purified and crystallized. The crystals belonged to space group P21, with unit-cell parameters a = 66.05, b = 79.41, c = 100.52 Å, β = 96.55°. The asymmetric unit contained two molecules and the structure has been determined and partially refined at 3.0 Å resolution. PMID:25372830

  17. 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. PMID:24118879

  18. 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.

  19. Characterization and cytotoxicity of L-amino acid oxidase from the venom of king cobra (Ophiophagus hannah).

    PubMed

    Ahn, M Y; Lee, B M; Kim, Y S

    1997-06-01

    The aim of this project was to determine the cytotoxic components from the venom of king cobra, Ophiophagus hannah. Venom was purified by a combination of gel-filtration, ion-exchange and reversed-phase chromatographic steps. The biochemical properties of the cytotoxic component were consistent with those of L-amino acid oxidase. The molecular weight of the enzyme was estimated to be 150,000 by gel filtration and 70,000 under the denaturing conditions of SDS-PAGE, indicating a dimer. It has an isoelectric point of 4.5 and is a glycoprotein. The N-terminal sequence of L-amino acid oxidase from the king cobra venom was determined to be SVINLEESFQEPEYE. The cytotoxicity of L-amino acid oxidase was observed in stomach cancer, murine melanoma, fibrosarcoma, colorectal cancer and Chinese hamster ovary cell lines. Cytotoxicity resulted in the loss of ability in attachment and inhibition of cell proliferation. The cytotoxic protein decreased the level of cell proliferation by 74% according to [3H]thymidine uptake assay. The mechanism of enzyme action may be related to the inhibition of thymidine incorporation and an interaction with DNA.

  20. Purification and characterization of L-amino acid oxidase from king cobra (Ophiophagus hannah) venom and its effects on human platelet aggregation.

    PubMed

    Li, Z Y; Yu, T F; Lian, E C

    1994-11-01

    Venoms of several snake species contain large amounts of L-amino acid oxidase but its effects on human plasma coagulation and platelet aggregation have not been explored. We have purified L-amino acid oxidase from king cobra venom through CM-Sephadex C-25, Sephadex G-100 and DEAE Sephadex A-50 chromatographies. The purified enzyme has a mol. wt of 135,000 as determined by gel filtration and 65,000 by SDS-PAGE under non-reducing and reducing conditions. Incubation of plasma with L-amino acid oxidase at 200 micrograms/ml did not affect prothrombin time, activated partial thromboplastin time, or thrombin time. Upon addition of L-amino acid oxidase, platelets in platelet-rich plasma were aggregated. The enzyme-induced aggregation was abolished by catalase. The aggregation was also inhibited by indomethacin, aspirin, ethylenediaminetetraacetate, sodium nitroprusside, prostaglandin E1, mepacrine and verapamil, but not by heparin, hirudin, creatine phosphate/creatine phosphokinase or antimycin/2-deoxy-D-glucose. These results suggest that L-amino acid oxidase induces human platelet aggregation through the formation of H2O2, and subsequent thromboxane A2 synthesis requiring Ca2+ but independent of ADP release. The platelet aggregation caused by L-amino acid oxidase is likely to contribute to toxicity inflicted by cobra venom.

  1. Evidence of caspase-mediated apoptosis induced by l-amino acid oxidase isolated from Bothrops atrox snake venom.

    PubMed

    Alves, Raquel Melo; Antonucci, Gilmara Ausech; Paiva, Helder Henrique; Cintra, Adélia Cristina Oliveira; Franco, João José; Mendonça-Franqueiro, Elaine Paula; Dorta, Daniel Junqueira; Giglio, José Roberto; Rosa, José César; Fuly, André Lopes; Dias-Baruffi, Marcelo; Soares, Andreimar Martins; Sampaio, Suely Vilela

    2008-12-01

    The aim of this work was to investigate the involvement of caspases in apoptosis induced by l-amino acid oxidase isolated from Bothrops atrox snake venom. The isolation of LAAO involved three chromatographic steps: molecular exclusion on a G-75 column; ion exchange column by HPLC and affinity chromatography on a Lentil Lectin column. SDS-PAGE was used to confirm the expected high purity level of BatroxLAAO. It is a glycoprotein with 12% sugar and an acidic character, as confirmed by its amino acid composition, rich in "Asp and Glu" residues. It displays high specificity toward hydrophobic l-amino acids. The N-terminal amino acid sequence and internal peptide sequences showed close structural homology to other snake venom LAAOs. This enzyme induces in vitro platelet aggregation, which may be due to H2O2 production by LAAOs, since the addition of catalase completely inhibited the aggregation effect. It also showed cytotoxicity towards several cancer cell lines: HL60, Jurkat, B16F10 and PC12. The cytotoxicity activity was abolished by catalase. A fluorescence microscopy evaluation revealed a significant increase in the apoptotic index of these cells after BatroxLAAO treatment. This observation was confirmed by phosphatidyl serine exposure and activation of caspases. BatroxLAAO is a protein with various biological functions that can be involved in envenomation. Further investigations of its function will contribute to toxicology advances. PMID:18804547

  2. p38 MAPK is involved in human neutrophil chemotaxis induced by L-amino acid oxidase from Calloselasma rhodosthoma.

    PubMed

    Pontes, Adriana S; Setúbal, Sulamita da S; Nery, Neriane Monteiro; da Silva, Francisquinha Souza; da Silva, Silvana D; Fernandes, Carla F C; Stábeli, Rodrigo G; Soares, Andreimar M; Zuliani, Juliana P

    2016-09-01

    The action of LAAO, an L-amino acid oxidase isolated from Calloselasma rhodosthoma snake venom, on isolated human neutrophil function was investigated. Cr-LAAO showed no toxicity on neutrophils. Cr-LAAO in its native form induced the neutrophil chemotaxis, suggesting that its primary structure is essential for stimulation the cell. p38 MAPK and PI3K have a role as signaling pathways of CR-LAAO induced chemotaxis. This toxin also induced the production of hydrogen peroxide and stimulated phagocytosis in neutrophils. Furthermore, Cr-LAAO was able to stimulate neutrophils to release IL-6, IL-8, MPO, LTB4 and PGE2. Together, the data showed that the Cr-LAAO triggers relevant proinflammatory events.

  3. Cloning, characterization and mutagenesis of Russell's viper venom L-amino acid oxidase: Insights into its catalytic mechanism.

    PubMed

    Chen, Hong-Sen; Wang, Ying-Ming; Huang, Wan-Ting; Huang, Kai-Fa; Tsai, Inn-Ho

    2012-02-01

    To investigate the structure-function relationships and geographic variations of L-amino acid oxidase (LAAO) from Daboia venoms, a single LAAO (designated as DrLAO) was purified from eastern Indian Daboia russelii venom and characterized. The purified DrLAO showed subunit molecular mass of 60-64kDa; its N-terminal sequence (1-20) was identical to those of several true viper LAAOs. Its preferred substrates were hydrophobic l-amino acids and the kinetic specificities were ordered as follows: Phe, Tyr, Met, Leu, and Trp. Enzyme assay and Western blotting showed that the venom LAAO contents of D. russelii were higher than those of Daboia siamensis. DrLAO dose-dependently inhibited ADP- and collagen-induced platelet aggregation with IC(50) values of 0.27 and 0.82μM, respectively. Apparently, DrLAO may synergize with other venom components to prolong and enhance bleeding symptoms after Daboia envenoming. The full sequence of DrLAO was deduced from its cDNA sequence and then confirmed by peptide mass fingerprinting. Molecular phylogenetic analysis revealed that SV-LAAO family members could be differentiated not only by snake taxonomy but also by the variations at position 223, and they divided into H223, S223, N223, and D223 subclasses. We have further prepared recombinant DrLAO and mutants by the Pichia expression system. Mutagenic analyses of DrLAO His223 revealed that this residue bound substrates instead of serving as an essential base in the catalytic steps. Our results suggest a direct hydride transfer from substrate to FAD as the mechanism for SV-LAAOs. PMID:21802487

  4. Antibacterial efficacy of recombinant Siganus oramin L-amino acid oxidase expressed in Pichia pastoris.

    PubMed

    Li, Ruijun; Li, Anxing

    2014-12-01

    Siganus oraminl-amino acid oxidase is a novel natural protein (named SR-LAAO) isolated from serum of the rabbitfish (S. oramin), which showed antibacterial activity against both Gram-positive and Gram-negative bacteria and had a lethal effect on the parasites Cryptocaryon irritans, Trypanosoma brucei brucei and Ichthyophthirius multifiliis. In order to test whether recombinant SR-LAAO (rSR-LAAO) produced by the eukaryotic expression system also has antimicrobial activity, the yeast Pichia pastoris was used as the expression host to obtain rSR-LAAO in vitro. Crude rSR-LAAO produced by P. pastoris integrated with the SR-LAAO gene had antibacterial activity against both Gram-positive and Gram-negative bacteria as shown by inhibition zone assay of the antibacterial spectrum on agar plates. The average diameter of the inhibition zone of crude rSR-LAAO against the Gram-positive bacteria Staphylococcus aureus and Streptococcus agalactiae was 1.040 ± 0.045 cm and 1.209 ± 0.085 cm, respectively. For the Gram-negative bacteria Aeromonas sobria, Escherichia coli, Vibrio alginolyticus, Vibrio cholera and Photobacterium damselae subsp. piscicida, the average diameter of inhibition zone was 1.291 ± 0.089 cm, 0.943 ± 0.061 cm, 0.756 ± 0.057 cm, 0.834 ± 0.023 cm and 1.211 ± 0.026 cm, respectively. These results were obtained at the logarithmic growth phase of S. agalactiae and A. sobria cell suspensions after incubation with 0.5 mg/mL crude rSR-LAAO for 24 h. The final bacterial growth rate was decreased significantly. The relative inhibition rate can reach 50% compared to crude products from P. pastoris integrated with an empty vector at the same concentration of protein. The antimicrobial activity of crude rSR-LAAO was likely associated with H2O2 formation, because its inhibition zones were disturbed significantly by catalase. Scanning electron microscopy results showed crude rSR-LAAO-treated bacterial surfaces became rough and particles were attached, cell walls were

  5. Antibacterial efficacy of recombinant Siganus oramin L-amino acid oxidase expressed in Pichia pastoris.

    PubMed

    Li, Ruijun; Li, Anxing

    2014-12-01

    Siganus oraminl-amino acid oxidase is a novel natural protein (named SR-LAAO) isolated from serum of the rabbitfish (S. oramin), which showed antibacterial activity against both Gram-positive and Gram-negative bacteria and had a lethal effect on the parasites Cryptocaryon irritans, Trypanosoma brucei brucei and Ichthyophthirius multifiliis. In order to test whether recombinant SR-LAAO (rSR-LAAO) produced by the eukaryotic expression system also has antimicrobial activity, the yeast Pichia pastoris was used as the expression host to obtain rSR-LAAO in vitro. Crude rSR-LAAO produced by P. pastoris integrated with the SR-LAAO gene had antibacterial activity against both Gram-positive and Gram-negative bacteria as shown by inhibition zone assay of the antibacterial spectrum on agar plates. The average diameter of the inhibition zone of crude rSR-LAAO against the Gram-positive bacteria Staphylococcus aureus and Streptococcus agalactiae was 1.040 ± 0.045 cm and 1.209 ± 0.085 cm, respectively. For the Gram-negative bacteria Aeromonas sobria, Escherichia coli, Vibrio alginolyticus, Vibrio cholera and Photobacterium damselae subsp. piscicida, the average diameter of inhibition zone was 1.291 ± 0.089 cm, 0.943 ± 0.061 cm, 0.756 ± 0.057 cm, 0.834 ± 0.023 cm and 1.211 ± 0.026 cm, respectively. These results were obtained at the logarithmic growth phase of S. agalactiae and A. sobria cell suspensions after incubation with 0.5 mg/mL crude rSR-LAAO for 24 h. The final bacterial growth rate was decreased significantly. The relative inhibition rate can reach 50% compared to crude products from P. pastoris integrated with an empty vector at the same concentration of protein. The antimicrobial activity of crude rSR-LAAO was likely associated with H2O2 formation, because its inhibition zones were disturbed significantly by catalase. Scanning electron microscopy results showed crude rSR-LAAO-treated bacterial surfaces became rough and particles were attached, cell walls were

  6. Exploring Regulation Genes Involved in the Expression of L-Amino Acid Oxidase in Pseudoalteromonas sp. Rf-1

    PubMed Central

    Wang, Ju; Lin, Jianxun; Zhao, Minyan

    2015-01-01

    Bacterial L-amino acid oxidase (LAAO) is believed to play important biological and ecological roles in marine niches, thus attracting increasing attention to understand the regulation mechanisms underlying its production. In this study, we investigated genes involved in LAAO production in marine bacterium Pseudoalteromonas sp. Rf-1 using transposon mutagenesis. Of more than 4,000 mutants screened, 15 mutants showed significant changes in LAAO activity. Desired transposon insertion was confirmed in 12 mutants, in which disrupted genes and corresponding functionswere identified. Analysis of LAAO activity and lao gene expression revealed that GntR family transcriptional regulator, methylase, non-ribosomal peptide synthetase, TonB-dependent heme-receptor family, Na+/H+ antiporter and related arsenite permease, N-acetyltransferase GCN5, Ketol-acid reductoisomerase and SAM-dependent methytransferase, and their coding genes may be involved in either upregulation or downregulation pathway at transcriptional, posttranscriptional, translational and/or posttranslational level. The nhaD and sdmT genes were separately complemented into the corresponding mutants with abolished LAAO-activity. The complementation of either gene can restore LAAO activity and lao gene expression, demonstrating their regulatory role in LAAO biosynthesis. This study provides, for the first time, insights into the molecular mechanisms regulating LAAO production in Pseudoalteromonas sp. Rf-1, which is important to better understand biological and ecological roles of LAAO. PMID:25815733

  7. Lactogenic hormone stimulation and epigenetic control of L-amino acid oxidase expression in lactating mammary glands.

    PubMed

    Fujii, Kazuki; Zhang, Haolin; Usuda, Kento; Watanabe, Gen; Nagaoka, Kentaro

    2015-11-01

    L-amino acid oxidase (LAO), a classic flavoprotein, shows antibacterial activity by producing hydrogen peroxide. LAO exists in many tissues such as salivary gland, thymus, spleen, small intestine and testis. In particular, LAO was highly expressed in mice milk and plays an important factor in innate immunity of mammary glands. However, the mechanism which LAO expression is regulated spatially and temporally in lactating mammary glands has been unclear. In this study, we showed the contribution of lactogenic hormone and epigenetic control on LAO gene expression. In monolayer of mammary epithelial cells, treatment of lactogenic hormone mixture, dexamethasone, insulin and prolactin, did not induce LAO mRNA expression and its promoter activity, even though one of milk protein β-casein expression was stimulated. However, increase of LAO expression was observed when the cells were treated with lactogenic hormones in a 3-dimensional culture. The results of chromatin immunoprecipitation analysis revealed that histone H3K18 acetylation increased and histone H3K27 tri-methylation decreased with lactation, which is associated with a period of high LAO expression. Moreover, the treatment of histone methylation inhibitor (DZNep) as well as histone deacetylation inhibitor (Trichostatine A) induced LAO expression in monolayer of mammary cells. Taken together, this is the first demonstration showing that LAO expression is induced in cell culture, and stimulation of lactogenic hormone and change of histone modification are promising signals to show highly expression of LAO in lactating mammary glands.

  8. Human spermatozoa possess an IL4I1 l-amino acid oxidase with a potential role in sperm function.

    PubMed

    Houston, B; Curry, B; Aitken, R J

    2015-06-01

    Reactive oxygen species (ROS) are known to play an important role in the regulation of human sperm function. In this study, we demonstrate for the first time that human spermatozoa possess interleukin-induced gene 1 (IL4I1), an l-amino acid oxidase (LAAO) which is capable of generating ROS on exposure to aromatic amino acids in the presence of oxygen. The preferred substrates were found to be phenylalanine and tryptophan while the enzyme was located in the acrosomal region and midpiece of these cells. In contrast to equine and bovine spermatozoa, enzyme activity was lost as soon as the spermatozoa became non-viable. On a cell-to-cell basis human spermatozoa were also shown to generate lower levels of hydrogen peroxide than their equine counterparts on exposure to phenylalanine. Stimulation of LAAO activity resulted in the induction of several hallmarks of capacitation including tyrosine phosphorylation of the sperm flagellum and concomitant activation of phospho-SRC expression. In addition, stimulation of LAAO resulted in an increase in the levels of acrosomal exocytosis in both the presence and absence of progesterone stimulation, via mechanisms that could be significantly reversed by the presence of catalase. As is often the case with free radical-mediated phenomena, prolonged exposure of human spermatozoa to phenylalanine resulted in the stimulation of apoptosis as indicated by significant increases in mitochondrial superoxide generation and the activation of intracellular caspases. These results confirm the existence of an LAAO in human spermatozoa with a potential role in driving the redox regulation of sperm capacitation and acrosomal exocytosis.

  9. Recombinant production and evaluation of an antibacterial L-amino acid oxidase derived from flounder Platichthys stellatus.

    PubMed

    Kasai, Kosuke; Hashiguchi, Kenro; Takahashi, Hiroki; Kasai, Ayano; Takeda, Sadanori; Nakano, Manabu; Ishikawa, Takashi; Nakamura, Toshiya; Miura, Tomisato

    2015-08-01

    Fish produce mucus substances as a defensive outer barrier against several bacterial infections. We have recently identified an antibacterial L-amino acid oxidase (psLAAO1) in the mucus layer of the flounder Platichthys stellate. In this study, the antibacterial protein psLAAO1 was expressed as a secretory bioactive recombinant protein in the methylotrophic yeast Pichia pastoris. The recombinant psLAAO1 inhibited the growth of bacteria to the same levels as native psLAAO1 present in mucus. In particular, Staphylococci and Yersinia were strongly suppressed, showing the highest growth retardation of the 21 species and strains tested. Moreover, Staphylococcus epidermidis was most sensitive to psLAAO1 with a minimum inhibitory concentration (MIC) of 0.078 μg/mL, whereas Escherichia coli was essentially resistant to psLAAO1 with a MIC of >10 μg/mL. Interestingly, psLAAO1-treated E. coli were found to upregulate the expression of the btuE gene, which encodes glutathione peroxidase (GPx). The biochemical function of GPx is to reduce free hydrogen peroxide and is induced under response to reactive oxygen species (ROS). Thus, E. coli confers resistance to the reduced free hydrogen peroxide produced by psLAAO1 by increasing GPx levels. Furthermore, the growth of Staphylococcus aureus was completely inhibited in the presence of recombinant psLAAO1. The morphology of psLAAO1-treated S. aureus showed cell surface damage, the formation of large aggregates and the cells showed severe deformations. Western blot analysis showed that psLAAO1 binds to the surface of S. aureus. Therefore, psLAAO1 binds to the surface of LAAO-sensitive S. aureus and directs peroxidative activity at the surface of the bacterial membrane.

  10. 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. PMID:25615711

  11. 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.

  12. Cytotoxic L-amino-acid oxidases from Amanita phalloides and Clitocybe geotropa induce caspase-dependent apoptosis

    PubMed Central

    Pišlar, A; Sabotič, J; Šlenc, J; Brzin, J; Kos, J

    2016-01-01

    L-amino-acid oxidases (LAO) purified from fungi induce cell death in various mammalian cells including human tumor cell lines. The mechanism, however, remains poorly understood. In this study, we aimed to define a precise mechanism of cell death induced in Jurkat and MCF7 cancer cell lines by ApLAO and CgLAO, LAOs isolated from Amanita phalloides and Clitocybe geotropa, respectively. Cell death induced by both LAOs is shown to be concentration- and time-dependent, with higher toxic effects in Jurkat cells. LAO activity is required for the cytotoxicity. Detailed study on Jurkat cells further demonstrated that ApLAO and CgLAO both induce the intrinsic mitochondrial pathway of apoptosis, accompanied by a time-dependent depolarization of the mitochondrial membrane through the generation of reactive oxygen species. Treatment with the LAOs resulted in an increased ratio of the expression of proapoptotic Bax to that of antiapoptotic Bcl-2, subsequently leading to the activation of caspase-9 and -3. However, the pancaspase inhibitor, Z-VAD-FMK, did not completely abolish the cell death induced by either ApLAO or CgLAO, suggesting an alternative pathway for LAO-induced apoptosis. Indeed, caspase-8 activity in ApLAO- and CgLAO-treated cells was increased. Further, Fas/FasL (Fas ligand) antagonist caused a slight reduction in toxin-induced cell death, supporting the involvement of ApLAO and CgLAO in death-receptor-mediated apoptosis. These results thus provide new evidence that ApLAO and CgLAO induce apoptosis in Jurkat cells via both the intrinsic and extrinsic pathways, although the significantly higher increase of caspase-9 over caspase-8 activity suggests that it is the intrinsic pathway that is the predominant mode of ApLAO- and CgLAO-induced apoptosis. PMID:27551514

  13. Cell cycle arrest evidence, parasiticidal and bactericidal properties induced by L-amino acid oxidase from Bothrops atrox snake venom.

    PubMed

    de Melo Alves Paiva, Raquel; de Freitas Figueiredo, Raquel; Antonucci, Gilmara Ausech; Paiva, Helder Henrique; de Lourdes Pires Bianchi, Maria; Rodrigues, Kelly C; Lucarini, Rodrigo; Caetano, Renato Cesar; Linhari Rodrigues Pietro, Rosemeire Cristina; Gomes Martins, Carlos Henrique; de Albuquerque, Sérgio; Sampaio, Suely Vilela

    2011-05-01

    The present article describes an l-amino acid oxidase from Bothrops atrox snake venom as with antiprotozoal activities in Trypanosoma cruzi and in different species of Leishmania (Leishmania braziliensis, Leishmania donovani and Leishmania major). Leishmanicidal effects were inhibited by catalase, suggesting that they are mediated by H(2)O(2) production. Leishmania spp. cause a spectrum of diseases, ranging from self-healing ulcers to disseminated and often fatal infections, depending on the species involved and the host's immune response. BatroxLAAO also displays bactericidal activity against both Gram-positive and Gram-negative bacteria. The apoptosis induced by BatroxLAAO on HL-60 cell lines and PBMC cells was determined by morphological cell evaluation using a mix of fluorescent dyes. As revealed by flow cytometry analysis, suppression of cell proliferation with BatroxLAAO was accompanied by the significant accumulation of cells in the G0/G1 phase boundary in HL-60 cells. BatroxLAAO at 25 μg/mL and 50 μg/mL blocked G0-G1 transition, resulting in G0/G1 phase cell cycle arrest, thereby delaying the progression of cells through S and G2/M phase in HL-60 cells. This was shown by an accentuated decrease in the proportion of cells in S phase, and the almost absence of G2/M phase cell population. BatroxLAAO is an interesting enzyme that provides a better understanding of the ophidian envenomation mechanism, and has biotechnological potential as a model for therapeutic agents. PMID:21300133

  14. Cytotoxic L-amino-acid oxidases from Amanita phalloides and Clitocybe geotropa induce caspase-dependent apoptosis.

    PubMed

    Pišlar, A; Sabotič, J; Šlenc, J; Brzin, J; Kos, J

    2016-01-01

    L-amino-acid oxidases (LAO) purified from fungi induce cell death in various mammalian cells including human tumor cell lines. The mechanism, however, remains poorly understood. In this study, we aimed to define a precise mechanism of cell death induced in Jurkat and MCF7 cancer cell lines by ApLAO and CgLAO, LAOs isolated from Amanita phalloides and Clitocybe geotropa, respectively. Cell death induced by both LAOs is shown to be concentration- and time-dependent, with higher toxic effects in Jurkat cells. LAO activity is required for the cytotoxicity. Detailed study on Jurkat cells further demonstrated that ApLAO and CgLAO both induce the intrinsic mitochondrial pathway of apoptosis, accompanied by a time-dependent depolarization of the mitochondrial membrane through the generation of reactive oxygen species. Treatment with the LAOs resulted in an increased ratio of the expression of proapoptotic Bax to that of antiapoptotic Bcl-2, subsequently leading to the activation of caspase-9 and -3. However, the pancaspase inhibitor, Z-VAD-FMK, did not completely abolish the cell death induced by either ApLAO or CgLAO, suggesting an alternative pathway for LAO-induced apoptosis. Indeed, caspase-8 activity in ApLAO- and CgLAO-treated cells was increased. Further, Fas/FasL (Fas ligand) antagonist caused a slight reduction in toxin-induced cell death, supporting the involvement of ApLAO and CgLAO in death-receptor-mediated apoptosis. These results thus provide new evidence that ApLAO and CgLAO induce apoptosis in Jurkat cells via both the intrinsic and extrinsic pathways, although the significantly higher increase of caspase-9 over caspase-8 activity suggests that it is the intrinsic pathway that is the predominant mode of ApLAO- and CgLAO-induced apoptosis. PMID:27551514

  15. Recombinant production and evaluation of an antibacterial L-amino acid oxidase derived from flounder Platichthys stellatus.

    PubMed

    Kasai, Kosuke; Hashiguchi, Kenro; Takahashi, Hiroki; Kasai, Ayano; Takeda, Sadanori; Nakano, Manabu; Ishikawa, Takashi; Nakamura, Toshiya; Miura, Tomisato

    2015-08-01

    Fish produce mucus substances as a defensive outer barrier against several bacterial infections. We have recently identified an antibacterial L-amino acid oxidase (psLAAO1) in the mucus layer of the flounder Platichthys stellate. In this study, the antibacterial protein psLAAO1 was expressed as a secretory bioactive recombinant protein in the methylotrophic yeast Pichia pastoris. The recombinant psLAAO1 inhibited the growth of bacteria to the same levels as native psLAAO1 present in mucus. In particular, Staphylococci and Yersinia were strongly suppressed, showing the highest growth retardation of the 21 species and strains tested. Moreover, Staphylococcus epidermidis was most sensitive to psLAAO1 with a minimum inhibitory concentration (MIC) of 0.078 μg/mL, whereas Escherichia coli was essentially resistant to psLAAO1 with a MIC of >10 μg/mL. Interestingly, psLAAO1-treated E. coli were found to upregulate the expression of the btuE gene, which encodes glutathione peroxidase (GPx). The biochemical function of GPx is to reduce free hydrogen peroxide and is induced under response to reactive oxygen species (ROS). Thus, E. coli confers resistance to the reduced free hydrogen peroxide produced by psLAAO1 by increasing GPx levels. Furthermore, the growth of Staphylococcus aureus was completely inhibited in the presence of recombinant psLAAO1. The morphology of psLAAO1-treated S. aureus showed cell surface damage, the formation of large aggregates and the cells showed severe deformations. Western blot analysis showed that psLAAO1 binds to the surface of S. aureus. Therefore, psLAAO1 binds to the surface of LAAO-sensitive S. aureus and directs peroxidative activity at the surface of the bacterial membrane. PMID:25661816

  16. SO-LAAO, a novel L-amino acid oxidase that enables Streptococcus oligofermentans to outcompete Streptococcus mutans by generating H2O2 from peptone.

    PubMed

    Tong, Huichun; Chen, Wei; Shi, Wenyuan; Qi, Fengxia; Dong, Xiuzhu

    2008-07-01

    We previously demonstrated that Streptococcus oligofermentans suppressed the growth of Streptococcus mutans, the primary cariogenic pathogen, by producing hydrogen peroxide (H(2)O(2)) through lactate oxidase activity. In this study, we found that the lox mutant of S. oligofermentans regained the inhibition while growing on peptone-rich plates. Further studies demonstrated that the H(2)O(2) produced on peptone by S. oligofermentans was mainly derived from seven L-amino acids, i.e., L-aspartic acid, L-tryptophan, L-lysine, L-isoleucine, L-arginine, L-asparagine, and L-glutamine, indicating the possible existence of L-amino acid oxidase (LAAO) that can produce H(2)O(2) from L-amino acids. Through searching the S. oligofermentans genome for open reading frames with a conserved flavin adenine dinucleotide binding motif that exists in the known LAAOs, including those of snake venom, fungi, and bacteria, a putative LAAO gene, assigned as aao(So), was cloned and overexpressed in Escherichia coli. The purified protein, SO-LAAO, showed a molecular mass of 43 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and catalyzed H(2)O(2) formation from the seven L-amino acids determined above, thus confirming its LAAO activity. The SO-LAAO identified in S. oligofermentans differed evidently from the known LAAOs in both substrate profile and sequence, suggesting that it could represent a novel LAAO. An aao(So) mutant of S. oligofermentans did lose H(2)O(2) formation from the seven L-amino acids, further verifying its function as an LAAO. Furthermore, the inhibition by S. oligofermentans of S. mutans in a peptone-rich mixed-species biofilm was greatly reduced for the aao(So) mutant, indicating the gene's importance in interspecies competition. PMID:18469105

  17. Evaluation of an Antimicrobial L-Amino Acid Oxidase and Peptide Derivatives from Bothropoides mattogrosensis Pitviper Venom

    PubMed Central

    Gomes, Diego G.; Porto, William F.; Batista, Carla L.; Ramos, Carmel S.; Holanda, Hortência H. S.; Dias, Simoni C.; Franco, Octavio L.; Moreno, Susana E.

    2012-01-01

    Healthcare-associated infections (HAIs) are causes of mortality and morbidity worldwide. The prevalence of bacterial resistance to common antibiotics has increased in recent years, highlighting the need to develop novel alternatives for controlling these pathogens. Pitviper venoms are composed of a multifaceted mixture of peptides, proteins and inorganic components. L-amino oxidase (LAO) is a multifunctional enzyme that is able to develop different activities including antibacterial activity. In this study a novel LAO from Bothrops mattogrosensis (BmLAO) was isolated and biochemically characterized. Partial enzyme sequence showed full identity to Bothrops pauloensis LAO. Moreover, LAO here isolated showed remarkable antibacterial activity against Gram-positive and -negative bacteria, clearly suggesting a secondary protective function. Otherwise, no cytotoxic activities against macrophages and erythrocytes were observed. Finally, some LAO fragments (BmLAO-f1, BmLAO-f2 and BmLAO-f3) were synthesized and further evaluated, also showing enhanced antimicrobial activity. Peptide fragments, which are the key residues involved in antimicrobial activity, were also structurally studied by using theoretical models. The fragments reported here may be promising candidates in the rational design of new antibiotics that could be used to control resistant microorganisms. PMID:22438972

  18. Quantitation of multiple pathways for the metabolism of nephrotoxic cysteine conjugates using selective inhibitors of L-alpha-hydroxy acid oxidase (L-amino acid oxidase) and cysteine conjugate beta-lyase

    SciTech Connect

    Stevens, J.L.; Hatzinger, P.B.; Hayden, P.J. )

    1989-05-01

    In this study, we have established the selectivity of inhibitors for rat kidney cysteine conjugate beta-lyase and L-alpha-hydroxy acid oxidase (L-amino acid oxidase) and have used these inhibitors to explore the relative roles of these two enzymes in the metabolism of nephrotoxic cysteine conjugates by rat kidney homogenate. In addition, we have investigated the relationship between structure and the metabolism of toxic cysteine conjugates by purified rat kidney L-alpha-hydroxy acid oxidase. With purified enzyme, S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine (PCBDC) was about four times more active than S(1,2-dichlorovinyl)-L-cysteine (DCVC). Three alkyl conjugates were less active than DCVC. Purified L-alpha-hydroxy acid oxidase was not inhibited by the beta-lyase inhibitor aminooxyacetic acid but was inactivated by 2-hydroxy-3-butynoate. PCBDC metabolism in rat kidney homogenate was inhibited 74% by aminooxyacetic acid and 42% by 2-hydroxy-3-butynoate, whereas DCVC metabolism was inhibited 77% by aminooxyacetic acid and 28% by 2-hydroxy-3-butynoate. However, only aminooxyacetic acid inhibited the binding of {sup 35}S label from ({sup 35}S)DCVC. Based on these results we have reached three conclusions. First, L-alpha-hydroxy acid oxidase plays a significant role in the metabolism of some cysteine conjugates. Second, metabolism of DCVC by L-alpha-hydroxy acid oxidase does not contribute directly to covalent binding. Third, as much as 65% of DCVC may be metabolized to its corresponding alpha-keto acid. The results are discussed with regard to the nephrotoxicity of cysteine conjugates.

  19. The L-amino acid oxidase from Calloselasma rhodostoma snake venom modulates apoptomiRs expression in Bcr-Abl-positive cell lines.

    PubMed

    Burin, Sandra Mara; Berzoti-Coelho, Maria Gabriela; Cominal, Juçara Gastaldi; Ambrosio, Luciana; Torqueti, Maria Regina; Sampaio, Suely Vilela; de Castro, Fabíola Attié

    2016-09-15

    Anti-apoptotic genes and apoptomiRs deregulated expression contribute to apoptosis resistance in chronic myeloid leukemia (CML) Bcr-Abl(+) cells. Here, the L-amino acid oxidase from Calloselasma rhodostoma (CR-LAAO) venom altered the apoptotic machinery regulation by modulating the expression of the miR-145, miR-26a, miR-142-3p, miR-21, miR-130a, and miR-146a, and of the apoptosis-related proteins Bid, Bim, Bcl-2, Ciap-2, c-Flip, and Mcl-1 in Bcr-Abl(+) cells. CR-LAAO is a potential tool to instigate apoptomiRs regulation that contributes to drive CML therapy. PMID:27421670

  20. Structural and functional properties of Bp-LAAO, a new L-amino acid oxidase isolated from Bothrops pauloensis snake venom.

    PubMed

    Rodrigues, Renata S; da Silva, Juliana F; Boldrini França, Johara; Fonseca, Fernando P P; Otaviano, Antônio R; Henrique Silva, Flávio; Hamaguchi, Amélia; Magro, Angelo J; Braz, Antônio Sérgio K; dos Santos, Juliana I; Homsi-Brandeburgo, Maria Inês; Fontes, Marcos R M; Fuly, André L; Soares, Andreimar M; Rodrigues, Veridiana M

    2009-04-01

    An L-amino acid oxidase (Bp-LAAO) from Bothrops pauloensis snake venom was highly purified using sequential chromatography steps on CM-Sepharose, Phenyl-Sepharose CL-4B, Benzamidine Sepharose and C18 reverse-phase HPLC. Purified Bp-LAAO showed to be a homodimeric acidic glycoprotein with molecular weight around 65kDa under reducing conditions in SDS-PAGE. The best substrates for Bp-LAAO were L-Met, L-Leu, L-Phe and L-Ile and the enzyme showed a strong reduction of its catalytic activity upon L-Met and L-Phe substrates at extreme temperatures. Bp-LAAO showed leishmanicidal, antitumoral and bactericidal activities dose dependently. Bp-LAAO induced platelet aggregation in platelet-rich plasma and this activity was inhibited by catalase. Bp-LAAO-cDNA of 1548bp codified a mature protein with 516 amino acid residues corresponding to a theoretical isoelectric point and molecular weight of 6.3 and 58kDa, respectively. Additionally, structural and phylogenetic studies identified residues under positive selection and their probable location in Bp-LAAO and other snake venom LAAOs (svLAAOs). Structural and functional investigations of these enzymes can contribute to the advancement of toxinology and to the elaboration of novel therapeutic agents. PMID:19135502

  1. Genetics Home Reference: aromatic l-amino acid decarboxylase deficiency

    MedlinePlus

    ... aromatic l-amino acid decarboxylase deficiency aromatic l-amino acid decarboxylase deficiency Enable Javascript to view the expand/ ... PDF Open All Close All Description Aromatic l-amino acid decarboxylase (AADC) deficiency is an inherited disorder that ...

  2. 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. PMID:21059402

  3. 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.

  4. The serum of rabbitfish (Siganus oramin) has antimicrobial activity to some pathogenic organisms and a novel serum L-amino acid oxidase is isolated.

    PubMed

    Wang, Fanghua; Li, Ruijun; Xie, Mingquan; Li, Anxing

    2011-01-01

    The serum of rabbitfish (Siganus oramin) has been confirmed previously to have killing effect to Cryptocaryon irritans, an important marine ciliate protozoan that causes a disease referred to as "marine white spot disease". Herein, we find the serum of the rabbitfish also shows antibacterial activity against both gram-positive and gram-negative bacteria and has killing effect on two other parasites: Trypanosoma brucei brucei, Ichthyophthirius multifiliis. Results of scanning electron microscopy indicated that after treating with rabbitfish serum, the surface of the Staphylococcus aureus was wrinkled and pores were formed on the surface of Escherichia coli. Serum of the rabbitfish possesses a strong killing effect to Ichthyophthirius multifiliis in vitro, causing a similar effect as to C. irritans. The serum of rabbitfish also showed strong killing effect to T. b. brucei in vitro, with the minimus trypanocidal titre (MTT) only to be 1.5% in 1 h. Results of laser confocal fluorescence microscopy indicated that rabbitfish serum could also induce cell rupture of T. b. brucei. A novel antimicrobial protein (SR-LAAO) was isolated from the serum of rabbitfish by using ultrafiltration, reversed phase high performance liquid chromatography (RP-HPLC) and Native polyacrylamide gel electrophoresis (Native-PAGE). Results of gel overlay assay showed that the protein could act alone to inhibit the growth of S. aureus and E. coli. Results of western blot and automated Edman degradation showed that it was the same as the antiparasitic protein (APP) reported before to have killing effect on C. irritans. Full length cDNA sequence of the SR-LAAO was cloned. BLAST research suggested that the cDNA of SR-LAAO has a close similarity with a number of L-amino acid oxidases (LAAOs) and possesses two conserved motifs that exist in LAAOs. Combined, these results demonstrate that this protein which has antimicrobial activity to some pathogenic organisms was a novel LAAO found in the serum of

  5. Isolation, characterization and screening of the in vitro cytotoxic activity of a novel L-amino acid oxidase (LAAOcdt) from Crotalus durissus terrificus venom on human cancer cell lines.

    PubMed

    Teixeira, Tuila Leveghim; Oliveira Silva, Viviane Aline; da Cunha, Daniel Batista; Polettini, Flávia Lino; Thomaz, Camila Daniele; Pianca, Ariana Aparecida; Zambom, Fabiana Letícia; da Silva Leitão Mazzi, Denise Pimenta; Reis, Rui Manuel; Mazzi, Maurício Ventura

    2016-09-01

    An L-amino acid oxidase (LAAOcdt) from Crotalus durissus terrificus venom was purified to homogeneity in a two-step procedure using molecular exclusion on Sephadex G-75, followed by Phenyl Sepharose FF chromatography. The molecular mass of the purified enzyme was 113 kDa, as determined by SDS-PAGE under reducing conditions. LAAOcdt showed amino acid homology to other L-amino acid oxidases isolated from different snake venoms. The comparative analysis of the internal peptide sequences of the NNPGILEYPVKPSEEGK fragments by LC-MS/MS spectrometry revealed 100% identity with C. durissus cumanensis LAAO. The purified protein catalyzed the oxidative deamination of L-amino acids, and the most specific substrates were L-Tyr and L-Phe. The enzyme presented optimum activity at pH 7.4 and at 44 °C. LAAOcdt also showed hemolytic activity (0.6-20 μg/μL) and induced both the formation plasma clots (5-100 μg/μL) and platelet aggregation (2.5 × 10(-3), 5.0 × 10(-3) and 10 × 10(-3) μg/mL), as well as bactericidal activity (2.5-10 μg/μL) against Staphylococcus aureus. Moreover, LAAOcdt exhibited cytotoxicity in distinct cancer cell lines, which presented a heterogeneous response profile. The mean IC50 value was 10.5 μg/mL. Glioma and pancreatic carcinoma cells were the most sensitive cell lines; they showed mean IC50 values of 7.2 μg/mL and 7.4 μg/mL, respectively. The exposure of the drug-sensitive cells to LAAOcdt for 24 h upregulated activated p-H2AX and efficiently decreased P42/P44 (ERK) activation in glioma cells (HCB151), which suggested an anti-proliferative effect. In addition, increased p21 expression was observed in SiHa cells, which showed a resistant phenotype. On the other hand, the flow cytometry and immunoblotting analyses showed that the enzyme did not induce cancer cell apoptosis. These results suggest that another cell death mechanism might contribute to the LAAOcdt-induced cytotoxicity. Taken together, this work may help to elucidate

  6. An investigation into the antigenic cross-reactivity of Ophiophagus hannah (king cobra) venom neurotoxin, phospholipase A2, hemorrhagin and L-amino acid oxidase using enzyme-linked immunosorbent assay.

    PubMed

    Tan, N H; Lim, K K; Jaafar, M I

    1993-07-01

    The antigenic cross-reactivity of four Ophiophagus hannah (king cobra) venom components, the neurotoxin (OH-NTX), phospholipase A2 (OH-PLA2), hemorrhagin (OH-HMG) and L-amino acid oxidase (OH-LAAO) were examined by indirect and double sandwich ELISAs. The indirect ELISAs for OH-NTX, OH-PLA2 and OH-HMG were very specific when assayed against the various heterologous snake venoms and O. hannah venom components, at 25 ng/ml antigen level. At higher antigen concentrations (100-400 ng/ml), there were moderate to strong indirect ELISA cross-reactions between anti-O. hannah neurotoxin and venoms from various species of cobra as well as two short neurotoxins. However, anti-O. hannah hemorrhagin did not cross-react with any of the venoms tested, even at these high antigen concentrations, indicating that O. hannah hemorrhagin is antigenically very different from other venom hemorrhagins. Examination of the indirect ELISA cross-reactions between anti-O. hannah PLA2 and several elapid PLA2 enzymes suggests that the elapid PLA2 antigenic class has more than two subgroups. The antibodies to O. hannah L-amino acid oxidase, however, yielded indirect ELISA cross-reactions with many venoms as well as with OH-NTX, OH-PLA2 and OH-HMG, indicating that OH-LAAO shares common epitopes even with unrelated proteins. The double sandwich ELISAs for the four anti-O. hannah venom components, on the other hand, generally exhibited a higher degree of selectivity than the indirect ELISA procedure.

  7. King cobra (Ophiophagus hannah) venom L-amino acid oxidase induces apoptosis in PC-3 cells and suppresses PC-3 solid tumor growth in a tumor xenograft mouse model.

    PubMed

    Lee, Mui Li; Fung, Shin Yee; Chung, Ivy; Pailoor, Jayalakshmi; Cheah, Swee Hung; Tan, Nget Hong

    2014-01-01

    King cobra (Ophiophagus hannah) venom L-amino acid oxidase (OH-LAAO), a heat stable enzyme, has been shown to exhibit very potent anti-proliferative activity against human breast and lung tumorigenic cells but not in their non-tumorigenic counterparts. We further examine its in vitro and in vivo anti-tumor activity in a human prostate adenocarcinoma (PC-3) model. OH-LAAO demonstrated potent cytotoxicity against PC-3 cells with IC50 of 0.05 µg/mL after 72 h incubation in vitro. It induced apoptosis as evidenced with an increase in caspase-3/7 cleavages and an increase in annexin V-stained cells. To examine its in vivo anti-tumor activity, we treated PC-3 tumor xenograft implanted subcutaneously in immunodeficient NU/NU (nude) mice with 1 µg/g OH-LAAO given intraperitoneally (i.p.). After 8 weeks of treatment, OH-LAAO treated PC-3 tumors were markedly inhibited, when compared to the control group (P <0.05). TUNEL staining analysis on the tumor sections showed a significantly increase of apoptotic cells in the LAAO-treated animals. Histological examinations of the vital organs in these two groups showed no significant differences with normal tissues, indicating no obvious tissue damage. The treatment also did not cause any significant changes on the body weight of the mice during the duration of the study. These observations suggest that OH-LAAO cytotoxic effects may be specific to tumor xenografts and less to normal organs. Given its potent anti-tumor activities shown in vitro as well as in vivo, the king cobra venom LAAO can potentially be developed to treat prostate cancer and other solid tumors. PMID:24782648

  8. King cobra (Ophiophagus hannah) venom L-amino acid oxidase induces apoptosis in PC-3 cells and suppresses PC-3 solid tumor growth in a tumor xenograft mouse model.

    PubMed

    Lee, Mui Li; Fung, Shin Yee; Chung, Ivy; Pailoor, Jayalakshmi; Cheah, Swee Hung; Tan, Nget Hong

    2014-01-01

    King cobra (Ophiophagus hannah) venom L-amino acid oxidase (OH-LAAO), a heat stable enzyme, has been shown to exhibit very potent anti-proliferative activity against human breast and lung tumorigenic cells but not in their non-tumorigenic counterparts. We further examine its in vitro and in vivo anti-tumor activity in a human prostate adenocarcinoma (PC-3) model. OH-LAAO demonstrated potent cytotoxicity against PC-3 cells with IC50 of 0.05 µg/mL after 72 h incubation in vitro. It induced apoptosis as evidenced with an increase in caspase-3/7 cleavages and an increase in annexin V-stained cells. To examine its in vivo anti-tumor activity, we treated PC-3 tumor xenograft implanted subcutaneously in immunodeficient NU/NU (nude) mice with 1 µg/g OH-LAAO given intraperitoneally (i.p.). After 8 weeks of treatment, OH-LAAO treated PC-3 tumors were markedly inhibited, when compared to the control group (P <0.05). TUNEL staining analysis on the tumor sections showed a significantly increase of apoptotic cells in the LAAO-treated animals. Histological examinations of the vital organs in these two groups showed no significant differences with normal tissues, indicating no obvious tissue damage. The treatment also did not cause any significant changes on the body weight of the mice during the duration of the study. These observations suggest that OH-LAAO cytotoxic effects may be specific to tumor xenografts and less to normal organs. Given its potent anti-tumor activities shown in vitro as well as in vivo, the king cobra venom LAAO can potentially be developed to treat prostate cancer and other solid tumors.

  9. Crystal structure of a membrane-bound l-amino acid deaminase from Proteus vulgaris.

    PubMed

    Ju, Yingchen; Tong, Shuilong; Gao, Yongxiang; Zhao, Wei; Liu, Qi; Gu, Qiong; Xu, Jun; Niu, Liwen; Teng, Maikun; Zhou, Huihao

    2016-09-01

    l-amino acid oxidases/deaminases (LAAOs/LAADs) are a class of oxidoreductases catalyzing the oxidative deamination of l-amino acids to α-keto acids. They are widely distributed in eukaryotic and prokaryotic organisms, and exhibit diverse substrate specificity, post-translational modifications and cellular localization. While LAAOs isolated from snake venom have been extensively characterized, the structures and functions of LAAOs from other species are largely unknown. Here, we reported crystal structure of a bacterial membrane-bound LAAD from Proteus vulgaris (pvLAAD) in complex with flavin adenine dinucleotide (FAD). We found that the overall fold of pvLAAD does not resemble typical LAAOs. Instead it, is similar to d-amino acid oxidases (DAAOs) with an additional hydrophobic insertion module on protein surface. Structural analysis and liposome-binding assays suggested that the hydrophobic module serves as an extra membrane-binding site for LAADs. Bacteria from genera Proteus and Providencia were found to encode two classes of membrane-bound LAADs. Based on our structure, the key roles of residues Q278 and L317 in substrate selectivity were proposed and biochemically analyzed. While LAADs on the membrane were proposed to transfer electrons to respiratory chain for FAD re-oxidization, we observed that the purified pvLAAD could generate a significant amount of hydrogen peroxide in vitro, suggesting it could use dioxygen to directly re-oxidize FADH2 as what typical LAAOs usually do. These findings provide a novel insights for a better understanding this class of enzymes and will help developing biocatalysts for industrial applications. PMID:27422658

  10. Aromatic L-amino acid decarboxylase deficiency with hyperdopaminuria. Clinical and laboratory findings in response to different therapies.

    PubMed

    Fiumara, A; Bräutigam, C; Hyland, K; Sharma, R; Lagae, L; Stoltenborg, B; Hoffmann, G F; Jaeken, J; Wevers, R A

    2002-08-01

    Aromatic L-amino acid decarboxylase (AADC - E.C. 4.1.1.28) converts L-dopa to dopamine and 5-hydroxytryptophan to serotonin. Inherited deficiency of this enzyme leads to decreased brain levels of these neurotransmitters. Clinically this results in the development of a progressive neurometabolic disorder characterized by severe hypotonia, dystonic and choreoathetoid movements, oculogyric crises, and hypothermia from infancy. Here we describe the clinical, biochemical and molecular details of two affected brothers, one of whom, despite the lack of AADC, presented with hyperdopaminuria. In addition, we detail his reactions to treatment with dopaminergic agonists, monoamine oxidase inhibitors and pyridoxine.

  11. Earth Microorganisms can Utilize D- and L-Amino Acids

    NASA Astrophysics Data System (ADS)

    Zhang, G.; Sun, H. J.

    2010-04-01

    Enantiomerically-selective amino acid consumption underpins the Mars Oxidant experiment and the claim that Atacama soils are Mars-like. This idea is problematic: D-, as well as L- enantionmers, are utilized by earth organisms.

  12. Production of Dopamine by Aromatic l-Amino Acid Decarboxylase Cells after Spinal Cord Injury.

    PubMed

    Ren, Li-Qun; Wienecke, Jacob; Hultborn, Hans; Zhang, Mengliang

    2016-06-15

    Aromatic l-amino acid decarboxylase (AADC) cells are widely distributed in the spinal cord, and their functions are largely unknown. We have previously found that AADC cells in the spinal cord could increase their ability to produce serotonin (5-hydroxytryptamine) from 5-hydroxytryptophan after spinal cord injury (SCI). Because AADC is a common enzyme catalyzing 5-hydroxytryptophan to serotonin and l-3,4-dihydroxyphenylalanine (l-dopa) to dopamine (DA), it seems likely that the ability of AADC cells using l-dopa to synthesize DA is also increased. To prove whether or not this is the case, a similar rat sacral SCI model and a similar experimental paradigm were adopted as that which we had used previously. In the chronic SCI rats (> 45 days), no AADC cells expressed DA if there was no exogenous l-dopa application. However, following administration of a peripheral AADC inhibitor (carbidopa) with or without a monoamine oxidase inhibitor (pargyline) co-application, systemic administration of l-dopa resulted in ∼94% of AADC cells becoming DA-immunopositive in the spinal cord below the lesion, whereas in normal or sham-operated rats none or very few of AADC cells became DA-immunopositive with the same treatment. Using tail electromyography, spontaneous tail muscle activity was increased nearly fivefold over the baseline level. When pretreated with a central AADC inhibitor (NSD-1015), further application of l-dopa failed to increase the motoneuron activity although the expression of DA in the AADC cells was not completely inhibited. These findings demonstrate that AADC cells in the spinal cord below the lesion gain the ability to produce DA from its precursor in response to SCI. This ability also enables the AADC cells to produce 5-HT and trace amines, and likely contributes to the development of hyperexcitability. These results might also be implicated for revealing the pathological mechanisms underlying l-dopa-induced dyskinesia in Parkinson's disease. PMID:26830512

  13. Production of Dopamine by Aromatic l-Amino Acid Decarboxylase Cells after Spinal Cord Injury.

    PubMed

    Ren, Li-Qun; Wienecke, Jacob; Hultborn, Hans; Zhang, Mengliang

    2016-06-15

    Aromatic l-amino acid decarboxylase (AADC) cells are widely distributed in the spinal cord, and their functions are largely unknown. We have previously found that AADC cells in the spinal cord could increase their ability to produce serotonin (5-hydroxytryptamine) from 5-hydroxytryptophan after spinal cord injury (SCI). Because AADC is a common enzyme catalyzing 5-hydroxytryptophan to serotonin and l-3,4-dihydroxyphenylalanine (l-dopa) to dopamine (DA), it seems likely that the ability of AADC cells using l-dopa to synthesize DA is also increased. To prove whether or not this is the case, a similar rat sacral SCI model and a similar experimental paradigm were adopted as that which we had used previously. In the chronic SCI rats (> 45 days), no AADC cells expressed DA if there was no exogenous l-dopa application. However, following administration of a peripheral AADC inhibitor (carbidopa) with or without a monoamine oxidase inhibitor (pargyline) co-application, systemic administration of l-dopa resulted in ∼94% of AADC cells becoming DA-immunopositive in the spinal cord below the lesion, whereas in normal or sham-operated rats none or very few of AADC cells became DA-immunopositive with the same treatment. Using tail electromyography, spontaneous tail muscle activity was increased nearly fivefold over the baseline level. When pretreated with a central AADC inhibitor (NSD-1015), further application of l-dopa failed to increase the motoneuron activity although the expression of DA in the AADC cells was not completely inhibited. These findings demonstrate that AADC cells in the spinal cord below the lesion gain the ability to produce DA from its precursor in response to SCI. This ability also enables the AADC cells to produce 5-HT and trace amines, and likely contributes to the development of hyperexcitability. These results might also be implicated for revealing the pathological mechanisms underlying l-dopa-induced dyskinesia in Parkinson's disease.

  14. Murine GPRC6A Mediates Cellular Responses to L-Amino Acids, but Not Osteocalcin Variants

    PubMed Central

    Rueda, Patricia; Harley, Elizabeth; Lu, Yao; Stewart, Gregory D.; Fabb, Stewart; Diepenhorst, Natalie; Cremers, Béatrice; Rouillon, Marie-Hélène; Wehrle, Isabelle; Geant, Anne; Lamarche, Gwladys; Leach, Katie; Charman, William N.; Christopoulos, Arthur; Summers, Roger J.; Sexton, Patrick M.; Langmead, Christopher J.

    2016-01-01

    Phenotyping of Gprc6a KO mice has shown that this promiscuous class C G protein coupled receptor is variously involved in regulation of metabolism, inflammation and endocrine function. Such effects are described as mediated by extracellular calcium, L-amino acids, the bone-derived peptide osteocalcin (OCN) and the male hormone testosterone, introducing the concept of a bone-energy-metabolism-reproduction functional crosstalk mediated by GPRC6A. However, whilst the calcium and L-amino acid-sensing properties of GPRC6A are well established, verification of activity of osteocalcin at both human and mouse GPRC6A in vitro has proven somewhat elusive. This study characterises the in vitro pharmacology of mouse GPRC6A in response to its putative ligands in both recombinant and endogenous GPRC6A-expressing cells. Using cell signalling, and glucagon-like peptide (GLP)-1 and insulin release assays, our results confirm that basic L-amino acids act as agonists of the murine GPRC6A receptor in both recombinant cells and immortalised entero-endocrine and pancreatic β-cells. In contrast, our studies do not support a role for OCN as a direct ligand for mouse GPRC6A, suggesting that the reported in vivo effects of OCN that require GPRC6A may be indirect, rather than via direct activation of the receptor. PMID:26785252

  15. D:L-AMINO Acids and the Turnover of Microbial Biomass

    NASA Astrophysics Data System (ADS)

    Lomstein, B. A.; Braun, S.; Mhatre, S. S.; Jørgensen, B. B.

    2015-12-01

    Decades of ocean drilling have demonstrated wide spread microbial life in deep sub-seafloor sediment, and surprisingly high microbial cell numbers. Despite the ubiquity of life in the deep biosphere, the large community sizes and the low energy fluxes in the vast buried ecosystem are still poorly understood. It is not know whether organisms of the deep biosphere are specifically adapted to extremely low energy fluxes or whether most of the observed cells are in a maintenance state. Recently we developed and applied a new culture independent approach - the D:L-amino acid model - to quantify the turnover times of living microbial biomass, microbial necromass and mean metabolic rates. This approach is based on the built-in molecular clock in amino acids that very slowly undergo chemical racemization until they reach an even mixture of L- and D- forms, unless microorganisms spend energy to keep them in the L-form that dominates in living organisms. The approach combines sensitive analyses of amino acids, the unique bacterial endospore marker (dipicolinic acid) with racemization dynamics of stereo-isomeric amino acids. Based on a heating experiment, we recently reported kinetic parameters for racemization of aspartic acid, glutamic acid, serine and alanine in bulk sediment from Aarhus Bay, Denmark. The obtained racemization rate constants were faster than the racemization rate constants of free amino acids, which we have previously applied in Holocene sediment from Aarhus Bay and in up to 10 mio yr old sediment from ODP Leg 201. Another important input parameter for the D:L-amino acid model is the cellular carbon content. It has recently been suggested that the cellular carbon content most likely is lower than previously thought. In recognition of these new findings, previously published data based on the D:L-amino acid model were recalculated and will be presented together with new data from an Arctic Holocene setting with constant sub-zero temperatures.

  16. Surface Enhanced Raman Scattering studies of L-amino acids adsorbed on silver nanoclusters

    NASA Astrophysics Data System (ADS)

    Botta, Raju; Rajanikanth, A.; Bansal, C.

    2015-01-01

    Silver nanocluster films were prepared using plasma inert gas phase condensation technique. These were used as Raman active substrates for Surface Enhanced Raman Scattering (SERS) studies of 19 standard L-amino acids adsorbed on the surface of Ag nanoclusters via Agsbnd N bonds. A detailed study of two essential aromatic amino acids viz. L-Phenylalanine and L-Tryptophan showed a correlation between the Raman intensity of the characteristic lines of phenol and indole side chains and their molar concentrations in the range 1 μM-1 mM. This indicates that Raman studies can be used for quantitative determination of the amino acids in proteins.

  17. Analysis of the Role of the Active Site Residue Arg98 in the Flavoprotein Tryptophan 2-Monooxygenase, a Member of the l-Amino Oxidase Family†

    PubMed Central

    Sobrado, Pablo; Fitzpatrick, Paul F.

    2006-01-01

    The flavoprotein tryptophan 2-monooxygenase catalyzes the oxidative decarboxylation of tryptophan to indoleacetamide. We have previously identified tryptophan 2-monooxygenase as a homologue of l-amino acid oxidase [Sobrado, P., and Fitzpatrick, P. F. (2002) Arch. Biochem. Biophys. 402, 24–30]. On the basis of the sequence comparisons of the different LAAO family members, Arg98 of tryptophan 2-monooxygenase can be identified as an active site residue which interacts with the carboxylate of the amino acid substrate. The catalytic properties of R98K and R98A tryptophan 2-monooxygenase have been characterized to evaluate the role of this residue. Mutation of Arg98 to lysine decreases the first-order rate constant for flavin reduction by 180-fold and the second-order rate constant for flavin oxidation by 26-fold, has no significant effect on the Kd value for tryptophan or the Ki value for the competitive inhibitor indoleacetamide, and increases the Ki value for indolepyruvate less than 2-fold. Mutation of this residue to alanine decreases the rate constants for reduction and oxidation an additional 5- and 2-fold, respectively, and increases the Kd value for tryptophan and the Ki value for indolepyruvate by 31- and 17-fold, respectively, while having an only 2-fold effect on the Ki value for indoleacetamide. Both mutations increase the value of the primary deuterium isotope effect with tryptophan as a substrate, consistent with a later transition state. Both mutant enzymes catalyze a simple oxidase reaction, producing indolepyruvate and hydrogen peroxide. The pH dependences of the V/Ktrp values for the mutant enzymes show that the anionic form of the substrate is preferred but that the zwitterionic form is a substrate. The results are consistent with the interaction between Arg98 and the carboxylate of the amino acid substrate being critical for correct positioning of the substrate in the active site for efficient catalysis. PMID:14636049

  18. Among the twenty classical L-amino acids, only glutamate directly activates metabotropic glutamate receptors.

    PubMed

    Frauli, Mélanie; Neuville, Pascal; Vol, Claire; Pin, Jean-Philippe; Prézeau, Laurent

    2006-02-01

    Under pathophysiological conditions, cellular amino acids can be profusely released from cells into the cerebral interstitial space. Because several class-C G protein coupled receptors (GPCRs) display a broad natural ligand spectrum, being sensitive to more than one endogenous ligand, we wondered whether the related metabotropic glutamate (mGlu) receptors could be modulated by various types of L-amino acids, allowing them to sense large increase in extracellular amino acid concentration. Here, the agonist, antagonist and allosteric effects of the twenty classical L-amino acids were evaluated on the eight mGlu receptor subtypes. We show that, in addition to glutamate (Glu), cysteine, aspartate and asparagine also lead to the activation of mGlu3, 4 and 5. Interestingly, our data demonstrate that the effect of these three amino acids did not result from a direct activation of the receptors, but from an indirect action involving Glu-transporters/exchangers. These data first demonstrate that mGlu receptors, unlike other class-C GPCRs, display an extremely high selectivity towards one ligand. Moreover, our results also show that Glu transport systems allow mGlu receptors to sense large increase in the extracellular concentration of some amino acids. Such a system will certainly lead to a large increase in some mGlu receptor activity under pathological conditions, such as seizure, ischemia or other brain injuries. PMID:16310227

  19. [Asymmetric synthesis of aromatic L-amino acids catalyzed by transaminase].

    PubMed

    Xia, Wenna; Sun, Yu; Min, Cong; Han, Wei; Wu, Sheng

    2012-11-01

    Aromatic L-Amino acids are important chiral building blocks for the synthesis of many drugs, pesticides, fine chemicals and food additives. Due to the high activity and steroselectivity, enzymatic synthesis of chiral building blocks has become the main research direction in asymmetric synthesis field. Guided by the phylogenetic analysis of transaminases from different sources, two representative aromatic transaminases TyrB and Aro8 in type I subfamily, from the prokaryote Escherichia coli and eukaryote Saccharomyces cerevisia, respectively, were applied for the comparative study of asymmetric transamination reaction process and catalytic efficiency of reversely converting keto acids to the corresponding aromatic L-amino acid. Both TyrB and Aro8 could efficiently synthesize the natural aromatic amino acids phenylalanine and tyrosine as well as non-natural amino acid phenylglycine. The chiral HPLC analysis showed the produced amino acids were L-configuration and the e.e value was 100%. L-alanine was the optimal amino donor, and the transaminase TyrB and Aro8 could not use D-amino acids as amino donor. The optimal molar ratio of amino donor (L-alanine) and amino acceptor (aromatic alpha-keto acids) was 4:1. Both of the substituted group on the aromatic ring and the length of fatty acid carbon chain part in the molecular structure of aromatic substrate alpha-keto acid have the significant impact on the enzyme-catalyzed transamination efficiency. In the experiments of preparative-scale transamination synthesis of L-phenylglycine, L-phenylalanine and L-tyrosine, the specific production rate catalyzed by TryB were 0.28 g/(g x h), 0.31 g/(g x h) and 0.60 g/(g x h) and the specific production rate catalyzed by Aro8 were 0.61 g/(g x h), 0.48 g/(g x h) and 0.59 g/(g x h). The results obtained here were useful for applying the transaminases to asymmetric synthesis of L-amino acids by reversing the reaction balance in industry.

  20. L-Amino Acids Elicit Diverse Response Patterns in Taste Sensory Cells: A Role for Multiple Receptors

    PubMed Central

    Pal Choudhuri, Shreoshi; Delay, Rona J.; Delay, Eugene R.

    2015-01-01

    Umami, the fifth basic taste, is elicited by the L-amino acid, glutamate. A unique characteristic of umami taste is the response potentiation by 5’ ribonucleotide monophosphates, which are also capable of eliciting an umami taste. Initial reports using human embryonic kidney (HEK) cells suggested that there is one broadly tuned receptor heterodimer, T1r1+T1r3, which detects L-glutamate and all other L-amino acids. However, there is growing evidence that multiple receptors detect glutamate in the oral cavity. While much is understood about glutamate transduction, the mechanisms for detecting the tastes of other L-amino acids are less well understood. We used calcium imaging of isolated taste sensory cells and taste cell clusters from the circumvallate and foliate papillae of C57BL/6J and T1r3 knockout mice to determine if other receptors might also be involved in detection of L-amino acids. Ratiometric imaging with Fura-2 was used to study calcium responses to monopotassium L-glutamate, L-serine, L-arginine, and L-glutamine, with and without inosine 5’ monophosphate (IMP). The results of these experiments showed that the response patterns elicited by L-amino acids varied significantly across taste sensory cells. L-amino acids other than glutamate also elicited synergistic responses in a subset of taste sensory cells. Along with its role in synergism, IMP alone elicited a response in a large number of taste sensory cells. Our data indicate that synergistic and non-synergistic responses to L-amino acids and IMP are mediated by multiple receptors or possibly a receptor complex. PMID:26110622

  1. L-Amino Acids Elicit Diverse Response Patterns in Taste Sensory Cells: A Role for Multiple Receptors.

    PubMed

    Pal Choudhuri, Shreoshi; Delay, Rona J; Delay, Eugene R

    2015-01-01

    Umami, the fifth basic taste, is elicited by the L-amino acid, glutamate. A unique characteristic of umami taste is the response potentiation by 5' ribonucleotide monophosphates, which are also capable of eliciting an umami taste. Initial reports using human embryonic kidney (HEK) cells suggested that there is one broadly tuned receptor heterodimer, T1r1+T1r3, which detects L-glutamate and all other L-amino acids. However, there is growing evidence that multiple receptors detect glutamate in the oral cavity. While much is understood about glutamate transduction, the mechanisms for detecting the tastes of other L-amino acids are less well understood. We used calcium imaging of isolated taste sensory cells and taste cell clusters from the circumvallate and foliate papillae of C57BL/6J and T1r3 knockout mice to determine if other receptors might also be involved in detection of L-amino acids. Ratiometric imaging with Fura-2 was used to study calcium responses to monopotassium L-glutamate, L-serine, L-arginine, and L-glutamine, with and without inosine 5' monophosphate (IMP). The results of these experiments showed that the response patterns elicited by L-amino acids varied significantly across taste sensory cells. L-amino acids other than glutamate also elicited synergistic responses in a subset of taste sensory cells. Along with its role in synergism, IMP alone elicited a response in a large number of taste sensory cells. Our data indicate that synergistic and non-synergistic responses to L-amino acids and IMP are mediated by multiple receptors or possibly a receptor complex.

  2. Metabotropic glutamate receptors are involved in the detection of IMP and L-amino acids by mouse taste sensory cells.

    PubMed

    Pal Choudhuri, S; Delay, R J; Delay, E R

    2016-03-01

    G-protein-coupled receptors are thought to be involved in the detection of umami and L-amino acid taste. These include the heterodimer taste receptor type 1 member 1 (T1r1)+taste receptor type 1 member 3 (T1r3), taste and brain variants of mGluR4 and mGluR1, and calcium sensors. While several studies suggest T1r1+T1r3 is a broadly tuned lLamino acid receptor, little is known about the function of metabotropic glutamate receptors (mGluRs) in L-amino acid taste transduction. Calcium imaging of isolated taste sensory cells (TSCs) of T1r3-GFP and T1r3 knock-out (T1r3 KO) mice was performed using the ratiometric dye Fura 2 AM to investigate the role of different mGluRs in detecting various L-amino acids and inosine 5' monophosphate (IMP). Using agonists selective for various mGluRs such as (RS)-3,5-dihydroxyphenylglycine (DHPG) (an mGluR1 agonist) and L-(+)-2-amino-4-phosphonobutyric acid (l-AP4) (an mGluR4 agonist), we evaluated TSCs to determine if they might respond to these agonists, IMP, and three L-amino acids (monopotassium L-glutamate, L-serine and L-arginine). Additionally, we used selective antagonists against different mGluRs such as (RS)-L-aminoindan-1,5-dicarboxylic acid (AIDA) (an mGluR1 antagonist), and (RS)-α-methylserine-O-phosphate (MSOP) (an mGluR4 antagonist) to determine if they can block responses elicited by these L-amino acids and IMP. We found that L-amino acid- and IMP-responsive cells also responded to each agonist. Antagonists for mGluR4 and mGluR1 significantly blocked the responses elicited by IMP and each of the L-amino acids. Collectively, these data provide evidence for the involvement of taste and brain variants of mGluR1 and mGluR4 in L-amino acid and IMP taste responses in mice, and support the concept that multiple receptors contribute to IMP and L-amino acid taste. PMID:26701297

  3. Metabotropic glutamate receptors are involved in the detection of IMP and L-amino acids by mouse taste sensory cells.

    PubMed

    Pal Choudhuri, S; Delay, R J; Delay, E R

    2016-03-01

    G-protein-coupled receptors are thought to be involved in the detection of umami and L-amino acid taste. These include the heterodimer taste receptor type 1 member 1 (T1r1)+taste receptor type 1 member 3 (T1r3), taste and brain variants of mGluR4 and mGluR1, and calcium sensors. While several studies suggest T1r1+T1r3 is a broadly tuned lLamino acid receptor, little is known about the function of metabotropic glutamate receptors (mGluRs) in L-amino acid taste transduction. Calcium imaging of isolated taste sensory cells (TSCs) of T1r3-GFP and T1r3 knock-out (T1r3 KO) mice was performed using the ratiometric dye Fura 2 AM to investigate the role of different mGluRs in detecting various L-amino acids and inosine 5' monophosphate (IMP). Using agonists selective for various mGluRs such as (RS)-3,5-dihydroxyphenylglycine (DHPG) (an mGluR1 agonist) and L-(+)-2-amino-4-phosphonobutyric acid (l-AP4) (an mGluR4 agonist), we evaluated TSCs to determine if they might respond to these agonists, IMP, and three L-amino acids (monopotassium L-glutamate, L-serine and L-arginine). Additionally, we used selective antagonists against different mGluRs such as (RS)-L-aminoindan-1,5-dicarboxylic acid (AIDA) (an mGluR1 antagonist), and (RS)-α-methylserine-O-phosphate (MSOP) (an mGluR4 antagonist) to determine if they can block responses elicited by these L-amino acids and IMP. We found that L-amino acid- and IMP-responsive cells also responded to each agonist. Antagonists for mGluR4 and mGluR1 significantly blocked the responses elicited by IMP and each of the L-amino acids. Collectively, these data provide evidence for the involvement of taste and brain variants of mGluR1 and mGluR4 in L-amino acid and IMP taste responses in mice, and support the concept that multiple receptors contribute to IMP and L-amino acid taste.

  4. Aldolase as a Chirality Intersection of L-Amino Acids and D-Sugars

    NASA Astrophysics Data System (ADS)

    Munegumi, Toratane

    2015-06-01

    Aldolase plays an important role in glycolysis and gluconeogenesis to produce D-fructose-1,6-bisphosphate (D-FBP) from dihydroxyacetone phosphate (DHP) and D-glyceraldehyde-3-phosphate (D-GAP). This reaction is stereoselective and retains the D-GAP 2R configuration and yields D-FBP (with the configuration: 3S, 4S, 5R). The 3- and 4-position carbons are the newly formed chiral carbons because the 5-position carbon of D-FBP comes from the 2-position of D-GAP. Although four diastereomeric products, ( 3S, 4R, 5R), ( 3R, 4R, 5R), ( 3R, 4S, 5R), ( 3S, 4S, 5R), are expected in the nonenzymatic reaction, only the ( 3S, 4S, 5R) diastereomer (D-FBP) is obtained. Therefore, the chirality in the 3- and 4-positions is induced by the chirality of the enzyme composed of L-amino acid residues. D-Glucose-6-phosphate (D-G6P), which is generated from D-FBP in the gluconeogenesis pathway, produces D-ribose-5-phosphate (D-R5P) in the pentose phosphate pathway. D-R5P is converted to PRPP (5-phosphoribosyl-α-pyrophosphate), which is used for the de novo synthesis of nucleotides. Ribonucleic acid (RNA) uses the nucleotides as building blocks. The configurations of the 4R-carbon and of the 3S-carbon are retained. The stereochemical structure of RNA is based on 3S as well as 4R (D). The consideration above suggests that aldolase is a key enzyme that determines the 3S configuration in D-R5P. It is thus a chirality intersection between amino acids and sugars, because the sugar chirality is determined by the chiral environment of an L-amino acid protein, aldolase, to produce D-FBP.

  5. Aldolase as a chirality intersection of L-amino acids and D-sugars.

    PubMed

    Munegumi, Toratane

    2015-06-01

    Aldolase plays an important role in glycolysis and gluconeogenesis to produce D-fructose-1,6-bisphosphate (D-FBP) from dihydroxyacetone phosphate (DHP) and D-glyceraldehyde-3-phosphate (D-GAP). This reaction is stereoselective and retains the D-GAP 2R configuration and yields D-FBP (with the configuration: 3S, 4S, 5R). The 3- and 4-position carbons are the newly formed chiral carbons because the 5-position carbon of D-FBP comes from the 2-position of D-GAP. Although four diastereomeric products, (3S, 4R, 5R), (3R, 4R, 5R), (3R, 4S, 5R), (3S, 4S, 5R), are expected in the nonenzymatic reaction, only the (3S, 4S, 5R) diastereomer (D-FBP) is obtained. Therefore, the chirality in the 3- and 4-positions is induced by the chirality of the enzyme composed of L-amino acid residues. D-Glucose-6-phosphate (D-G6P), which is generated from D-FBP in the gluconeogenesis pathway, produces D-ribose-5-phosphate (D-R5P) in the pentose phosphate pathway. D-R5P is converted to PRPP (5-phosphoribosyl-α-pyrophosphate), which is used for the de novo synthesis of nucleotides. Ribonucleic acid (RNA) uses the nucleotides as building blocks. The configurations of the 4R-carbon and of the 3S-carbon are retained. The stereochemical structure of RNA is based on 3S as well as 4R (D). The consideration above suggests that aldolase is a key enzyme that determines the 3S configuration in D-R5P. It is thus a chirality intersection between amino acids and sugars, because the sugar chirality is determined by the chiral environment of an L-amino acid protein, aldolase, to produce D-FBP.

  6. Amino acid oxidase of leukocytes in relation to H2O2-mediated bacterial killing

    PubMed Central

    Eckstein, Marlene R.; Baehner, Robert L.; Nathan, David G.

    1971-01-01

    D-Amino acid oxidase and L-amino acid oxidase have been measured in sucrose homogenates of polymorphonuclear leukocytes (PMN) obtained from guinea pigs and humans. Subcellular distribution patterns and studies on latency indicate that these oxidases are soluble enzymes. Their hydrogen peroxide-generating capacity was verified. Chronic granulomatous disease PMN, which lack a respiratory burst and fail to generate H2O2 during phagocytosis and do not kill catalase positive bacteria, had peroxide-generating amino acid oxidase activity equal to that found in PMN homogenates from patients with bacterial infections. The precise metabolic and bactericidal role of amino acid oxidases in PMN remains uncertain. PMID:4397948

  7. Structure-Function Relationships in l-Amino Acid Deaminase, a Flavoprotein Belonging to a Novel Class of Biotechnologically Relevant Enzymes.

    PubMed

    Motta, Paolo; Molla, Gianluca; Pollegioni, Loredano; Nardini, Marco

    2016-05-13

    l-Amino acid deaminase from Proteus myxofaciens (PmaLAAD) is a membrane flavoenzyme that catalyzes the deamination of neutral and aromatic l-amino acids into α-keto acids and ammonia. PmaLAAD does not use dioxygen to re-oxidize reduced FADH2 and thus does not produce hydrogen peroxide; instead, it uses a cytochrome b-like protein as an electron acceptor. Although the overall fold of this enzyme resembles that of known amine or amino acid oxidases, it shows the following specific structural features: an additional novel α+β subdomain placed close to the putative transmembrane α-helix and to the active-site entrance; an FAD isoalloxazine ring exposed to solvent; and a large and accessible active site suitable to bind large hydrophobic substrates. In addition, PmaLAAD requires substrate-induced conformational changes of part of the active site, particularly in Arg-316 and Phe-318, to achieve the correct geometry for catalysis. These studies are expected to pave the way for rationally improving the versatility of this flavoenzyme, which is critical for biocatalysis of enantiomerically pure amino acids.

  8. The Venus Fly Trap domain of the extracellular Ca2+ -sensing receptor is required for L-amino acid sensing.

    PubMed

    Mun, Hee-Chang; Franks, Alison H; Culverston, Emma L; Krapcho, Karen; Nemeth, Edward F; Conigrave, Arthur D

    2004-12-10

    We previously demonstrated that the human calcium-sensing receptor (CaR) is allosterically activated by L-amino acids (Conigrave, A. D., Quinn, S. J., and Brown, E. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 4814-4819). However, the domain-based location of amino acid binding has been uncertain. We now show that the Venus Fly Trap (VFT) domain of CaR, but none of its other major domains, is required for amino acid sensing. Several constructs were informative when expressed in HEK293 cells. First, the wild-type CaR exhibited allosteric activation by L-amino acids as previously observed. Second, two CaR-mGlu chimeric receptor constructs that retained the VFT domain of CaR, one containing the extracellular Cys-rich region of CaR and the other containing the Cys-rich region of the rat metabotropic glutamate type-1 (mGlu-1) receptor, together with the rat mGlu-1 transmembrane region and C-terminal tail, retained amino acid sensing. Third, a CaR lacking residues 1-599 of the N-terminal extracellular head but retaining an intact CaR transmembrane region and a functional but truncated C terminus (headless-T903 CaR) failed to respond to L-amino acids but retained responsiveness to the type-II calcimimetic NPS R-467. Finally, a T903 CaR control that retained an intact N terminus also retained L-amino acid sensing. Taken together, the data indicate that the VFT domain of CaR is necessary for L-amino acid sensing and are consistent with the hypothesis that the VFT domain is the site of L-amino acid binding. The findings support the concept that the mGlu-1 amino acid binding site for L-glutamate is conserved as an L-amino acid binding site in its homolog, the CaR.

  9. A mutant leucine aminopeptidase from Streptomyces cinnamoneus with enhanced L-aspartyl L-amino acid methyl ester synthetic activity.

    PubMed

    Arima, Jiro; Kono, Mirai; Kita, Manami; Mori, Nobuhiro

    2012-06-01

    L-aspartyl L-amino acid methyl ester was synthesized using a mutant of a thermostable leucine aminopeptidase from Streptomyces cinnamoneus, D198 K SSAP, obtained in previously. A peptide of high-intensity sweetener, L-aspartyl-L-phenylalanine methyl ester, was selected as a model for demonstrating the synthesis of L-aspartyl L-amino acid methyl ester. The hydrolytic activities of D198 K SSAP toward L-aspartyl-L-phenylalanine and its methyl ester were, respectively, 74-fold and fourfold higher than those of wild type. Similarly, the initial rate of the enzyme for L-aspartyl-L-phenylalanine methyl ester synthesis was over fivefold higher than that of wild-type SSAP in 90% methanol (v/v) in a one-pot reaction. Furthermore, other L-aspartyl L-amino acid methyl esters were synthesized efficiently using D198 K SSAP. Results show that the substitution of Asp198 of SSAP with Lys is effective for synthesizing L-aspartyl L-amino acid methyl ester.

  10. Aromatic L-amino acid decarboxylase deficiency diagnosed by clinical metabolomic profiling of plasma.

    PubMed

    Atwal, Paldeep S; Donti, Taraka R; Cardon, Aaron L; Bacino, C A; Sun, Qin; Emrick, L; Reid Sutton, V; Elsea, Sarah H

    2015-01-01

    Aromatic L-amino acid decarboxylase (AADC) deficiency is an inborn error of metabolism affecting the biosynthesis of serotonin, dopamine, and catecholamines. We report a case of AADC deficiency that was detected using the Global MAPS platform. This is a novel platform that allows for parallel clinical testing of hundreds of metabolites in a single plasma specimen. It uses a state-of-the-art mass spectrometry platform, and the resulting spectra are compared against a library of ~2500 metabolites. Our patient is now a 4 year old boy initially seen at 11 months of age for developmental delay and hypotonia. Multiple tests had not yielded a diagnosis until exome sequencing revealed compound heterozygous variants of uncertain significance (VUS), c.286G>A (p.G96R) and c.260C>T (p.P87L) in the DDC gene, causal for AADC deficiency. CSF neurotransmitter analysis confirmed the diagnosis with elevated 3-methoxytyrosine (3-O-methyldopa). Metabolomic profiling was performed on plasma and revealed marked elevation in 3-methoxytyrosine (Z-score +6.1) consistent with the diagnosis of AADC deficiency. These results demonstrate that the Global MAPS platform is able to diagnose AADC deficiency from plasma. In summary, we report a novel and less invasive approach to diagnose AADC deficiency using plasma metabolomic profiling.

  11. Aromatic L-amino acid decarboxylase (AADC) is crucial for brain development and motor functions.

    PubMed

    Shih, De-Fen; Hsiao, Chung-Der; Min, Ming-Yuan; Lai, Wen-Sung; Yang, Chianne-Wen; Lee, Wang-Tso; Lee, Shyh-Jye

    2013-01-01

    Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare pediatric neuro-metabolic disease in children. Due to the lack of an animal model, its pathogenetic mechanism is poorly understood. To study the role of AADC in brain development, a zebrafish model of AADC deficiency was generated. We identified an aadc gene homolog, dopa decarboxylase (ddc), in the zebrafish genome. Whole-mount in situ hybridization analysis showed that the ddc gene is expressed in the epiphysis, locus caeruleus, diencephalic catecholaminergic clusters, and raphe nuclei of 36-h post-fertilization (hpf) zebrafish embryos. Inhibition of Ddc by AADC inhibitor NSD-1015 or anti-sense morpholino oligonucleotides (MO) reduced brain volume and body length. We observed increased brain cell apoptosis and loss of dipencephalic catecholaminergic cluster neurons in ddc morphants (ddc MO-injected embryos). Seizure-like activity was also detected in ddc morphants in a dose-dependent manner. ddc morphants had less sensitive touch response and impaired swimming activity that could be rescued by injection of ddc plasmids. In addition, eye movement was also significantly impaired in ddc morphants. Collectively, loss of Ddc appears to result in similar phenotypes as that of ADCC deficiency, thus zebrafish could be a good model for investigating pathogenetic mechanisms of AADC deficiency in children. PMID:23940784

  12. Induction of aromatic-L-amino acid decarboxylase by decarboxylase inhibitors in idiopathic parkinsonism.

    PubMed

    Boomsma, F; Meerwaldt, J D; Man in 't Veld, A J; Hovestadt, A; Schalekamp, M A

    1989-06-01

    We evaluated the effect of administration of L-dopa, alone or in combination with a peripheral decarboxylase inhibitor, on plasma levels of aromatic-L-amino acid decarboxylase (ALAAD). After single-dose administration of L-dopa plus benserazide (Madopar) in healthy subjects and in chronically treated patients with parkinsonism, plasma ALAAD followed for 2 to 3 hours fell, but returned to predosing levels within 90 minutes. Four groups of patients with idiopathic parkinsonism were studied during chronic treatment: Group I, no L-dopa treatment (n = 31); Group II, L-dopa alone (n = 15); Group III, L-dopa plus benserazide (n = 28); and Group IV, L-dopa plus carbidopa (Sinemet, n = 30). Plasma ALAAD 2 hours after dosing was normal in Groups I and II. ALAAD was increased threefold in Groups III and IV, suggesting induction of ALAAD by the coadministration of a peripheral decarboxylase inhibitor. In a study of 3 patients in whom L-dopa/benserazide was started, plasma ALAAD rose gradually over 3 to 4 weeks. Further detailed pharmacokinetic studies of L-dopa, dopamine, and ALAAD in plasma and cerebrospinal fluid are required to determine if the apparent ALAAD induction by a peripheral decarboxylase inhibitor may be related to the loss of clinical efficacy of combination therapy in some patients and how it is related to end-of-dose deterioration and on-off phenomena. PMID:2742363

  13. Induction of aromatic-L-amino acid decarboxylase by decarboxylase inhibitors in idiopathic parkinsonism.

    PubMed

    Boomsma, F; Meerwaldt, J D; Man in 't Veld, A J; Hovestadt, A; Schalekamp, M A

    1989-06-01

    We evaluated the effect of administration of L-dopa, alone or in combination with a peripheral decarboxylase inhibitor, on plasma levels of aromatic-L-amino acid decarboxylase (ALAAD). After single-dose administration of L-dopa plus benserazide (Madopar) in healthy subjects and in chronically treated patients with parkinsonism, plasma ALAAD followed for 2 to 3 hours fell, but returned to predosing levels within 90 minutes. Four groups of patients with idiopathic parkinsonism were studied during chronic treatment: Group I, no L-dopa treatment (n = 31); Group II, L-dopa alone (n = 15); Group III, L-dopa plus benserazide (n = 28); and Group IV, L-dopa plus carbidopa (Sinemet, n = 30). Plasma ALAAD 2 hours after dosing was normal in Groups I and II. ALAAD was increased threefold in Groups III and IV, suggesting induction of ALAAD by the coadministration of a peripheral decarboxylase inhibitor. In a study of 3 patients in whom L-dopa/benserazide was started, plasma ALAAD rose gradually over 3 to 4 weeks. Further detailed pharmacokinetic studies of L-dopa, dopamine, and ALAAD in plasma and cerebrospinal fluid are required to determine if the apparent ALAAD induction by a peripheral decarboxylase inhibitor may be related to the loss of clinical efficacy of combination therapy in some patients and how it is related to end-of-dose deterioration and on-off phenomena.

  14. Spinal cord injury enables aromatic L-amino acid decarboxylase cells to synthesize monoamines.

    PubMed

    Wienecke, Jacob; Ren, Li-Qun; Hultborn, Hans; Chen, Meng; Møller, Morten; Zhang, Yifan; Zhang, Mengliang

    2014-09-01

    Serotonin (5-HT), an important modulator of both sensory and motor functions in the mammalian spinal cord, originates mainly in the raphe nuclei of the brainstem. However, following complete transection of the spinal cord, small amounts of 5-HT remain detectable below the lesion. It has been suggested, but not proven, that this residual 5-HT is produced by intraspinal 5-HT neurons. Here, we show by immunohistochemical techniques that cells containing the enzyme aromatic l-amino acid decarboxylase (AADC) occur not only near the central canal, as reported by others, but also in the intermediate zone and dorsal horn of the spinal gray matter. We show that, following complete transection of the rat spinal cord at S2 level, AADC cells distal to the lesion acquire the ability to produce 5-HT from its immediate precursor, 5-hydroxytryptophan. Our results indicate that this phenotypic change in spinal AADC cells is initiated by the loss of descending 5-HT projections due to spinal cord injury (SCI). By in vivo and in vitro electrophysiology, we show that 5-HT produced by AADC cells increases the excitability of spinal motoneurons. The phenotypic change in AADC cells appears to result from a loss of inhibition by descending 5-HT neurons and to be mediated by 5-HT1B receptors expressed by AADC cells. These findings indicate that AADC cells are a potential source of 5-HT at spinal levels below an SCI. The production of 5-HT by AADC cells, together with an upregulation of 5-HT2 receptors, offers a partial explanation of hyperreflexia below a chronic SCI. PMID:25186745

  15. /sup 13/N-labeled L-amino acids for in vivo assessment of local myocardial metabolism

    SciTech Connect

    Baumgartner, F.J.; Barrio, J.R.; Henze, E.; Schelbert, H.R.; MacDonald, N.S.; Phelps, M.E.; Kuhl, D.E.

    1981-06-01

    The hot cell synthesis of sterile, pyrogen-free /sup 13/N-labeled L-amino acids was accomplished by employing the appropriate immobilized enzymes on a CNBr-activated Sepharose support and using remote, semiautomated systems. The syntheses were completed 6-12 min after cyclotron production of (/sup 13/N)ammonia. Myocardial time-activity curves after intracoronary injection of /sup 13/N-labeled L-amino acids in dogs were triexponential in both normal and ischemic myocardium. Higher retention of /sup 13/N activity was observed in ischemic segments. Positron computed tomography imaging also showed increased uptake of /sup 13/N-labeled L-glutamate and L-alanine in ischemic segments compared with normal myocardium when blood flow corrections were made. Myocardial transaminases are primarily responsible for the observed retention fractions. It suggests the participation of the carbon skeletons of these amino acids in the Krebs cycle.

  16. Identification of a novel system L amino acid transporter structurally distinct from heterodimeric amino acid transporters.

    PubMed

    Babu, Ellappan; Kanai, Yoshikatsu; Chairoungdua, Arthit; Kim, Do Kyung; Iribe, Yuji; Tangtrongsup, Sahatchai; Jutabha, Promsuk; Li, Yuewei; Ahmed, Nesar; Sakamoto, Shinichi; Anzai, Naohiko; Nagamori, Seishi; Endou, Hitoshi

    2003-10-31

    A cDNA that encodes a novel Na+-independent neutral amino acid transporter was isolated from FLC4 human hepatocarcinoma cells by expression cloning. When expressed in Xenopus oocytes, the encoded protein designated LAT3 (L-type amino acid transporter 3) transported neutral amino acids such as l-leucine, l-isoleucine, l-valine, and l-phenylalanine. The LAT3-mediated transport was Na+-independent and inhibited by 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid, consistent with the properties of system L. Distinct from already known system L transporters LAT1 and LAT2, which form heterodimeric complex with 4F2 heavy chain, LAT3 was functional by itself in Xenopus oocytes. The deduced amino acid sequence of LAT3 was identical to the gene product of POV1 reported as a prostate cancer-up-regulated gene whose function was not determined, whereas it did not exhibit significant similarity to already identified transporters. The Eadie-Hofstee plots of LAT3-mediated transport were curvilinear, whereas the low affinity component is predominant at physiological plasma amino acid concentration. In addition to amino acid substrates, LAT3 recognized amino acid alcohols. The transport of l-leucine was electroneutral and mediated by a facilitated diffusion. In contrast, l-leucinol, l-valinol, and l-phenylalaninol, which have a net positive charge induced inward currents under voltage clamp, suggesting these compounds are transported by LAT3. LAT3-mediated transport was inhibited by the pretreatment with N-ethylmaleimide, consistent with the property of system L2 originally characterized in hepatocyte primary culture. Based on the substrate selectivity, affinity, and N-ethylmaleimide sensitivity, LAT3 is proposed to be a transporter subserving system L2. LAT3 should denote a new family of organic solute transporters. PMID:12930836

  17. Mixtures of l-Amino Acids as Reaction Medium for Formation of Iron Nanoparticles: The Order of Addition into a Ferrous Salt Solution Matters

    PubMed Central

    Šišková, Karolína M.; Machala, Libor; Tuček, Jiři; Kašlík, Josef; Mojzeš, Peter; Zbořil, Radek

    2013-01-01

    Owing to Mössbauer spectroscopy, an advanced characterization technique for iron-containing materials, the present study reveals previously unknown possibilities using l-amino acids for the generation of magnetic particles. Based on our results, a simple choice of the order of l-amino acids addition into a reaction mixture containing ferrous ions leads to either superparamagnetic ferric oxide/oxyhydroxide particles, or magnetically strong Fe0-Fe2O3/FeOOH core-shell particles after chemical reduction. Conversely, when ferric salts are employed with the addition of selected l-amino acids, only Fe0-Fe2O3/FeOOH core-shell particles are observed, regardless of the addition order. We explain this phenomenon by a specific transient/intermediate complex formation between Fe2+ and l-glutamic acid. This type of complexation prevents ferrous ions from spontaneous oxidation in solutions with full air access. Moreover, due to surface-enhanced Raman scattering spectroscopy we show that the functional groups of l-amino acids are not destroyed during the borohydride-induced reduction. These functionalities can be further exploited for (i) attachment of l-amino acids to the as-prepared magnetic particles, and (ii) for targeted bio- and/or environmental applications where the surface chemistry needs to be tailored and directed toward biocompatible species. PMID:24071943

  18. Endospore abundance and D:L-amino acid modeling of bacterial turnover in holocene marine sediment (Aarhus Bay)

    NASA Astrophysics Data System (ADS)

    Langerhuus, Alice T.; Røy, Hans; Lever, Mark A.; Morono, Yuki; Inagaki, Fumio; Jørgensen, Bo B.; Lomstein, Bente Aa.

    2012-12-01

    In order to study bacterial activity, and turnover times of bacterial necromass and biomass in marine sediment, two stations from the Aarhus Bay, Denmark were analyzed. Sediment cores were up to 11 m deep and covered a timescale from the present to ˜11,000 years ago. Sediment was analyzed for total hydrolysable amino acids (THAA), total hydrolysable amino sugars, the bacterial endospore marker dipicolinic acid (DPA), and amino acid enantiomers (L- and D-form) of aspartic acid. Turnover times of bacterial necromass and vegetative cells, as well as carbon oxidation rates were estimated by use of the D:L-amino acid racemization model. Diagenetic indicators were applied to evaluate the diagenetic state of the sedimentary organic matter. The contribution of amino acids to total organic carbon, and the ratio between the amino acids aspartic acid and glutamic acid, and their respective non protein degradation products, β-alanine and γ-amino butyric acid, all indicated increasing degradation state of the organic matter with sediment depth and age. Quantification of DPA showed that endospores were abundant, and increased with depth relative to vegetative cells. Most of the amino acids (97%) could be ascribed to microbial necromass, i.e. the remains of dead bacterial cells. Model estimates showed that the turnover times of microbial necromass were in the range of 0.5-1 × 105 years, while turnover times of vegetative cells were in the range of tens to hundreds of years. The turnover time of the TOC pool increased with depth in the sediment, indicating that the TOC pool became progressively more refractory and unavailable to microorganisms with depth and age of the organic matter.

  19. Aromatic L-amino acids activate the calcium-sensing receptor.

    PubMed

    Conigrave, Arthur D; Mun, Hee-Chang; Lok, Hiu-Chuen

    2007-06-01

    The calcium-sensing receptor (CaR) is recognized as a member of class 3 of the G-protein coupled receptor superfamily. Members of this subgroup, which have large N-terminal extracellular domains, include receptors that respond specifically to the amino acid glutamate; receptors that respond to the glutamate analogue, gamma-amino butyric acid; and several receptors that act as broad-spectrum amino acid sensors. The CaR is one of these broad-spectrum amino acid sensors that, along with several other members of the subgroup, also responds to extracellular Ca2+. In this mini-review, we consider evidence that the CaR is a sensor of aromatic amino acids, that it has broad-spectrum amino acid sensing properties, that it provides an amino acid binding site in its extracellular N-terminal Venus Fly Trap domain, and that amino acids have a physiological impact on systems in which the CaR is expressed.

  20. Use of immobilized glutamate dehydrogenase to synthesize /sup 13/N-labeled L-amino acids

    SciTech Connect

    Cooper, A.J.L.; Gelbard, A.S.

    1981-02-01

    By utilizing glutamate dehydrogenase immobilized onto CNBr-activated Sepharose it is possible to synthesize six L-/sup 13/N-amino acids in high radiochemical yield (5-140 mCi) and in high (> 99%) radiochemical purity. These /sup 13/N-amino acid solutions are potentially suitable for whole body and organ imaging in large animals and man.

  1. Comparison of polarimetry and crown ether-based HPLC chiral stationary phase method to determine (L)-amino acid optical purity.

    PubMed

    Lee, Mee Sung; Khan, F Nawaz; Shin, Sung Chul; Jeong, Euh Duck; Kim, Hae Gyeong; Kim, Mi Jin; Cho, Yoon Jae; Cha, Jae-Young; Hyun, Myung Ho; Jin, Jong Sung

    2012-11-15

    Although various pharmacopoeias provide titration methods to assay (L)-amino acid content, none of these methods distinguish between (L)- and (D)-amino acids and do not consider the presence of enantiomeric impurities. Consequently, these methods are limited in scope to describe the relationship between content and specific rotation, [α]. In this study, the US Pharmacopoeia method was compared with the crown ether-based high performance liquid chromatographic (HPLC) chiral stationary phase (CSP) method to determine (L)-amino acid content and specific rotation. The (L)-amino acid content specified by the US Pharmacopoeia method was not consistent with the specific rotation in the presence of enantiomeric impurities, whereas the HPLC-CSP method was very effective for determining the (L)-amino acid content and the optical purity. The other advantage is that the HPLC-CSP method requires amino acid samples of quite low concentration (as low as 1 μg/mL), whereas the pharmacopoeia method requires higher concentrations (20-110 mg/mL).

  2. Comparison of polarimetry and crown ether-based HPLC chiral stationary phase method to determine (L)-amino acid optical purity.

    PubMed

    Lee, Mee Sung; Khan, F Nawaz; Shin, Sung Chul; Jeong, Euh Duck; Kim, Hae Gyeong; Kim, Mi Jin; Cho, Yoon Jae; Cha, Jae-Young; Hyun, Myung Ho; Jin, Jong Sung

    2012-11-15

    Although various pharmacopoeias provide titration methods to assay (L)-amino acid content, none of these methods distinguish between (L)- and (D)-amino acids and do not consider the presence of enantiomeric impurities. Consequently, these methods are limited in scope to describe the relationship between content and specific rotation, [α]. In this study, the US Pharmacopoeia method was compared with the crown ether-based high performance liquid chromatographic (HPLC) chiral stationary phase (CSP) method to determine (L)-amino acid content and specific rotation. The (L)-amino acid content specified by the US Pharmacopoeia method was not consistent with the specific rotation in the presence of enantiomeric impurities, whereas the HPLC-CSP method was very effective for determining the (L)-amino acid content and the optical purity. The other advantage is that the HPLC-CSP method requires amino acid samples of quite low concentration (as low as 1 μg/mL), whereas the pharmacopoeia method requires higher concentrations (20-110 mg/mL). PMID:22868097

  3. Siganus oramin recombinant L-amino acid oxidase is lethal to Cryptocaryon irritans.

    PubMed

    Li, Ruijun; Dan, Xueming; Li, Anxing

    2013-12-01

    A novel antimicrobial and antiparasitic protein (APP/SR-LAAO) isolated from serum of the rabbitfish (Siganus oramin) was confirmed to be lethal to Cryptocaryon irritans, an important marine parasitic ciliate that causes marine white spot disease in a variety of wild and cultured fish. In this study, a recombinant SR-LAAO (rSR-LAAO) was expressed on a large scale in Escherichia coli Rosetta-gami™(DE3)pLysS cells. rSR-LAAO was expressed as an inactive form in the inclusion bodies. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that after purification, refolding and ultrafiltration, rSR-LAAO had a significantly cytotoxic effect on C. irritans theronts. Using light microscopy, scanning electron microscopy and fluorescence microscopy, we found that theronts rapidly became weakly motile, cilia became detached, cells became rounded, membranes eventually lysed in different cell positions and cytoplasmic contents leaked out of the cell. These results suggested the recombinant SR-LAAO was significantly lethal to C. irritans and the death process of the parasite incubated with rSR-LAAO was remarkably similar compared to the SR-LAAO group as reported earlier.

  4. The role of aromatic L-amino acid decarboxylase in bacillamide C biosynthesis by Bacillus atrophaeus C89.

    PubMed

    Yuwen, Lei; Zhang, Feng-Li; Chen, Qi-Hua; Lin, Shuang-Jun; Zhao, Yi-Lei; Li, Zhi-Yong

    2013-01-01

    For biosynthesis of bacillamide C by Bacillus atrophaeus C89 associated with South China sea sponge Dysidea avara, it is hypothesized that decarboxylation from L-tryptophan to tryptamine could be performed before amidation by the downstream aromatic L-amino acid decarboxylase (AADC) to the non-ribosomal peptide synthetases (NRPS) gene cluster for biosynthesizing bacillamide C. The structural analysis of decarboxylases' known substrates in KEGG database and alignment analysis of amino acid sequence of AADC have suggested that L-tryptophan and L-phenylalanine are the potential substrates of AADC. The enzymatic kinetic experiment of the recombinant AADC proved that L-tryptophan is a more reactive substrate of AADC than L-phenylalanine. Meanwhile, the AADC-catalyzed conversion of L-tryptophan into tryptamine was confirmed by means of HPLC and LC/MS. Thus during bacillamide C biosynthesis, the decarboxylation of L-tryptophan to tryptamine is likely conducted first under AADC catalysis, followed by the amidation of tryptamine with the carboxylic product of NRPS gene cluster.

  5. Characterization of bovine aromatic L-amino acid decarboxylase expressed in a mouse cell line: comparison with native enzyme.

    PubMed

    Park, D H; Kim, K T; Choi, M U; Samanta, H; Joh, T H

    1992-12-01

    Bovine aromatic L-amino acid decarboxylase (AADC) was expressed in a mouse cell line, using a bovine papilloma virus-derived expression vector containing the full coding region of bovine AADC. The recombinant bovine AADC was characterized biochemically and immunochemically and compared with the native bovine AADC. The specific activity of crude recombinant bovine AADC was 30-fold higher than that of crude native AADC. With regard to optimal pH, effects of pyridoxal phosphate concentration and Km for 3,4-dihydroxyphenylalanine as a substrate, both native and recombinant enzymes were essentially identical. Rabbit polyclonal antiserum directed against bovine adrenal AADC recognized on Western blot a single protein band (molecular mass = 55,000 Dalton) in both native and recombinant bovine AADC crude extracts. Furthermore, double immunodiffusion analysis showed a single precipitin line of confluence with both enzyme preparations, indicating immunological identity of native and recombinant bovine AADC. Northern blot analysis identified a single mRNA species (2.2 kb) from native and recombinant bovine AADC preparations. The recombinant bovine AADC has two charge isozymes corresponding to those of the native bovine enzyme, although their relative abundances are different between native and recombinant enzymes. Taken together, our results show that recombinant bovine AADC, expressed from bovine AADC cDNA in a mouse cell line is not only enzymatically active, but also shares many biochemical and immunochemical common features with native bovine AADC.

  6. Enantiomeric purity determination of (L)-amino acids with pre-column derivatization and chiral stationary phase: development and validation of the method.

    PubMed

    Cui, Yan; Jiang, Zhen; Sun, Jiayi; Yu, Jia; Li, Minghua; Li, Mingjie; Liu, Mingxia; Guo, Xingjie

    2014-09-01

    A simple, efficient and general HPLC method for the determination of enantiomeric purity of a series of (L)-amino acids was developed. In order to improve the detection sensitivity, pre-column derivatization was adopted and 7-chloro-4-nitrobenzoxadiazole (NBD-Cl) was selected as derivatization reagent. NBD-amino acid enantiomers were then enantioseparated on a Pirkle-type chiral stationary phase, Sumichiral OA-2500S (250 mm × 4.6 mm, 5 μm), using a mobile phase composed of acetonitrile-methanol (50:50, v/v) containing 5 mmol L(-1) citric acid at the flow rate of 0.5 mL min(-1). The detection wavelength was 470 nm. All the eleven pairs of tested amino acid enantiomers were well separated, and trace amounts of (D)-amino acids (0.5%) in the presence of a large excess of corresponding (L)-enantiomers could be quantified. The proposed method was validated in terms of selectivity, precision, linearity range, LOD, LOQ and accuracy, and then successfully applied to the determination of enantiomeric purity in bulk samples of (L)-amino acids.

  7. Spatiotemporal Localization of d-Amino Acid Oxidase and d-Aspartate Oxidases during Development in Caenorhabditis elegans

    PubMed Central

    Saitoh, Yasuaki; Katane, Masumi; Kawata, Tomonori; Maeda, Kazuhiro; Sekine, Masae; Furuchi, Takemitsu; Kobuna, Hiroyuki; Sakamoto, Taro; Inoue, Takao; Arai, Hiroyuki; Nakagawa, Yasuhito

    2012-01-01

    Recent investigations have shown that a variety of d-amino acids are present in living organisms and that they possibly play important roles in physiological functions in the body. d-Amino acid oxidase (DAO) and d-aspartate oxidase (DDO) are degradative enzymes stereospecific for d-amino acids. They have been identified in various organisms, including mammals and the nematode Caenorhabditis elegans, although the significance of these enzymes and the relevant functions of d-amino acids remain to be elucidated. In this study, we investigated the spatiotemporal localization of C. elegans DAO and DDOs (DDO-1, DDO-2, and DDO-3) and measured the levels of several d- and l-amino acids in wild-type C. elegans and four mutants in which each gene for DAO and the DDOs was partially deleted and thereby inactivated. Furthermore, several phenotypes of these mutant strains were characterized. The results reported in this study indicate that C. elegans DAO and DDOs are involved in egg-laying events and the early development of C. elegans. In particular, DDOs appear to play important roles in the development and maturation of germ cells. This work provides novel and useful insights into the physiological functions of these enzymes and d-amino acids in multicellular organisms. PMID:22393259

  8. Microbial Reworking Organic Matter in the Hauraki Gulf, New Zealand: Evidence from the Stable Isotopic Composition of Sedimentary D- and L-Amino Acids

    NASA Astrophysics Data System (ADS)

    Uhle, M. E.; Sikes, E. L.; Nodder, S. D.; Hage, M. M.; Howard, M. E.

    2004-05-01

    Amino acids are one of the more labile classes of organic matter in marine sediments. These compounds are the structural components of proteins and constitute the largest reservoir of organic nitrogen in most organisms. Their distribution and abundance have been used to assess diagenetic status of organic matter and their isotopic compositions have been linked to organic matter source identification. Organic matter in marine environments is derived from both allochthonous and autochthonous sources. Detailed source apportionment is difficult using bulk chemical characterization owing to the contribution of organic matter from multiple sources. Chemical and isotopic characterization of individual organic compounds, however, can yield detailed information on organic matter sources in complex systems. Sediment samples were recovered in 1999 from the Hauraki Gulf of New Zealand aboard the HMS Tangaroa. Near-shore and shelf environments were sampled along major currents to investigate the source and distribution of organic matter in the gulf. Amino acids were isolated from sediments by acid hydrolysis and quantified by gas chromatography/mass spectrometry following derivatization to their respective N-TFA-isopropyl esters. Isotopic compositions of the stereoisomers were determined by gas chromatography/isotope ratio mass spectrometry. Amino acid distributions from several sites throughout the gulf show a predominance of D- over L-amino acids, which reflects microbial input of amino acids in these sediments. At the Firth of Thames, the concentration of L-amino acids is greater than the D- isomers, which may be due to input of fresh algal material at this site. The isotopic compositions of many amino acids at this site reflect a marine source, which is consistent with the stereoisomer distributions. The carbon isotopic compositions of glycine and leucine at all sites are significantly depleted in 13C relative to typical values for marine and terrestrial sources and appear

  9. Bacterial Growth, Necromass Turnover, And Endospore Abundance In The Deep Subseafloor Sediments Of The Greenland Shelf Using D:L Amino Acid Model.

    NASA Astrophysics Data System (ADS)

    Mhatre, S. S.; Braun, S.; Jaussi, M.; Røy, H.; Jørgensen, B. B.; Lomstein, B. A.

    2015-12-01

    The subsurface realm is colonized by a large number of microorganisms- about 3 × 1029. Microbial cells in these very stable and oligotrophic settings catabolize at a much slower rate than model organisms in nutrient rich cultures. The aim of this work was to use recently developed D:L-amino acid racemization model for studying the turnover times of microbial biomass and microbial necromass in a ~12,000 years old Greenland shelf marine sediment samples. Sediments were analyzed for total hydrolysable amino acids (THAA), the bacterial endospore marker dipicolinic acid (DPA), and amino acid enantiomers of aspartic acid. The percentage amino acid carbon content (%TAAC) and the percentage amino acid nitrogen content (%TAAN) were used for determining the degradation state of the organic matter. Endospores quantified using DPA quantification method were found to be as abundant as vegetative cells. The microbial necromass turnover times were thousand years, and biomass turnover times were in the range of tens to hundred years. Studies with deeper sediment cores will further improve our understanding of the energetic limits of life in the deep biosphere.

  10. Production of phenylpyruvic acid from L-phenylalanine using an L-amino acid deaminase from Proteus mirabilis: comparison of enzymatic and whole-cell biotransformation approaches.

    PubMed

    Hou, Ying; Hossain, Gazi Sakir; Li, Jianghua; Shin, Hyun-Dong; Liu, Long; Du, Guocheng

    2015-10-01

    Phenylpyruvic acid (PPA) is an important organic acid that has a wide range of applications. In this study, the membrane-bound L-amino acid deaminase (L-AAD) gene from Proteus mirabilis KCTC 2566 was expressed in Escherichia coli BL21(DE3) and then the L-AAD was purified. After that, we used the purified enzyme and the recombinant E. coli whole-cell biocatalyst to produce PPA via a one-step biotransformation from L-phenylalanine. L-AAD was solubilized from the membrane and purified 52-fold with an overall yield of 13 %, which corresponded to a specific activity of 0.94 ± 0.01 μmol PPA min(-1)·mg(-1). Then, the biotransformation conditions for the pure enzyme and the whole-cell biocatalyst were optimized. The maximal production was 2.6 ± 0.1 g·L(-1) (specific activity of 1.02 ± 0.02 μmol PPA min(-1)·mg(-1) protein, 86.7 ± 5 % mass conversion rate, and 1.04 g·L(-1)·h(-1) productivity) and 3.3 ± 0.2 g L(-1) (specific activity of 0.013 ± 0.003 μmol PPA min(-1)·mg(-1) protein, 82.5 ± 4 % mass conversion rate, and 0.55 g·L(-1)·h(-1) productivity) for the pure enzyme and whole-cell biocatalyst, respectively. Comparative studies of the enzymatic and whole-cell biotransformation were performed in terms of specific activity, production, conversion, productivity, stability, need of external cofactors, and recycling. We have developed two eco-friendly and efficient approaches for PPA production. The strategy described herein may aid the biotransformational synthesis of other α-keto acids from their corresponding amino acids.

  11. Telmisartan prevents hepatic fibrosis and enzyme-altered lesions in liver cirrhosis rat induced by a choline-deficient L-amino acid-defined diet

    SciTech Connect

    Jin Haiyan; Yamamoto, Naoki; Uchida, Koichi; Terai, Shuji; Sakaida, Isao

    2007-12-28

    Rennin-angiotensin system is involved in liver fibrogenesis through activating hepatic stellate cells (HSCs). Telmisartan (Tel) is an angiotensin II type 1 receptor antagonist, could function as a selective peroxisome proliferator-activated receptor {gamma} activator. Here we studied the effect of Tel on liver fibrosis, pre-neoplastic lesions in vivo and primary HSCs in vitro. In vivo study, we used the choline-deficient L-amino acid-defined (CDAA)-diet induced rat NASH model. The rats were fed the CDAA diet for 8 weeks to induce liver fibrosis and pre-neoplastic lesions, and then co-administrated with Tel for another 10 weeks. Tel prevented liver fibrogenesis and pre-neoplastic lesions by down-regulating TGF{beta}1 and TIMP-1, 2 and increasing MMP-13 expression. Tel inhibited HSCs activation and proliferation. These results suggested that Tel could be a promising drug for NASH related liver fibrosis.

  12. NF-Y binding is required for transactivation of neuronal aromatic L-amino acid decarboxylase gene promoter by the POU-domain protein Brn-2.

    PubMed

    Dugast, C; Weber, M J

    2001-04-18

    We have previously characterized binding sites for the NF-Y transcription factor (-71/-52) and Brn-2 POU-domain protein (-92/-71) in the neuronal promoter of the human aromatic L-amino acid decarboxylase gene [Mol. Brain Res. 56 (1998) 227]. We have now explored the functional role of these binding sites in transfected SK-N-BE neuroblastoma cells. Mutations of the NF-Y site that abolish binding depressed expression of a luciferase reporter gene up to 25-fold. The overexpression of a dominant negative mutant of NF-YA subunit depressed expression by 60%. Promoter activity was increased by the overexpression of Brn-2. Mutations or deletion of the binding site of Brn-2 did not suppress transcriptional activation by overexpressed Brn-2, while promoters defective in NF-Y binding were not transactivated by Brn-2. A GST-pulldown experiment showed that recombinant human Brn-2 protein weakly interacts with recombinant NF-Y outside of DNA. Cooperative binding of recombinant NF-Y and GST--Brn-2 proteins on the neuronal promoter was evidenced by an electrophoretic mobility shift assay. The POU-domain of Brn-2 was sufficient for such interaction. The results thus suggest that the activation of the neuronal promoter of the aromatic L-amino acid decarboxylase gene requires a direct interaction between the ubiquitous NF-Y factor and a cell-specific POU-domain protein. The NF-Y, but not the Brn-2 binding site, is essential for the recruitment of the NF-Y/Brn-2 complex on the promoter. PMID:11311976

  13. Thermodynamical characteristics of the reaction of pyridoxal-5'-phosphate with L-amino acids in aqueous buffer solution

    NASA Astrophysics Data System (ADS)

    Barannikov, V. P.; Badelin, V. G.; Venediktov, E. A.; Mezhevoi, I. N.; Guseinov, S. S.

    2011-01-01

    The reaction of pyridoxal-5'-phosphate with L-isomers of alanine, lysine, arginine, aspartic acid, glutamic acid, and glycine in phosphate buffer solution was studied by absorption spectroscopy and the calorimetry of dissolution at physiological acidity of the medium (pH 7.35). The formation constants of Schiff bases during reactions and changes in Gibbs energy, enthalpy, and entropy were determined. It was shown that the formation constant of the Schiff base and its spectral properties depend on the nature of the bound amino acid. The progress of the reaction with a majority of amino acids is governed by the entropy factor due to the predominant role of the dehydration effect of the reaction center of amino acids during chemical reactions. The intramolecular electrostatic interaction of an ionized phosphate group with the positively charged amino group on the end of the chain of amino acid residue stabilizes the Schiff bases formed by lysine and arginine. The extinction coefficient of the base, equilibrium constant, and the exothermic effect of the reaction then increase. The excess negative charge on the end of the chain of amino acid residues of aspartic and glutamic acids destabilizes the molecule of the Schiff base. In this case, the equilibrium constant decreases and the endothermic effect of the reaction increases.

  14. Enantioselective analysis of D- and L-amino acids from mouse macrophages using high performance liquid chromatography.

    PubMed

    Kato, Shiro; Masuda, Yuki; Konishi, Morichika; Oikawa, Tadao

    2015-12-10

    The intrinsic D-amino acid profile of mouse macrophages extracted from the peritoneal cavity was analyzed using high performance liquid chromatography. Six D-amino acids (D-Asp, D-Ser, D-Ala, D-Leu, D-Gln and D-Lys) were detected in cell lysates of mouse macrophages. The content and the D/D+L ratio differed depending on the type of D-amino acid and were approximately 3.5-22 nmol/g cells, and approximately 1-20%, respectively. The D-amino acid composition of RAW 264.7 cells, which is a model macrophage cell line, was similar to that of the mouse macrophage. These results suggest that macrophages and RAW 264.7 cells with macrophage-like functions have a similar D-amino acid profile.

  15. Optical activity and ultraviolet absorbance detection of dansyl L-amino acids separated by gradient liquid chromatography

    SciTech Connect

    Not Available

    1987-04-01

    Many scientific investigations (e.g., geochronology, pharmaceuticals) have the need to determine enantiometric ratios of amino acids and other compounds. It has been reported that OA/UV or OA/RI (refractive index) are ideal methods for the determination of enantiomeric ratios without the need for chiral columns, chiral eluents, or diasteromer preparation. Unfortunately, only three amino acids are naturally UV absorbing (254 nm), and RI sensitivity for amino acids is low. Derivatization by several methods (o-phthalaldehyde, dansyl, phenylisothiocyanate, fluorescamine, 2,4-dinitrofluorobenzene, and phenylthiohydantoin) renders all amino acids UV absorbing and makes UV or fluorescence viable techniques for amino acids determinations. A previously neglected aspect of derivatization is the effect on optical activity. These highly polar groups influence the chiral center of amino acids drastically (electronic and steric effects). The shifting of the absorption band to the proximity of the wavelength used for OA measurements further enhances the importance of the substituent. The authors report here the determination of 17 dansyl amino acids in a mixture by UV absorbance and optical activity. This involves gradient elution. Previously, the optical activity detector (OAD) has been used only with isocratic HPLC.

  16. Differentiation of Positional Isomers of Hybrid Peptides Containing Repeats of β-Nucleoside Derived Amino Acid (β-Nda-) and L-Amino Acids by Positive and Negative Ion Electrospray Ionization Tandem Mass Spectrometry (ESI-MS n )

    NASA Astrophysics Data System (ADS)

    Raju, B.; Ramesh, M.; Srinivas, R.; Chandrasekhar, S.; Kiranmai, N.; Sarma, V. U. M.

    2011-04-01

    A new class of positional isomeric pairs of -Boc protected oligopeptides comprised of alternating nucleoside derived β-amino acid (β-Nda-) and L-amino acid residues (alanine, valine, and phenylalanine) have been differentiated by both positive and negative ion electrospray ionization ion-trap tandem mass spectrometry (ESI-MS n ). The protonated dipeptide positional isomers with β-Nda- at the N-terminus lose CH3OH, NH3, and C2H4O2, whereas these processes are absent for the peptides with L-amino acids at the N-terminus. Instead, the presence of L-amino acids at the N-terminus results in characteristic retro-Mannich reaction involving elimination of imine. A good correlation has been observed between the conformational structure of the peptides and the abundance of y{n/+} and b{n/+} ions in MS n spectra. In the case of tetrapeptide isomers that are reported to form helical structures in solution phase, no y{n/+} and b{n/+} ions are observed when the corresponding amide -NH- participates in the helical structures. In contrast, significant y{n/+} and b{n/+} ions are formed when the amide -NH- is not involved in the H-bonding. In the case of tetra- and hexapeptides, it is observed that abundant b{n/+} ions are formed, presumably with stable oxazolone structures when the C-terminus of the b{n/+} ions possessed L-amino acid and the β-Nda- at the C-terminus appears to prevent the cyclization process leading to the absence of corresponding b{n/+} ions.

  17. Alteration of the substrate specificity of l-amino acid ligase and selective synthesis of Met-Gly as a salt taste enhancer.

    PubMed

    Kino, Haruka; Kino, Kuniki

    2015-01-01

    Dipeptides have unique physiological functions. This study focused on the salt-taste-enhancing dipeptide Met-Gly. BL00235, an l-amino acid ligase from Bacillus licheniformis NBRC12200, synthesizes Met-Gly as a major product as well as Met-Met as a by-product. To alter the substrate specificity of BL00235 and synthesize Met-Gly selectively, we chose to alter Pro85 residue based on the BL00235 crystal structure. We predicted that Met might be not recognized as a C-terminal substrate by occupying the space around C-terminal substrate. Pro85 was replaced with Phe, Tyr, and Trp, which have bulky aromatic side chains, by site-directed mutagenesis. These mutants lost the capacity to synthesize Met-Met, during the synthesis of Met-Gly. Furthermore, they did not synthesize Met-Met, even when methionine was used as a substrate. These results show that the amino acid residue at position 85 has a key role in C-terminal substrate specificity. PMID:26088155

  18. Molecular cloning of genomic DNA and chromosomal assignment of the gene for human aromatic L-amino acid decarboxylase, the enzyme for catecholamine and serotonin biosynthesis

    SciTech Connect

    Sumi-Ichinose, Chiho ); Ichinose, Hiroshi; Nagatsu, Toshiharu ); Takahashi, Eiichi; Hori, Tadaaki )

    1992-03-03

    Aromatic L-amino acid decarboxylase (AADC) catalyzes the decarboxylation of both L-3,4-dihydroxyphenylalanine and L-5-hydroxytryptophan to dopamine and serotonin, respectively, which are major mammalian neurotransmitters and hormones belonging to catecholamines and indoleamines. This report describes the organization of the human AADC gene. The authors proved that the gene of human AADC consists of 15 exons spanning more than 85 kilobases and exists as a single copy in the haploid genome. The boundaries between exon and intron followed the AG/GT rule. The sizes of exons and introns ranged from 20 to 400 bp and from 1.0 to 17.7 kb, respectively, while the sizes of four introns were not determined. Untranslated regions located in the 5{prime} region of mRNA were encoded by two exons, exons 1 and 2. The transcriptional starting point was determined around G at position {minus}111 by primer extension and S1 mapping. There were no typical TATA box' and CAAT box' within 540 bp from the transcriptional starting point. The human AADC gene was mapped to chromosome band 7p12.1-p12.3 by fluorescence in situ hybridization. This is the first report on the genomic structure and chromosomal localization of the AADC gene in mammals.

  19. The role of the cell cycle in the cellular uptake of folate-modified poly(l-amino acid) micelles in a cell population

    NASA Astrophysics Data System (ADS)

    Tang, Jihui; Liu, Ziwei; Ji, Fenqi; Li, Yao; Liu, Junjie; Song, Jian; Li, Jun; Zhou, Jianping

    2015-12-01

    Nanoparticles are widely recognized as a vehicle for tumor-targeted therapies. There are many factors that can influence the uptake of nanoparticles, such as the size of the nanoparticles, and/or their shape, elasticity, surface charge and even the cell cycle phase. However, the influence of the cell cycle on the active targeting of a drug delivery system has been unknown until now. In this study, we initially investigated the folate receptor α (FR-α) expression in different phases of HeLa cells by flow cytometric and immunocytochemical methods. The results obtained showed that FR-α expression was cell cycle-dependent, i.e. the S cells' folate receptor expression was the highest as the cell progressed through its cycle. Then, we used folate modified poly(l-amino acid) micelles (FA-PM) as an example to investigate the influence of the cell cycle on the active targeting drug delivery system. The results obtained indicated that the uptake of FA-PM by cells was influenced by the cell cycle phase, and the S cells took up the greatest number of folate conjugated nanoparticles. Our findings suggest that future studies on ligand-mediated active targeting preparations should consider the cell cycle, especially when this system is used for a cell cycle-specific drug.

  20. D-amino acid-induced expression of D-amino acid oxidase in the yeast Schizosaccharomyces pombe.

    PubMed

    Takahashi, Shouji; Okada, Hirotsune; Abe, Katsumasa; Kera, Yoshio

    2012-12-01

    We investigated D-amino acid oxidase (DAO) induction in the popular model yeast Schizosaccharomyces pombe. The product of the putative DAO gene of the yeast expressed in E. coli displayed oxidase activity to neutral and basic D-amino acids, but not to an L-amino acid or acidic D-amino acids, showing that the putative DAO gene encodes catalytically active DAO. DAO activity was weakly detected in yeast cells grown on a culture medium without D-amino acid, and was approximately doubled by adding D-alanine. The elimination of ammonium chloride from culture medium induced activity by up to eight-fold. L-Alanine also induced the activity, but only by about half of that induced by D-alanine. The induction by D-alanine reached a maximum level at 2 h cultivation; it remained roughly constant until cell growth reached a stationary phase. The best inducer was D-alanine, followed by D-proline and then D-serine. Not effective were N-carbamoyl-D,L-alanine (a better inducer of DAO than D-alanine in the yeast Trigonopsis variabilis), and both basic and acidic D-amino acids. These results showed that S. pombe DAO could be a suitable model for analyzing the regulation of DAO expression in eukaryotic organisms. PMID:22986818

  1. Synthesis of variants of Marfey's reagent having d-amino acids as chiral auxiliaries and liquid-chromatographic enantioseparation of (RS)-Mexiletine in spiked plasma: assessment and comparison with L-amino acid analogs.

    PubMed

    Bhushan, Ravi; Vashistha, Vinod Kumar

    2015-01-30

    Five d-amino acids have been used for the first time to synthesize chiral derivatizing reagents (as variants of Marfey's reagent) by nucleophilic displacement of one of the fluorine atoms in 1,5-difluoro-2,4-dinitrobenzene as against the literature reports on application of only l-amino acids or their amides as chiral auxiliaries in dinitrobenzene (DNB) moiety. Five other DNB based reagents were also prepared by nucleophilic substitution of fluorine atom with the set of the same amino acids in l-configuration, as chiral auxiliaries. These reagents were characterized and used for synthesis of diastereomers of (RS)-Mexiletine spiked in human plasma. Diastereomers were synthesized employing microwave irradiation and were separated on reversed-phase C18 column. Performance of the two types of chiral derivatizing reagents was compared. The reagents containing d-amino acids provided enhanced separation of diastereomers than those containing l-amino acids. The best resolution was obtained using mobile phase consisting of acetonitrile and 0.1% trifluoroacetic acid in gradient mode. The detection was carried out at 340nm. The method so developed was validated for linearity, accuracy and precision. The limit of quantitation was found to be approximately 25.2ngmL(-1) in human plasma. PMID:25576038

  2. Synthesis of variants of Marfey's reagent having d-amino acids as chiral auxiliaries and liquid-chromatographic enantioseparation of (RS)-Mexiletine in spiked plasma: assessment and comparison with L-amino acid analogs.

    PubMed

    Bhushan, Ravi; Vashistha, Vinod Kumar

    2015-01-30

    Five d-amino acids have been used for the first time to synthesize chiral derivatizing reagents (as variants of Marfey's reagent) by nucleophilic displacement of one of the fluorine atoms in 1,5-difluoro-2,4-dinitrobenzene as against the literature reports on application of only l-amino acids or their amides as chiral auxiliaries in dinitrobenzene (DNB) moiety. Five other DNB based reagents were also prepared by nucleophilic substitution of fluorine atom with the set of the same amino acids in l-configuration, as chiral auxiliaries. These reagents were characterized and used for synthesis of diastereomers of (RS)-Mexiletine spiked in human plasma. Diastereomers were synthesized employing microwave irradiation and were separated on reversed-phase C18 column. Performance of the two types of chiral derivatizing reagents was compared. The reagents containing d-amino acids provided enhanced separation of diastereomers than those containing l-amino acids. The best resolution was obtained using mobile phase consisting of acetonitrile and 0.1% trifluoroacetic acid in gradient mode. The detection was carried out at 340nm. The method so developed was validated for linearity, accuracy and precision. The limit of quantitation was found to be approximately 25.2ngmL(-1) in human plasma.

  3. Separation of alpha-hydroxy acid enantiomers by high performance capillary electrophoresis using copper(II)-L-amino acid and copper(II)-aspartame complexes as chiral selectors in the background electrolyte.

    PubMed

    Desiderio, C; Aturki, Z; Fanali, S

    1994-06-01

    Optical isomers of some alpha-hydroxy acids, namely 2-, 3-phenyllactic acid, mandelic, p-hydroxy-, m-hydroxy and 3,4-di-hydroxymandelic acid, were separated by means of capillary zone electrophoresis in free solution, using copper (II) complexes with L-amino acid or aspartame ligands in the background electrolyte. The concentration and the pH dependence of the enantiomer separations have been studied in the cases of different chiral ligands and/or analytes. With the use of L-proline as ligand only the optical isomers of 3-phenyllactic acid were resolvable, whereas using L-hydroxyproline the D and L forms of all compounds, except for 2-phenyllactic acid, were separated. Better results were obtained with aspartame as chiral ligand. PMID:7982412

  4. Separation of alpha-hydroxy acid enantiomers by high performance capillary electrophoresis using copper(II)-L-amino acid and copper(II)-aspartame complexes as chiral selectors in the background electrolyte.

    PubMed

    Desiderio, C; Aturki, Z; Fanali, S

    1994-06-01

    Optical isomers of some alpha-hydroxy acids, namely 2-, 3-phenyllactic acid, mandelic, p-hydroxy-, m-hydroxy and 3,4-di-hydroxymandelic acid, were separated by means of capillary zone electrophoresis in free solution, using copper (II) complexes with L-amino acid or aspartame ligands in the background electrolyte. The concentration and the pH dependence of the enantiomer separations have been studied in the cases of different chiral ligands and/or analytes. With the use of L-proline as ligand only the optical isomers of 3-phenyllactic acid were resolvable, whereas using L-hydroxyproline the D and L forms of all compounds, except for 2-phenyllactic acid, were separated. Better results were obtained with aspartame as chiral ligand.

  5. Bioconversion of l-glutamic acid to α-ketoglutaric acid by an immobilized whole-cell biocatalyst expressing l-amino acid deaminase from Proteus mirabilis.

    PubMed

    Hossain, Gazi Sakir; Li, Jianghua; Shin, Hyun-dong; Chen, Rachel R; Du, Guocheng; Liu, Long; Chen, Jian

    2014-01-01

    The goal of this work was to develop an immobilized whole-cell biocatalytic process for the environment-friendly synthesis of α-ketoglutaric acid (α-KG) from l-glutamic acid. We compared the suitability of Escherichia coli and Bacillus subtilis strains overexpressing Proteus mirabilisl-amino acid deaminase (l-AAD) as potential biocatalysts. Although both recombinant strains were biocatalytically active, the performance of B. subtilis was superior to that of E. coli. With l-glutamic acid as the substrate, α-KG production levels by membranes isolated from B. subtilis and E. coli were 55.3±1.73 and 21.7±0.39μg/mg protein/min, respectively. The maximal conversion ratio of l-glutamic acid to α-KG was 31% (w/w) under the following optimal conditions: 15g/L l-glutamic acid, 20g/L whole-cell biocatalyst, 5mM MgCl2, 40°C, pH 8.0, and 24-h incubation. Immobilization of whole cells with alginate increased the recyclability by an average of 23.33% per cycle. This work established an efficient one-step biotransformation process for the production of α-KG using immobilized whole B. subtilis overexpressing P. mirabilisl-AAD. Compared with traditional multistep chemical synthesis, the biocatalytic process described here has the advantage of reducing environmental pollution and thus has great potential for the large-scale production of α-KG.

  6. In vivo pharmacokinetics, biodistribution and antitumor effect of amphiphilic poly(L-amino acids) micelles loaded with a novel all-trans retinoic acid derivative.

    PubMed

    Tang, Jihui; Wang, Xinqun; Wang, Ting; Chen, Feihu; Zhou, Jianping

    2014-01-23

    Poly(amino acid)s are well-known as biodegradable and environmentally acceptable materials. In this study, a series of poly(L-aspartic acid)-b-poly(L-phenylalanine) (PAA-PPA) compounds with different degrees of polymerization were used to prepare copolymer micelles for a poorly water-soluble drug 4-amino-2-trifluoromethyl-phenyl retinate (ATPR, a novel all-trans retinoic acid derivative) and in vivo pharmacokinetics, biodistribution and antitumor efficacy of ATPR delivered by PAA-PPA micelles were evaluated. The area under the plasma concentration time curve AUC0→∞ of ATPR-loaded PAA20PPA20 micelles was 2.23 and 1.97 times higher than that of ATPR solution and ATPR CrmEL solution, respectively; In addition, the mean residence time (MRT) was increased 1.67 and 1.97-fold, respectively and the total body clearance (CL) was reduced 2.25 and 1.98-fold, respectively. The biodistribution study indicated that most of the ATPR in the ATPR-M group was distributed in the liver and there was delayed liver aggregation compared with the ATPR solution and ATPR CrmEL solution groups. Furthermore, the antitumor efficacy of ATPR-loaded PAA20PPA20 micelles was demonstrated in in vivo antitumor models involving mice inoculated with the human gastric cancer cell line SGC-7901. At the same dose of 7mg/kg, the ATPR-loaded micelles group demonstrated a better tumor growth inhibition and induced differentiation than the groups given ATPR solution and ATPR CrmEL solution. Therefore, the ATPR-loaded PAA-PPA micelles appear to be a potentially useful drug delivery system for ATPR and suitable for the chemotherapy of gastric cancer.

  7. Distribution in Different Organisms of Amino Acid Oxidases with FAD or a Quinone As Cofactor and Their Role as Antimicrobial Proteins in Marine Bacteria.

    PubMed

    Campillo-Brocal, Jonatan C; Lucas-Elío, Patricia; Sanchez-Amat, Antonio

    2015-12-16

    Amino acid oxidases (AAOs) catalyze the oxidative deamination of amino acids releasing ammonium and hydrogen peroxide. Several kinds of these enzymes have been reported. Depending on the amino acid isomer used as a substrate, it is possible to differentiate between l-amino acid oxidases and d-amino acid oxidases. Both use FAD as cofactor and oxidize the amino acid in the alpha position releasing the corresponding keto acid. Recently, a novel class of AAOs has been described that does not contain FAD as cofactor, but a quinone generated by post-translational modification of residues in the same protein. These proteins are named as LodA-like proteins, after the first member of this group described, LodA, a lysine epsilon oxidase synthesized by the marine bacterium Marinomonas mediterranea. In this review, a phylogenetic analysis of all the enzymes described with AAO activity has been performed. It is shown that it is possible to recognize different groups of these enzymes and those containing the quinone cofactor are clearly differentiated. In marine bacteria, particularly in the genus Pseudoalteromonas, most of the proteins described as antimicrobial because of their capacity to generate hydrogen peroxide belong to the group of LodA-like proteins.

  8. Distribution in Different Organisms of Amino Acid Oxidases with FAD or a Quinone As Cofactor and Their Role as Antimicrobial Proteins in Marine Bacteria

    PubMed Central

    Campillo-Brocal, Jonatan C.; Lucas-Elío, Patricia; Sanchez-Amat, Antonio

    2015-01-01

    Amino acid oxidases (AAOs) catalyze the oxidative deamination of amino acids releasing ammonium and hydrogen peroxide. Several kinds of these enzymes have been reported. Depending on the amino acid isomer used as a substrate, it is possible to differentiate between l-amino acid oxidases and d-amino acid oxidases. Both use FAD as cofactor and oxidize the amino acid in the alpha position releasing the corresponding keto acid. Recently, a novel class of AAOs has been described that does not contain FAD as cofactor, but a quinone generated by post-translational modification of residues in the same protein. These proteins are named as LodA-like proteins, after the first member of this group described, LodA, a lysine epsilon oxidase synthesized by the marine bacterium Marinomonas mediterranea. In this review, a phylogenetic analysis of all the enzymes described with AAO activity has been performed. It is shown that it is possible to recognize different groups of these enzymes and those containing the quinone cofactor are clearly differentiated. In marine bacteria, particularly in the genus Pseudoalteromonas, most of the proteins described as antimicrobial because of their capacity to generate hydrogen peroxide belong to the group of LodA-like proteins. PMID:26694422

  9. A simple purification procedure of D-amino-acid oxidase from Candida guilliermondii H(see symbol)-4.

    PubMed

    Gevorgyan, G K; Davtyan, M A; Hambardzumyan, A A

    2012-01-01

    D-amino-acid oxidase (EC 1.4.3.3) was purified about 1480-fold from the yeast Candida guilliermondii H(see symbol)-4 using chromatofocusing method. The purification procedure gave an enzyme preparation which is greater than 90% homogenous on SDS-polyacrylamide gels with a specific activity of 11.54 U/mg at 30 degrees C with D-proline as substrate with the yield of total activity 9.3%. The molecular weights of subunit and native enzyme were determined to be 38.4 and 78.6 kDa by SDS-polyacrylamide gel electrophoresis and gel-filtration, respectively, suggesting that the native enzyme exists as a homodimer. A single molecular form with an isoelectric point of 6.85 was detected in analytical isoelectrofocusing. The optimum pH and temperature were 8.0 and 33 degrees C. An enzyme shows stability in the pH range from 7.4 to 9.0 and at the temperature no higher than 38 degrees C. Activation energy for D-amino-acid oxidase reaction was calculated to be 60 kJ/mol at 30 degrees C. The strict D-isomer specificity of the enzyme is confirmed, since no reaction could be detected with L-amino acids, and a large number of D-amino acids could be substrates for this enzyme. K(m) and V(max) values were determined for D-proline and D-alanine, which, among 22 tested, were the best substrates of the enzyme. D-amino-acid oxidase from the yeast C. guilliermondii is a flavoprotein oxidase in which the prosthetic group is tightly, but not covalently, bound FAD. The enzyme is completely inhibited by sodium benzoate, SH-oxidizing agents, but not by sodium azide, toluene or chloroform. PMID:23156699

  10. Persistent fibrosis in the liver of choline-deficient and iron-supplemented L-amino acid-defined diet-induced nonalcoholic steatohepatitis rat due to continuing oxidative stress after choline supplementation

    SciTech Connect

    Takeuchi-Yorimoto, Ayano; Noto, Takahisa; Yamada, Atsushi; Miyamae, Yoichi; Oishi, Yuji; Matsumoto, Masahiro

    2013-05-01

    Nonalcoholic steatohepatitis (NASH) is characterized by combined pathology of steatosis, lobular inflammation, fibrosis, and hepatocellular degeneration, with systemic symptoms of diabetes or hyperlipidemia, all in the absence of alcohol abuse. Given the therapeutic importance and conflicting findings regarding the potential for healing the histopathologic features of NASH in humans, particularly fibrosis, we investigated the reversibility of NASH-related findings in Wistar rats fed a choline-deficient and iron-supplemented L-amino acid-defined (CDAA) diet for 12 weeks, with a recovery period of 7 weeks, during which the diets were switched to a choline-sufficient and iron-supplemented L-amino acid-defined (CSAA) one. Analysis showed that steatosis and inflammation were significantly resolved by the end of the recovery period, along with decreases in AST and ALT activities within 4 weeks. In contrast, fibrosis remained even after the recovery period, to an extent similar to that in continuously CDAA-fed animals. Real-time reverse transcriptase-polymerase chain reaction, Western blot, and immunohistochemical investigations revealed that expression of some factors indicating oxidative stress (CYP2E1, 4-HNE, and iNOS) were elevated, whereas catalase and SOD1 were decreased, and a hypoxic state and CD34-positive neovascularization were evident even after the recovery period, although the fibrogenesis pathway by activated α-SMA-positive hepatic stellate cells via TGF-β and TIMPs decreased to the CSAA group level. In conclusion, persistent fibrosis was noted after the recovery period of 7 weeks, possibly due to sustained hypoxia and oxidative stress supposedly caused by capillarization. Otherwise, histopathological features of steatosis and inflammation, as well as serum AST and ALT activities, were recovered. - Highlights: ► NASH-like liver lesions are induced in rats by feeding a CDAA diet. ► Steatosis and lobular inflammation are resolved after switching to a

  11. One-Step Biosynthesis of α-Keto-γ-Methylthiobutyric Acid from L-Methionine by an Escherichia coli Whole-Cell Biocatalyst Expressing an Engineered L-Amino Acid Deaminase from Proteus vulgaris

    PubMed Central

    Shin, Hyun-dong; Du, Guocheng; Wang, Miao; Liu, Long; Chen, Jian

    2014-01-01

    α-Keto-γ-methylthiobutyric acid (KMTB), a keto derivative of l-methionine, has great potential for use as an alternative to l-methionine in the poultry industry and as an anti-cancer drug. This study developed an environment friendly process for KMTB production from l-methionine by an Escherichia coli whole-cell biocatalyst expressing an engineered l-amino acid deaminase (l-AAD) from Proteus vulgaris. We first overexpressed the P. vulgaris l-AAD in E. coli BL21 (DE3) and further optimized the whole-cell transformation process. The maximal molar conversion ratio of l-methionine to KMTB was 71.2% (mol/mol) under the optimal conditions (70 g/L l-methionine, 20 g/L whole-cell biocatalyst, 5 mM CaCl2, 40°C, 50 mM Tris-HCl [pH 8.0]). Then, error-prone polymerase chain reaction was used to construct P. vulgaris l-AAD mutant libraries. Among approximately 104 mutants, two mutants bearing lysine 104 to arginine and alanine 337 to serine substitutions showed 82.2% and 80.8% molar conversion ratios, respectively. Furthermore, the combination of these mutations enhanced the catalytic activity and molar conversion ratio by 1.3-fold and up to 91.4% with a KMTB concentration of 63.6 g/L. Finally, the effect of immobilization on whole-cell transformation was examined, and the immobilized whole-cell biocatalyst with Ca2+ alginate increased reusability by 41.3% compared to that of free cell production. Compared with the traditional multi-step chemical synthesis, our one-step biocatalytic production of KMTB has an advantage in terms of environmental pollution and thus has great potential for industrial KMTB production. PMID:25531756

  12. One-step biosynthesis of α-keto-γ-methylthiobutyric acid from L-methionine by an Escherichia coli whole-cell biocatalyst expressing an engineered L-amino acid deaminase from Proteus vulgaris.

    PubMed

    Hossain, Gazi Sakir; Li, Jianghua; Shin, Hyun-dong; Du, Guocheng; Wang, Miao; Liu, Long; Chen, Jian

    2014-01-01

    α-Keto-γ-methylthiobutyric acid (KMTB), a keto derivative of l-methionine, has great potential for use as an alternative to l-methionine in the poultry industry and as an anti-cancer drug. This study developed an environment friendly process for KMTB production from l-methionine by an Escherichia coli whole-cell biocatalyst expressing an engineered l-amino acid deaminase (l-AAD) from Proteus vulgaris. We first overexpressed the P. vulgaris l-AAD in E. coli BL21 (DE3) and further optimized the whole-cell transformation process. The maximal molar conversion ratio of l-methionine to KMTB was 71.2% (mol/mol) under the optimal conditions (70 g/L l-methionine, 20 g/L whole-cell biocatalyst, 5 mM CaCl2, 40°C, 50 mM Tris-HCl [pH 8.0]). Then, error-prone polymerase chain reaction was used to construct P. vulgaris l-AAD mutant libraries. Among approximately 104 mutants, two mutants bearing lysine 104 to arginine and alanine 337 to serine substitutions showed 82.2% and 80.8% molar conversion ratios, respectively. Furthermore, the combination of these mutations enhanced the catalytic activity and molar conversion ratio by 1.3-fold and up to 91.4% with a KMTB concentration of 63.6 g/L. Finally, the effect of immobilization on whole-cell transformation was examined, and the immobilized whole-cell biocatalyst with Ca2+ alginate increased reusability by 41.3% compared to that of free cell production. Compared with the traditional multi-step chemical synthesis, our one-step biocatalytic production of KMTB has an advantage in terms of environmental pollution and thus has great potential for industrial KMTB production.

  13. Left-handed helical preference in an achiral peptide chain is induced by an L-amino acid in an N-terminal type II β-turn.

    PubMed

    De Poli, Matteo; De Zotti, Marta; Raftery, James; Aguilar, Juan A; Morris, Gareth A; Clayden, Jonathan

    2013-03-15

    Oligomers of the achiral amino acid Aib adopt helical conformations in which the screw-sense may be controlled by a single N-terminal residue. Using crystallographic and NMR techniques, we show that the left- or right-handed sense of helical induction arises from the nature of the β-turn at the N terminus: the tertiary amino acid L-Val induces a left-handed type II β-turn in both the solid state and in solution, while the corresponding quaternary amino acid L-α-methylvaline induces a right-handed type III β-turn.

  14. Molecular aggregation in selected crystalline 1:1 complexes of hydrophobic D- and L-amino acids. IV. The L-phenylalanine series.

    PubMed

    Görbitz, Carl Henrik; Rissanen, Kari; Valkonen, Arto; Husabø, Asmund

    2009-06-01

    The amino acid L-phenylalanine has been cocrystallized with D-2-aminobutyric acid, C(9)H(11)NO(2).C(4)H(9)NO(2), D-norvaline, C(9)H(11)NO(2).C(5)H(11)NO(2), and D-methionine, C(9)H(11)NO(2).C(5)H(11)NO(2)S, with linear side chains, as well as with D-leucine, C(9)H(11)NO(2).C(6)H(13)NO(2), D-isoleucine, C(9)H(11)NO(2).C(6)H(13)NO(2), and D-allo-isoleucine, C(9)H(11)NO(2).C(6)H(13)NO(2), with branched side chains. The structures of these 1:1 complexes fall into two classes based on the observed hydrogen-bonding pattern. From a comparison with other L:D complexes involving hydrophobic amino acids and regular racemates, it is shown that the structure-directing properties of phenylalanine closely parallel those of valine and isoleucine but not those of leucine, which shares side-chain branching at C(gamma) with phenylalanine and is normally considered to be the most closely related non-aromatic amino acid.

  15. Unique and selective expression of L-amino acid transporter 1 in human tissue as well as being an aspect of oncofetal protein.

    PubMed

    Nakada, Norihiro; Mikami, Tetuo; Hana, Kiyomi; Ichinoe, Masaaki; Yanagisawa, Nobuyuki; Yoshida, Tsutomu; Endou, Hitoshi; Okayasu, Isao

    2014-02-01

    Dysregulated expression of L-type amino acid transporter 1 (LAT1), which transports large neutral amino acids, is a characteristic of various human cancers and possibly offers a molecular target for chemotherapy. LAT2, in contrast, shows lower expression in neoplasms. LAT1 is presumed to be a biomarker of many cancers, suggesting a kind of oncoprotein. However, no precise analysis of LAT1 and LAT2 expression has been performed in systemic normal tissues. To see characteristics of LAT1 and LAT2, immunohistochemical expression of LAT1 and LAT2 was assessed and compared in normal human systemic organs and tissues from 3 adults, 3 children and 3 fetuses in the present study. Cardiac muscles, hepatocytes, thymic epithelial cells and primitive neuroectodermal cells in fetus were positive with LAT1, whereas no expression was found in the respective adult tissues, indicating an aspect of oncofetal protein. In adult tissues, LAT1 was found to be expressed proximal to proliferative zones in gastrointestinal mucosa by double immunostaining of LAT1 and Ki-67. Testicular Sertoli cells, ovarian follicular cells, and pancreatic islet cells showed strong expression. Although the systemic capillary endothelium did not express LAT1, but did express LAT2, capillaries corresponding to the blood-brain, blood-follicle, and blood-retinal barriers demonstrated strong LAT1 immunoreactions. In conclusion, LAT1 was expressed in gonad tissues and several kinds of cells having special functions, as well as being discovered to be an aspect of oncofetal protein. In addition, ubiquitous LAT2 expression was confirmed immunohistochemically in systemic tissues, indicating constitutional function.

  16. Occurrence of N-phenylpropenoyl-L-amino acid amides in different herbal drugs and their influence on human keratinocytes, on human liver cells and on adhesion of Helicobacter pylori to the human stomach.

    PubMed

    Hensel, A; Deters, A M; Müller, G; Stark, T; Wittschier, N; Hofmann, T

    2007-02-01

    Thirty commonly used medicinal plants were screened by a selective and specific LC-MS/MS method for the occurrence of N-phenylpropenoyl- L-amino acid amides, a new homologous class of secondary products. In 15 plants, one or more of the respective derivatives (1 to 12) were found and quantitated. Especially roots from Angelica archangelica, fruits of Cassia angustifolia, C. senna, Coriandrum sativum, leaves from Hedera helix, flowers from Lavandula spec. and from Sambucus nigra contained high amounts (1 to 11 microg/g) of mixtures of the different amides 1 to 12. For functional investigations on potential activity in cellular physiology, two amides with an aliphatic (8) and an aromatic amino acid residue (5) were used. N-(E)-Caffeic acid L-aspartic acid amide (8) and N-(E)-caffeic acid L-tryptophan amide (5) stimulated mitochondrial activity as well as the proliferation rate of human liver cells (HepG2) at 10 microg/mL significantly. When monitoring the influence of selected phase I and II metabolizing enzymes, both compounds did not influence CYP3A4 gene expression, but stimulated CYP1A2 gene expression and inhibited GST expression. Also, the proliferation of human keratinocytes (NHK) was increased up to 150% by both amides 5 and 8; this stimulation was also detectable on the level of gene expression by an up-regulation of the transcription factor STAT6. The aliphatic aspartic compound 8 showed strong antiadhesive properties on the adhesion of Helicobacter pylori to human stomach tissue. PMID:17295182

  17. Conformational and functional effects induced by D- and L-amino acid epimerization on a single gene encoded peptide from the skin secretion of Hypsiboas punctatus.

    PubMed

    de Magalhães, Mariana T Q; Barbosa, Eder A; Prates, Maura V; Verly, Rodrigo M; Munhoz, Victor Hugo O; de Araújo, Ivan E; Bloch, Carlos

    2013-01-01

    Skin secretion of Hypsiboas punctatus is the source of a complex mixture of bioactive compounds where peptides and small proteins prevail, similarly to many other amphibians. Among dozens of molecules isolated from H. punctatus in a proteomic based approach, we report here the structural and functional studies of a novel peptide named Phenylseptin (FFFDTLKNLAGKVIGALT-NH2) that was purified as two naturally occurring D- and L-Phes configurations. The amino acid epimerization and C-terminal amidation for both molecules were confirmed by a combination of techniques including reverse-phase UFLC, ion mobility mass spectrometry, high resolution MS/MS experiments, Edman degradation, cDNA sequencing and solid-phase peptide synthesis. RMSD analysis of the twenty lowest-energy (1)H NMR structures of each peptide revealed a major 90° difference between the two backbones at the first four N-terminal residues and substantial orientation changes of their respective side chains. These structural divergences were considered to be the primary cause of the in vitro quantitative differences in antimicrobial activities between the two molecules. Finally, both molecules elicited equally aversive reactions in mice when delivered orally, an effect that depended entirely on peripheral gustatory pathways. PMID:23565145

  18. One-step biosynthesis of α-ketoisocaproate from l-leucine by an Escherichia coli whole-cell biocatalyst expressing an l-amino acid deaminase from Proteus vulgaris

    PubMed Central

    Song, Yang; Li, Jianghua; Shin, Hyun-dong; Du, Guocheng; Liu, Long; Chen, Jian

    2015-01-01

    This work aimed to develop a whole-cell biotransformation process for the production of α-ketoisocaproate from L-leucine. A recombinant Escherichia coli strain was constructed by expressing an L-amino acid deaminase from Proteus vulgaris. To enhance α-ketoisocaproate production, the reaction conditions were optimized as follows: whole-cell biocatalyst 0.8 g/L, leucine concentration 13.1 g/L, temperature 35 °C, pH 7.5, and reaction time 20 h. Under the above conditions, the α-ketoisocaproate titer reached 12.7 g/L with a leucine conversion rate of 97.8%. In addition, different leucine feeding strategies were examined to increase the α-ketoisocaproate titer. When 13.1 g/L leucine was added at 2-h intervals (from 0 to 22 h, 12 addition times), the α-ketoisocaproate titer reached 69.1 g/L, while the leucine conversion rate decreased to 50.3%. We have developed an effective process for the biotechnological production of α-ketoisocaproate that is more environmentally friendly than the traditional petrochemical synthesis approach. PMID:26217895

  19. Detection and substrate selectivity of new microbial D-amino acid oxidases.

    PubMed

    Gabler; Hensel; Fischer

    2000-11-01

    In order to screen for new microbial D-amino acid oxidase activities a selective and sensitive peroxidase/o-dianisidine assay, detecting the formation of hydrogen peroxide was developed. Catalase, which coexists with oxidases in the peroxisomes or the microsomes and, which competes with peroxidase for hydrogen peroxide, was completely inhibited by o-dianisidine up to a catalase activity of 500 nkat ml(-)(1). Thus, using the peroxidase/o-dianisidine assay and employing crude extracts of microorganisms in a microplate reader, a detection sensitivity for oxidase activity of 0.6 nkat ml(-)(1) was obtained.Wild type colonies which were grown on a selective medium containing D-alanine as carbon, energy and nitrogen source were examined for D-amino acid oxidase activity by the peroxidase/o-dianisidine assay. The oxidase positive colonies possessing an apparent oxidase activity > 2 nkat g dry biomass(-)(1) were isolated. Among them three new D-amino acid oxidase-producers were found and identified as Fusarium oxysporum, Verticilium lutealbum and Candida parapsilosis. The best new D-amino oxidase producer was the fungus F. oxysporum with a D-amino acid oxidase activity of about 900 nkat g dry biomass(-)(1) or 21 nkat mg protein(-)(1). With regard to the use as a biocatalytic tool in biotechnology the substrate specificities of the three new D-amino acid oxidases were compared with those of the known D-amino acid oxidases from Trigonopsis variabilis, Rhodotorula gracilis and pig kidney under the same conditions. All six D-amino acid oxidases accepted the D-enantiomers of alanine, valine, leucine, proline, phenylalanine, serine and glutamine as substrates and, except for the D-amino acid oxidase from V. luteoalbum, D-tryptophane, D-tyrosine, D-arginine and D-histidine were accepted as well. The relative highest activities (>95%) were measured versus D-alanine (C. parapsilosis, F. oxysporum, T. variabilis), D-methionine (V. luteoalbum, R. gracilis), D-valine (T. variabilis, R

  20. Locomotor response to L-DOPA in reserpine-treated rats following central inhibition of aromatic L-amino acid decarboxylase: further evidence for non-dopaminergic actions of L-DOPA and its metabolites.

    PubMed

    Alachkar, Amal; Brotchie, Jonathan M; Jones, Owen T

    2010-09-01

    L-DOPA is the most widely used treatment for Parkinson's disease. The anti-parkinsonian and pro-dyskinetic actions of L-DOPA are widely attributed to its conversion, by the enzyme aromatic L-amino acid decarboxylase (AADC), to dopamine. We investigated the hypothesis that exogenous L-DOPA can induce behavioural effects without being converted to dopamine in the reserpine-treated rat-model of Parkinson's disease. A parkinsonian state was induced with reserpine (3 mg/kg s.c.). Eighteen hours later, the rats were administered L-DOPA plus the peripherally acting AADC inhibitor benserazide (25 mg/kg), with or without the centrally acting AADC inhibitor NSD1015 (100 mg/kg). L-DOPA/benserazide alone reversed reserpine-induced akinesia (4158+/-1125 activity counts/6 h, cf vehicle 1327+/-227). Addition of NSD1015 elicited hyperactive behaviour that was approximately 7-fold higher than L-DOPA/benserazide (35755+/-5226, P<0.001). The hyperactivity induced by L-DOPA and NSD1015 was reduced by the alpha(2C) antagonist rauwolscine (1 mg/kg) and the 5-HT(2C) agonist MK212 (5 mg/kg), but not by the D2 dopamine receptor antagonist remoxipride (3 mg/kg) or the D1 dopamine receptor antagonist SCH23390 (1 mg/kg). These data suggest that L-DOPA, or metabolites produced via routes not involving AADC, might be responsible for the generation of at least some L-DOPA actions in reserpine-treated rats. PMID:20542064

  1. Synthesis of kojic acid derivatives as secondary binding site probes of D-amino acid oxidase

    PubMed Central

    Raje, Mithun; Hin, Niyada; Duvall, Bridget; Ferraris, Dana V.; Berry, James F.; Thomas, Ajit G.; Alt, Jesse; Rojas, Camilo; Slusher, Barbara S.; Tsukamoto, Takashi

    2013-01-01

    A series of kojic acid (5-hydroxy-2-hydroxymethyl-4H-pyran-4-one) derivatives were synthesized and tested for their ability to inhibit D-amino acid oxidase (DAAO). Various substituents were incorporated into kojic acid at its 2-hydroxymethyl group. These analogs serve as useful molecular probes to explore the secondary binding site, which can be exploited in designing more potent DAAO inhibitors. PMID:23683589

  2. Interplay between microbial d-amino acids and host d-amino acid oxidase modifies murine mucosal defence and gut microbiota.

    PubMed

    Sasabe, Jumpei; Miyoshi, Yurika; Rakoff-Nahoum, Seth; Zhang, Ting; Mita, Masashi; Davis, Brigid M; Hamase, Kenji; Waldor, Matthew K

    2016-01-01

    L-Amino acids are the building blocks for proteins synthesized in ribosomes in all kingdoms of life, but d-amino acids (d-aa) have important non-ribosome-based functions(1). Mammals synthesize d-Ser and d-Asp, primarily in the central nervous system, where d-Ser is critical for neurotransmission(2). Bacteria synthesize a largely distinct set of d-aa, which become integral components of the cell wall and are also released as free d-aa(3,4). However, the impact of free microbial d-aa on host physiology at the host-microbial interface has not been explored. Here, we show that the mouse intestine is rich in free d-aa that are derived from the microbiota. Furthermore, the microbiota induces production of d-amino acid oxidase (DAO) by intestinal epithelial cells, including goblet cells, which secrete the enzyme into the lumen. Oxidative deamination of intestinal d-aa by DAO, which yields the antimicrobial product H2O2, protects the mucosal surface in the small intestine from the cholera pathogen. DAO also modifies the composition of the microbiota and is associated with microbial induction of intestinal sIgA. Collectively, these results identify d-aa and DAO as previously unrecognized mediators of microbe-host interplay and homeostasis on the epithelial surface of the small intestine. PMID:27670111

  3. The pea gene NA encodes ent-kaurenoic acid oxidase.

    PubMed

    Davidson, Sandra E; Elliott, Robert C; Helliwell, Chris A; Poole, Andrew T; Reid, James B

    2003-01-01

    The gibberellin (GA)-deficient dwarf na mutant in pea (Pisum sativum) has severely reduced internode elongation, reduced root growth, and decreased leaflet size. However, the seeds develop normally. Two genes, PsKAO1 and PsKAO2, encoding cytochrome P450 monooxygenases of the subfamily CYP88A were isolated. Both PsKAO1 and PsKAO2 had ent-kaurenoic acid oxidase (KAO) activity, catalyzing the three steps of the GA biosynthetic pathway from ent-kaurenoic acid to GA(12) when expressed in yeast (Saccharomyces cerevisiae). In addition to the intermediates ent-7alpha-hydroxykaurenoic acid and GA(12)-aldehyde, some additional products of the pea KAO activity were detected, including ent-6alpha,7alpha-dihydroxykaurenoic acid and 7beta-hydroxykaurenolide. The NA gene encodes PsKAO1, because in two independent mutant alleles, na-1 and na-2, PsKAO1 had altered sequences and the five-base deletion in PsKAO1 associated with the na-1 allele cosegregated with the dwarf na phenotype. PsKAO1 was expressed in the stem, apical bud, leaf, pod, and root, organs in which GA levels have previously been shown to be reduced in na plants. PsKAO2 was expressed only in seeds and this may explain the normal seed development and normal GA biosynthesis in seeds of na plants.

  4. 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)).

  5. Deletion of glucose oxidase changes the pattern of organic acid production in Aspergillus carbonarius.

    PubMed

    Yang, Lei; Lübeck, Mette; Lübeck, Peter S

    2014-01-01

    Aspergillus carbonarius has potential as a cell factory for the production of different organic acids. At pH 5.5, A.carbonarius accumulates high amounts of gluconic acid when it grows on glucose based medium whereas at low pH, it produces citric acid. The conversion of glucose to gluconic acid is carried out by secretion of the enzyme, glucose oxidase. In this work, the gene encoding glucose oxidase was identified and deleted from A. carbonarius with the aim of changing the carbon flux towards other organic acids. The effect of genetic engineering was examined by testing glucose oxidase deficient (Δgox) mutants for the production of different organic acids in a defined production medium. The results obtained showed that the gluconic acid accumulation was completely inhibited and increased amounts of citric acid, oxalic acid and malic acid were observed in the Δgox mutants.

  6. Involvement of phospholipase D and NADPH-oxidase in salicylic acid signaling cascade.

    PubMed

    Kalachova, Tetiana; Iakovenko, Oksana; Kretinin, Sergii; Kravets, Volodymyr

    2013-05-01

    Salicylic acid is associated with the primary defense responses to biotic stress and formation of systemic acquired resistance. However, molecular mechanisms of early cell reactions to phytohormone application are currently undisclosed. The present study investigates the participation of phospholipase D and NADPH-oxidase in salicylic acid signal transduction cascade. The activation of lipid signaling enzymes within 15 min of salicylic acid application was shown in Arabidopsis thaliana plants by measuring the phosphatidic acid accumulation. Adding of primary alcohol (1-butanol) to the incubation medium led to phosphatidylbutanol accumulation as a result of phospholipase D (PLD) action in wild-type and NADPH-oxidase RbohD deficient plants. Salicylic acid induced rapid increase in NADPH-oxidase activity in histochemical assay with nitroblue tetrazolium but the reaction was not observed in presence of 1-butanol and NADPH-oxidase inhibitor diphenylene iodide (DPI). The further physiological effect of salicylic acid and inhibitory analysis of the signaling cascade were made in the guard cell model. Stomatal closure induced by salicylic acid was inhibited by 1-butanol and DPI treatment. rbohD transgenic plants showed impaired stomatal reaction upon phytohormone effect, while the reaction to H2O2 did not differ from that of wild-type plants. Thus a key role of NADPH-oxidase D-isoform in the process of stomatal closure in response to salicylic acid has been postulated. It has enabled to predict a cascade implication of PLD and NADPH oxidase to salicylic acid signaling pathway.

  7. Replacement of a terminal cytochrome c oxidase by ubiquinol oxidase during the evolution of acetic acid bacteria.

    PubMed

    Matsutani, Minenosuke; Fukushima, Kota; Kayama, Chiho; Arimitsu, Misato; Hirakawa, Hideki; Toyama, Hirohide; Adachi, Osao; Yakushi, Toshiharu; Matsushita, Kazunobu

    2014-10-01

    The bacterial aerobic respiratory chain has a terminal oxidase of the heme-copper oxidase superfamily, comprised of cytochrome c oxidase (COX) and ubiquinol oxidase (UOX); UOX evolved from COX. Acetobacter pasteurianus, an α-Proteobacterial acetic acid bacterium (AAB), produces UOX but not COX, although it has a partial COX gene cluster, ctaBD and ctaA, in addition to the UOX operon cyaBACD. We expressed ctaB and ctaA genes of A. pasteurianus in Escherichia coli and demonstrated their function as heme O and heme A synthases. We also found that the absence of ctaD function is likely due to accumulated mutations. These COX genes are closely related to other α-Proteobacterial COX proteins. However, the UOX operons of AAB are closely related to those of the β/γ-Proteobacteria (γ-type UOX), distinct from the α/β-Proteobacterial proteins (α-type UOX), but different from the other γ-type UOX proteins by the absence of the cyoE heme O synthase. Thus, we suggest that A. pasteurianus has a functional γ-type UOX but has lost the COX genes, with the exception of ctaB and ctaA, which supply the heme O and A moieties for UOX. Our results suggest that, in AAB, COX was replaced by β/γ-Proteobacterial UOX via horizontal gene transfer, while the COX genes, except for the heme O/A synthase genes, were lost. PMID:24862920

  8. Methanol and ethanol oxidase respiratory chains of the methylotrophic acetic acid bacterium, Acetobacter methanolicus.

    PubMed

    Matsushita, K; Takahashi, K; Takahashi, M; Ameyama, M; Adachi, O

    1992-06-01

    Acetobacter methanolicus is a unique acetic acid bacterium which has a methanol oxidase respiratory chain, as seen in methylotrophs, in addition to its ethanol oxidase respiratory chain. In this study, the relationship between methanol and ethanol oxidase respiratory chains was investigated. The organism is able to grow by oxidizing several carbon sources, including methanol, glycerol, and glucose. Cells grown on methanol exhibited a high methanol-oxidizing activity and contained large amounts of methanol dehydrogenase and soluble cytochromes c. Cells grown on glycerol showed higher oxygen uptake rate and dehydrogenase activity with ethanol but little methanol-oxidizing activity. Furthermore, two different terminal oxidases, cytochrome c and ubiquinol oxidases, have been shown to be involved in the respiratory chain; cytochrome c oxidase predominates in cells grown on methanol while ubiquinol oxidase predominates in cells grown on glycerol. Both terminal oxidases could be solubilized from the membranes and separated from each other. The cytochrome c oxidase and the ubiquinol oxidase have been shown to be a cytochrome co and a cytochrome bo, respectively. Methanol-oxidizing activity was diminished by several treatments that disrupt the integrity of the cells. The activity of the intact cells was inhibited with NaCl and/or EDTA, which disturbed the interaction between methanol dehydrogenase and cytochrome c. Ethanol-oxidizing activity in the membranes was inhibited with 2-heptyl-4-hydroxyquinoline N-oxide, which inhibited ubiquinol oxidase but not cytochrome c oxidase. Alcohol dehydrogenase has been purified from the membranes of glycerol-grown cells and shown to reduce ubiquinone-10 as well as a short side-chain homologue in detergent solution.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. Expression of ascorbic acid oxidase in zucchini squash (Cucurbita pepo L. )

    SciTech Connect

    Lin, Liangshiou; Varner, J.E. )

    1991-05-01

    The expression of ascorbic acid oxidase was studied in zuchini squash (Cucurbita pepo L.), one of the most abundant natural sources of the enzyme. In the developing fruit, specific activity of ascorbic acid oxidase was highest between 4 and 6 days after anthesis. Protein and mRNA levels followed the same trend as enzyme activity. Highest growth rate of the fruit occurred before 6 days after anthesis. Within a given fruit, ascorbic acid oxidase activity was higher in young leaves, and very low in old leaves. Within a given leaf, enzyme activity was highest in the fast-growing region (approximately the lower third of the blade), and lowest in the central placental region. In leaf tissue, ascorbic acid oxidase activity was higher in young leaves, and very low in old leaves. Within a given leaf, enzyme activity was highest in the fast-growing region (approximately the lower third of the blade), and lowest in the slow-growing region (near leaf apex). High expression of ascorbic acid oxidase at a stage when rapid growth is occurring (in both fruits and leaves), and localization of the enzyme in the fruit epidermis, where cells are under greatest tension during rapid growth in girth, suggest that ascorbic acid oxidase might be involved in reorganization of the cell wall to allow for expansion. Based on the known chemistry of dehydroascorbic acid, the end product of the ascorbic acid oxidase-catalyzed reaction, the authors have proposed several hypotheses to explain how dehydroascorbic acid might cause cell wall loosening.

  10. Phytanic acid alpha-oxidase deficiency (Refsum disease) presenting in infancy.

    PubMed

    Herbert, M A; Clayton, P T

    1994-01-01

    This report describes a patient with high serum phytanic acid concentration due to phytanic acid alpha-oxidase deficiency (classical Refsum disease). He presented unusually early, hypotonia and developmental delay being apparent by 7 months. A generalized peroxisomal disorder (so-called 'infantile Refsum disease') was excluded by analyses of pristanic acid, very long-chain fatty acids, bile acids and plasmalogen synthesis. The early presentation raises the possibility of in utero exposure to phytanate.

  11. Oxidase-peroxidase enzymes of Datura innoxia. Oxidation of formylphenylacetic acid ethyl ester.

    PubMed Central

    Kalyanaraman, V S; Mahadevan, S; Kumar, S A

    1975-01-01

    An enzyme system from Datura innoxia roots oxidizing formylphenylacetic acid ethyl ester was purified 38-fold by conventional methods such as (NH4)2SO4 fractionation, negative adsorption on alumina Cy gel and chromatography on DEAE-cellulose. The purified enzyme was shown to catalyse the stoicheiometric oxidation of formylphenylacetic acid ethyl ester to benzoylformic acid ethyl ester and formic acid, utilizing molecular O2. Substrate analogues such as phenylacetaldehyde and phenylpyruvate were oxidized at a very low rate, and formylphenylacetonitrile was an inhilating agents, cyanide, thiol compounds and ascorbic acid. This enzyme was identical with an oxidase-peroxidase isoenzyme. Another oxidase-peroxidase isoenzyme which separated on DEAE-chromatography also showed formylphenylacetic acid ethyl ester oxidase activity, albeit to a lesser extent. The properties of the two isoenzymes of the oxidase were compared and shown to differ in their oxidation and peroxidation properties. The oxidation of formylphenylacetic acid ethyl ester was also catalysed by horseradish peroxidase. The Datura isoenzymes exhibited typical haemoprotein spectra. The oxidation of formylphenylacetic acid ethyl ester was different from other peroxidase-catalysed reactions in not being activated by either Mn2+ or monophenols. The oxidation was inhibited by several mono- and poly-phenols and by catalase. A reaction mechanism for the oxidation is proposed. PMID:997

  12. NADPH oxidase-dependent acid production in airway epithelial cells.

    PubMed

    Schwarzer, Christian; Machen, Terry E; Illek, Beate; Fischer, Horst

    2004-08-27

    The purpose of this study was to determine the role of NADPH oxidase in H(+) secretion by airway epithelia. In whole cell patch clamp recordings primary human tracheal epithelial cells (hTE) and the human serous gland cell line Calu-3 expressed a functionally similar zinc-blockable plasma membrane H(+) conductance. However, the rate of H(+) secretion of confluent epithelial monolayers measured in Ussing chambers was 9-fold larger in hTE compared with Calu-3. In hTE H(+) secretion was blocked by mucosal ZnCl(2) and the NADPH oxidase blockers acetovanillone and 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), whereas these same blockers had no effect in Calu-3. We determined levels of transcripts for the NADPH oxidase transmembrane isoforms (Nox1 through -5, Duox1 and -2, and p22(phox)) and found Duox1, -2, and p22(phox) to be highly expressed in hTE, as well as the intracellular subunits p40(phox), p47(phox), and p67(phox). In contrast, Calu-3 lacked transcripts for Duox1, p40(phox), and p47(phox). Anti-Duox antibody staining resulted in prominent apical staining in hTE but no significant staining in Calu-3. When treated with amiloride to block the Na(+)/H(+) exchanger, intracellular pH in hTE acidified at significantly higher rates than in Calu-3, and treatment with AEBSF blocked acidification. These data suggest a role for an apically located Duox-based NADPH oxidase during intracellular H(+) production and H(+) secretion, but not in H(+) conduction.

  13. Simultaneous production of catalase, glucose oxidase and gluconic acid by Aspergillus niger mutant.

    PubMed

    Fiedurek, J; Gromada, A; Pielecki, J

    1998-01-01

    The production of gluconic acid, extracellular glucose oxidase and catalase in submerged culture by a number of biochemical mutants has been evaluated. Optimization of stirrer speed, time cultivation and buffering action of some chemicals on glucose oxidase, catalase and gluconic acid production by the most active mutant, AM-11, grown in a 3-L glass bioreactor was investigated. Three hundred rpm appeared to be optimum to ensure good growth and best glucose oxidase production, but gluconic acid or catalase activity obtained maximal value at 500 or 900 rpm, respectively. Significant increase of dissolved oxygen concentration in culture (16-21%) and extracellular catalase activity were obtained when the traditional aeration was employed together with automatic dosed hydrogen peroxide.

  14. Time dependent inhibition of xanthine oxidase in irradiated solutions of folic acid, aminopterin and methotrexate

    SciTech Connect

    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 in solution and the ability of these solutions to inhibit xanthine oxidase. The relative potency of the respective photolysis products were estimated.

  15. Resolution and isolation of enantiomers of (±)-isoxsuprine using thin silica gel layers impregnated with L-glutamic acid, comparison of separation of its diastereomers prepared with chiral derivatizing reagents having L-amino acids as chiral auxiliaries.

    PubMed

    Bhushan, Ravi; Nagar, Hariom

    2015-03-01

    Thin silica gel layers impregnated with optically pure l-glutamic acid were used for direct resolution of enantiomers of (±)-isoxsuprine in their native form. Three chiral derivatizing reagents, based on DFDNB moiety, were synthesized having l-alanine, l-valine and S-benzyl-l-cysteine as chiral auxiliaries. These were used to prepare diastereomers under microwave irradiation and conventional heating. The diastereomers were separated by reversed-phase high-performance liquid chromatography on a C18 column with detection at 340 nm using gradient elution with mobile phase containing aqueous trifluoroacetic acid and acetonitrile in different compositions and by thin-layer chromatography (TLC) on reversed phase (RP) C18 plates. Diastereomers prepared with enantiomerically pure (+)-isoxsuprine were used as standards for the determination of the elution order of diastereomers of (±)-isoxsuprine. The elution order in the experimental study of RP-TLC and RP-HPLC supported the developed optimized structures of diastereomers based on density functional theory. The limit of detection was 0.1-0.09 µg/mL in TLC while it was in the range of 22-23 pg/mL in HPLC and 11-13 ng/mL in RP-TLC for each enantiomer. The conditions of derivatization and chromatographic separation were optimized. The method was validated for accuracy, precision, limit of detection and limit of quantification.

  16. The role of superoxide in xanthine oxidase-induced autooxidation of linoleic acid.

    PubMed

    Thomas, M J; Mehl, K S; Pryor, W A

    1982-07-25

    The effect of hydroxyperoxyoctadecadienoic acid, e.g. 13-hydroperoxy-cis,9,trans-11-octadecadienoic acid, on the autooxidation of linoleic acid induced by superoxide radical was examined in a system containing xanthine oxidase, acetaldehyde, and diethylenetriaminepentaacetic acid dissolved in an aqueous phosphate buffer containing 10% ethanol. The superoxide radical is required for autooxidation, as shown by essentially complete inhibition on the addition of superoxide dismutase. Pure linoleic acid was not readily oxidized, but the addition of lipid hydroperoxide markedly stimulated the autooxidation. Addition of 2.8 microM FeCl3 did not produce an increase in the rate of xanthine oxidase-induced autooxidation. Spontaneous autooxidation, a process slower than xanthine oxidase-induced autooxidation, was detectable on the time scale of these observations but was slower than the xanthine oxidase-induced autooxidation. Initiation of linoleic acid autooxidation is postulated to result from a reaction between superoxide and lipid hydroperoxide. The nature of this reaction is uncertain, but it does not appear to depend on iron catalysis. PMID:6282880

  17. Effect of high pressure on peanut allergens in the presence of polyphenol oxidase and caffeic acid

    Technology Transfer Automated Retrieval System (TEKTRAN)

    High pressure (HP) enhances enzymatic reactions. Because polyphenol oxidase (PPO) is an enzyme, and reduces IgE binding of peanut allergens in presence of caffeic acid (CA), we postulated that a further reduction in IgE binding can be achieved, using HP together with PPO and CA. Peanut extracts cont...

  18. D-Amino acid oxidase and presence of D-proline in Xenopus laevis.

    PubMed

    Soma, Hiroki; Furuya, Ryuji; Kaneko, Ryo; Tsukamoto, Ayaka; Shirasu, Kazumitsu; Tanigawa, Minoru; Nagata, Yoko

    2013-10-01

    We purified D-amino acid oxidase (EC 1.4.3.3, DAO) from Xenopus laevis tadpoles. The optimal temperature and pH for enzyme activity were 35-40 °C and 8.3-9.0, respectively, depending on the substrate amino acids available to the enzyme; the highest activity was observed with D-proline followed by D-phenylalanine. Activity was significantly inhibited by p-hydroxymercuribenzoate, but only moderately by p-chloromercuribenzoate or benzoate. Enzyme activity was increased until the final tadpole stage, but was reduced to one-third in the adult and was localized primarily in the kidney. The tadpoles contained high concentrations of D-proline close to the final developmental stage and nearly no D-amino acids were detected in the adult frog, indicating that D-amino acid oxidase functions in metamorphosis.

  19. Electrochemical L-lactic acid sensor based on immobilized ZnO nanorods with lactate oxidase.

    PubMed

    Ibupoto, Zafar Hussain; Shah, Syed Muhammad Usman Ali; Khun, Kimleang; Willander, Magnus

    2012-01-01

    In this work, fabrication of gold coated glass substrate, growth of ZnO nanorods and potentiometric response of lactic acid are explained. The biosensor was developed by immobilizing the lactate oxidase on the ZnO nanorods in combination with glutaraldehyde as a cross linker for lactate oxidase enzyme. The potentiometric technique was applied for the measuring the output (EMF) response of l-lactic acid biosensor. We noticed that the present biosensor has wide linear detection range of concentration from 1 × 10(-4)-1 × 10(0) mM with acceptable sensitivity about 41.33 ± 1.58 mV/decade. In addition, the proposed biosensor showed fast response time less than 10 s, a good selectivity towards l-lactic acid in presence of common interfering substances such as ascorbic acid, urea, glucose, galactose, magnesium ions and calcium ions. The present biosensor based on immobilized ZnO nanorods with lactate oxidase sustained its stability for more than three weeks. PMID:22736960

  20. Renaturation studies of free and immobilized D-amino-acid oxidase.

    PubMed

    Carrea, G; Pasta, P; Curti, B

    1983-06-15

    The renaturation of free and Sepharose-immobilized D-amino-acid oxidase (D-amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.3), after its denaturation with 6 M guanidine hydrochloride, was investigated. No reactivation, or extremely limited reactivation (less than or equal to 4+), was obtained with the free enzyme, is spite of various attempts including the use of dialysis or buffers containing cofactors, different types of anions, surfactants and low concentrations of denaturing agents. The main obstacle to renaturation appeared to be the interaction among denatured or partially renatured monomers giving rise to inactive aggregates. In contrast, using the immobilized enzyme approach, substantial renaturation (up to 50%) of D-amino-acid oxidase was achieved. The denaturation-renaturation process was followed by monitoring the catalytic activity as well as the intrinsic protein fluorescence. An inverse correlation was found to exist between the degree of matrix activation by CNBr and the yield of enzyme reactivation. The anions of the lyotropic series markedly influenced the reactivation, showing an effectiveness opposite to their salting-out potential (thiocyanate congruent to iodide greater than chloride greater than phosphate congruent to sulphate congruent to citrate). Instead, the anions considerably increased the activity and stability of free and immobilized enzyme, according to their salting-out potential. Immobilized monomers of D-amino-acid oxidase, which in solution undergoes self-association, showed poor capacity to interact with the free enzyme: thus they appear unsuitable for analytical and preparative purposes.

  1. Electrochemical l-Lactic Acid Sensor Based on Immobilized ZnO Nanorods with Lactate Oxidase

    PubMed Central

    Ibupoto, Zafar Hussain; Ali Shah, Syed Muhammad Usman; Khun, Kimleang; Willander, Magnus

    2012-01-01

    In this work, fabrication of gold coated glass substrate, growth of ZnO nanorods and potentiometric response of lactic acid are explained. The biosensor was developed by immobilizing the lactate oxidase on the ZnO nanorods in combination with glutaraldehyde as a cross linker for lactate oxidase enzyme. The potentiometric technique was applied for the measuring the output (EMF) response of l-lactic acid biosensor. We noticed that the present biosensor has wide linear detection range of concentration from 1 × 10−4–1 × 100 mM with acceptable sensitivity about 41.33 ± 1.58 mV/decade. In addition, the proposed biosensor showed fast response time less than 10 s, a good selectivity towards l-lactic acid in presence of common interfering substances such as ascorbic acid, urea, glucose, galactose, magnesium ions and calcium ions. The present biosensor based on immobilized ZnO nanorods with lactate oxidase sustained its stability for more than three weeks. PMID:22736960

  2. Electrochemical L-lactic acid sensor based on immobilized ZnO nanorods with lactate oxidase.

    PubMed

    Ibupoto, Zafar Hussain; Shah, Syed Muhammad Usman Ali; Khun, Kimleang; Willander, Magnus

    2012-01-01

    In this work, fabrication of gold coated glass substrate, growth of ZnO nanorods and potentiometric response of lactic acid are explained. The biosensor was developed by immobilizing the lactate oxidase on the ZnO nanorods in combination with glutaraldehyde as a cross linker for lactate oxidase enzyme. The potentiometric technique was applied for the measuring the output (EMF) response of l-lactic acid biosensor. We noticed that the present biosensor has wide linear detection range of concentration from 1 × 10(-4)-1 × 10(0) mM with acceptable sensitivity about 41.33 ± 1.58 mV/decade. In addition, the proposed biosensor showed fast response time less than 10 s, a good selectivity towards l-lactic acid in presence of common interfering substances such as ascorbic acid, urea, glucose, galactose, magnesium ions and calcium ions. The present biosensor based on immobilized ZnO nanorods with lactate oxidase sustained its stability for more than three weeks.

  3. Endoplasmic reticulum thiol oxidase deficiency leads to ascorbic acid depletion and noncanonical scurvy in mice.

    PubMed

    Zito, Ester; Hansen, Henning Gram; Yeo, Giles S H; Fujii, Junichi; Ron, David

    2012-10-12

    Endoplasmic reticulum (ER) thiol oxidases initiate a disulfide relay to oxidatively fold secreted proteins. We found that combined loss-of-function mutations in genes encoding the ER thiol oxidases ERO1α, ERO1β, and PRDX4 compromised the extracellular matrix in mice and interfered with the intracellular maturation of procollagen. These severe abnormalities were associated with an unexpectedly modest delay in disulfide bond formation in secreted proteins but a profound, 5-fold lower procollagen 4-hydroxyproline content and enhanced cysteinyl sulfenic acid modification of ER proteins. Tissue ascorbic acid content was lower in mutant mice, and ascorbic acid supplementation improved procollagen maturation and lowered sulfenic acid content in vivo. In vitro, the presence of a sulfenic acid donor accelerated the oxidative inactivation of ascorbate by an H(2)O(2)-generating system. Compromised ER disulfide relay thus exposes protein thiols to competing oxidation to sulfenic acid, resulting in depletion of ascorbic acid, impaired procollagen proline 4-hydroxylation, and a noncanonical form of scurvy.

  4. Kinetics of Inhibition of Monoamine Oxidase Using Curcumin and Ellagic Acid

    PubMed Central

    Khatri, Dharmendra Kumar; Juvekar, Archana Ramesh

    2016-01-01

    Background: Curcumin and ellagic are the natural polyphenols having a wide range of pharmacological actions. They have been reported to have their use in various neurological disorders. Objective: This study was aimed to evaluate the effect of curcumin and ellagic acid on the activity of monoamine oxidase (MAO), the enzyme responsible for metabolism of monoamine neurotransmitters which are pivotal for neuronal development and function. Materials and Methods: The in vitro effects of these selected polyphenols on MAO activities in mitochondria isolated from rat brains were examined. Brain mitochondria were assayed for MAO type-B (MAO-B) using benzylamine as substrates. Rat brain mitochondrial MAO preparation was used to study the kinetics of enzyme inhibition using double reciprocal Lineweaver–Burk plot. Results: MAO activity was inhibited by curcumin and ellagic acid; however, higher half maximal inhibitory concentrations of curcumin (500.46 nM) and ellagic acid (412.24 nM) were required compared to the known MAO-B inhibitor selegiline. It is observed that the curcumin and ellagic acid inhibit the MAO activity with both the competitive and noncompetitive type of inhibitions. Conclusions: Curcumin and ellagic acid can be considered a possible source of MAO inhibitor used in the treatment of Parkinson's and other neurological disorders. SUMMARY Monoamine oxidase (MAO) is involved in a variety of neurological disorders including Parkinson's disease (PD)Curcumin and ellagic acid inhibit the monoamine oxidase activityEllagic acid revealed more potent MAO type-B (MAO-B) inhibitory activity than curcuminKinetic studies of MAO inhibition using different concentrations of curcumin and ellagic acid were plotted as double reciprocal Lineweaver–Burk plotThe mode of inhibition of both compounds toward MAO-B is mixed (competitive and uncompetitive) type of inhibition with both the competitive and noncompetitive type of inhibitions. Abbreviations used: MAO: Monoamine oxidase

  5. Prevention by 1'-acetoxychavicol acetate of the induction but not growth of putative preneoplastic, glutathione S-transferase placental form-positive, focal lesions in the livers of rats fed a choline-deficient, L-amino acid-defined diet.

    PubMed

    Kobayashi, Y; Nakae, D; Akai, H; Kishida, H; Okajima, E; Kitayama, W; Denda, A; Tsujiuchi, T; Murakami, A; Koshimizu, K; Ohigashi, H; Konishi, Y

    1998-10-01

    The effects of 1'-acetoxychavicol acetate (ACA) on endogenous rat liver carcinogenesis because of chronic feeding of a choline-deficient, L-amino acid-defined (CDAA) diet were examined. Male Fischer 344 rats, 6 weeks old, received the CDAA diet containing ACA at doses of 0, 0.005, 0.010 and 0.050% for 12 weeks and were then killed. ACA decreased the numbers of putative preneoplastic, glutathione S-transferase placental form (GST-P)-positive, focal lesions developing in the livers of rats fed the CDAA diet but did not alter their sizes. At the same time, ACA reduced the levels of 8-hydroxyguanine, a parameter of oxidative DNA damage, but did not significantly affect generation of 2-thiobarbituric acid-reacting substances, indicators of oxidative extra-DNA damage, or hepatocyte proliferation. Furthermore, ACA did not exert any significant effects on the numbers or sizes of GST-P-positive lesions in the livers of rats when administered between weeks 2 and 8 after initiation with a single i.p. dose of 200 mg/kg body wt of N-nitrosodiethylamine. These results indicate that ACA prevents the CDAA diet-associated induction of putative preneoplastic lesions by reduction of oxidative DNA damage but does not affect their subsequent growth.

  6. On-column labeling technique and chiral ligand-exchange CE with zinc(II)-L-arginine complex as a chiral selector for assay of dansylated D,L-amino acids.

    PubMed

    Qi, Li; Yang, Gengliang

    2009-08-01

    A novel on-column labeling method of amino acid (AA) enantiomers by using dansyl chloride (Dns-Cl) has been explored combined with chiral ligand-exchange CE (CLE-CE) technique and UV detection. Efficient labeling was achieved by sequential injection of buffer, Dns-Cl, AA enantiomers, Dns-Cl and buffer at 0.2 psi for 10.0, 3.0, 24.0, 3.0, and 10.0 s, respectively. After injection, the sandwich sections were allowed to react at room temperature for 35.0 min. With this procedure, successful on-column labeling and CLE-CE separation of 17 pairs AA enantiomers have been achieved with a buffer of 100.0 mM boric acid, 5.0 mM ammonium acetate, 3.0 mM ZnSO4 and 6.0 mM L-Arg at pH 8.4, giving nine pairs fully enantioresolved with resolution in between 2.0 and 5.1. CLE-CE of some individual and mixed pairs was also demonstrated, much the same as using pre-column labeling. As validated by both artificially prepared solutions and serum samples, this new method was shown to be applicable to the quantitative analysis, with a linear range between 14.0 muM and 3.7 mM, correlation coefficient above 0.99 and recovery in between 90.4% and 111.7%. It was also demonstrated that the migration time-temperature based curve allows for temperature determination in CE by using this new method. PMID:19655328

  7. Involvement of Gluconic Acid and Glucose Oxidase in the Pathogenicity of Penicillium expansum in Apples.

    PubMed

    Hadas, Yoav; Goldberg, Israel; Pines, Ophry; Prusky, Dov

    2007-03-01

    ABSTRACT The contribution of gluconic acid secretion to the colonization of apple tissue by Penicillium expansum was analyzed by modulation (increase or decrease) of gluconic acid accumulation at the infection court. P. expansum isolates that express the most gox2 transcripts and concomitant glucose oxidase (GOX) activity and that secrete the most gluconic acid cause disease of apple at the fastest rate. Cultures grown under reduced oxygen concentration generated fewer gox2 transcripts, produced less gluconic acid, and led to a 15% reduction in disease. Furthermore, the detection of significantly high levels of transcripts of gox2 and GOX activity at the edge of the decaying tissue emphasize the involvement of GOX in tissue acidification of the decaying tissue. Taken together, these results emphasize the importance of GOX in the production of the gluconic acid that leads, in turn, to host tissue acidification. This acidification enhanced the expression of pectolytic enzymes and the establishment of conditions for necrotrophic development of P. expansum.

  8. Oxalic acid degradation by a novel fungal oxalate oxidase from Abortiporus biennis.

    PubMed

    Grąz, Marcin; Rachwał, Kamila; Zan, Radosław; Jarosz-Wilkołazka, Anna

    2016-01-01

    Oxalate oxidase was identified in mycelial extracts of a basidiomycete Abortiporus biennis strain. Intracellular enzyme activity was detected only after prior lowering of the pH value of the fungal cultures by using oxalic or hydrochloric acids. This enzyme was purified using size exclusion chromatography (Sephadex G-25) and ion-exchange chromatography (DEAE-Sepharose). This enzyme exhibited optimum activity at pH 2 when incubated at 40°C, and the optimum temperature was established at 60°C. Among the tested organic acids, this enzyme exhibited specificity only towards oxalic acid. Molecular mass was calculated as 58 kDa. The values of Km for oxalate and Vmax for the enzyme reaction were 0.015 M and 30 mmol min(-1), respectively. PMID:27337220

  9. A thermostable L-aspartate oxidase: a new tool for biotechnological applications.

    PubMed

    Bifulco, Davide; Pollegioni, Loredano; Tessaro, Davide; Servi, Stefano; Molla, Gianluca

    2013-08-01

    L-Amino acid oxidases (LAAOs) are homodimeric flavin adenine dinucleotide (FAD)-containing flavoproteins that catalyze the stereospecific oxidative deamination of L-amino acids to α-keto acids, ammonia, and hydrogen peroxide. Unlike the D-selective counterpart, the biotechnological application of LAAOs has not been thoroughly advanced because of the difficulties in their expression as recombinant protein in prokaryotic hosts. In this work, L-aspartate oxidase from the thermophilic archea Sulfolobus tokodaii (StLASPO, specific for L-aspartate and L-asparagine only) was efficiently produced as recombinant protein in E. coli in the active form as holoenzyme. This recombinant flavoenzyme shows the classical properties of FAD-containing oxidases. Indeed, StLASPO shows distinctive features that makes it attractive for biotechnological applications: high thermal stability (it is fully stable up to 80 °C) and high temperature optimum, stable activity in a broad range of pH (7.0-10.0), weak inhibition by the product oxaloacetate and by D-aspartate, and tight binding of the FAD cofactor. This latter property significantly distinguishes StLASPO from the E. coli counterpart. StLASPO represents an appropriate novel biocatalyst for the production of D-aspartate and a well-suited protein scaffold to evolve a LAAO activity by protein engineering.

  10. Xanthine oxidase-mediated denitrosation of N-nitroso-tryptophan by superoxide and uric acid.

    PubMed

    Viles, Kimberley; Mathai, Clinton; Jourd'heuil, Frances L; Jourd'heuil, David

    2013-01-15

    Recent studies indicate the formation of protein nitrosamines in vivo and tryptophan residues in proteins might represent important targets of nitrosative and oxidative stress. In the present work, we examined the mechanism by which xanthine oxidase (XO) denitrosates N-nitroso Trp residues and determined the applicability of the reactions involved to the detection of nitrosated Trp residues by tri-iodide-based chemiluminescence. We found that - in addition to superoxide - denitrosation of N-acetyl-nitroso Trp (NANT) by hypoxanthine and XO occurred via the intermediacy of uric acid. Zero-order dependence of NANT decay rate with uric acid was achieved with increasing concentrations of uric acid (k(0)∼6.0×10(-4)s(-1)) and generated nitric oxide. In contrast, S-nitrosoglutathione and nitrosyl-myoglobin were stable in the presence of uric acid. NANT decomposition by uric acid could be reproducibly measured using the tri-iodide-based chemiluminescence assay in the presence of excess nitrite upon pre-treatment with acidified sulfanilamide. N-nitrosated albumin was sensitive to uric acid-induced decomposition only after proteolytic degradation. In conclusion, XO decomposes nitrosated Trp through superoxide and uric acid pathways and in the case of uric acid generates free nitric oxide. Site-specificity of this reaction may possibly be used in combination with the tri-iodide-based chemiluminescence assay to discern between nitrosated Trp, S-nitrosothiols, and nitrosylated heme proteins. PMID:23099296

  11. In vitro and in vivo studies on adlay-derived seed extracts: phenolic profiles, antioxidant activities, serum uric acid suppression, and xanthine oxidase inhibitory effects.

    PubMed

    Zhao, Mouming; Zhu, Dashuai; Sun-Waterhouse, Dongxiao; Su, Guowan; Lin, Lianzhu; Wang, Xiao; Dong, Yi

    2014-08-01

    This study aimed to explore the potential of polished adlay, brown adlay, adlay bran, and adlay hull to prevent and treat hyperuricemia. Brown adlay extract effectively decreased the serum uric acid levels of oxonate-induced hyperuricemic rats. Free and bound phenolic extracts from these materials contained significant amounts of phenolics, with free phenolics dominated by chlorogenic acid and p-coumaric acid while bound phenolics dominated by p-coumaric acid and ferulic acid. Free and bound phenolics of adlay bran exhibited significant xanthine oxidase inhibition activities, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities, oxygen radical absorbance capacities, and superoxide radical scavenging activities. Adlay bran phenolics could be effective xanthine oxidase inhibitors and radical scavengers. p-Coumaric acid is a xanthine oxidase inhibitor with strong superoxide radical scavenging activity. However, ferulic acid is a xanthine oxidase inhibitor with weak superoxide radical scavenging activity. Chlorogenic acid is a superoxide radical scavenger with weak xanthine oxidase inhibitory activity.

  12. Use of Glucose Oxidase in a Membrane Reactor for Gluconic Acid Production

    NASA Astrophysics Data System (ADS)

    Das Neves, Luiz Carlos Martins; Vitolo, Michele

    This article aims at the evaluation of the catalytic performance of glucose oxidase (GO) (EC.1.1.3.4) for the glucose/gluconic acid conversion in the ultrafiltration cell type membrane reactor (MB-CSTR). The reactor was coupled with a Millipore ultrafiltration-membrane (cutoff of 100 kDa) and operated for 24 h under agitation of 100 rpm, pH 5.5, and 30°C. The experimental conditions varied were the glucose concentration (2.5, 5.0, 10.0, 20.0, and 40.0 mM), the feeding rate (0.5, 1.0, 3.0, and 6.0/h), dissolved oxygen (8.0 and 16.0 mg/L), GO concentration (2.5, 5.0, 10.0, and 20.0 UGO/mL), and the glucose oxidase/catalase activity ratio (UGO/UCAT)(1∶0, 1∶10, 1∶20, and 1∶30). A conversion yield of 80% and specific reaction rate of 40×10-4 mmol/h·UGO were attained when the process was carried out under the following conditions: D=3.0/h, dissolved oxygen=16.0 mg/L, [G]=40 mM, and (UGO/UCAT)=1∶20. A simplified model for explaining the inhibition of GO activity by hydrogen peroxide, formed during the glucose/gluconic acid conversion, was presented.

  13. [Enzymatic production of α-ketoglutaric acid by L-glutamate oxidase from L-glutamic acid].

    PubMed

    Niu, Panqing; Zhang, Zhenyu; Liu, Liming

    2014-08-01

    We produced α-ketoglutaric acid (α-KG) from L-glutamic acid, using enzymatic transformation approach with L-glutamate oxidase (LGOX). First, wild strain Streptomyces sp. FMME066 was mutated with NTG, a genetically stable mutant Streptomyces sp. FMME067 was obtained. Under the optimal nutrition conditions with fructose 10 g/L, peptone 7.5 g/L, KH2PO4 1 g/L and CaCl2 0.05 g/L, the maximum LGOX activity reached 0.14 U/mL. The LGOX was stable to pH and temperature, and Mn2+ had a stimulating effect. Finally, after 24 h enzymatic conversion under the optimal conditions, the maximum titer of α-KG reached 38.1 g/L from 47 g/L L-glutamic acid. Enzymatic transformation by LGOX is a potential approach for α-KG production.

  14. Oleic, Linoleic and Linolenic Acids Increase ROS Production by Fibroblasts via NADPH Oxidase Activation

    PubMed Central

    Hatanaka, Elaine; Dermargos, Alexandre; Hirata, Aparecida Emiko; Vinolo, Marco Aurélio Ramirez; Carpinelli, Angelo Rafael; Newsholme, Philip; Armelin, Hugo Aguirre; Curi, Rui

    2013-01-01

    The effect of oleic, linoleic and γ-linolenic acids on ROS production by 3T3 Swiss and Rat 1 fibroblasts was investigated. Using lucigenin-amplified chemiluminescence, a dose-dependent increase in extracellular superoxide levels was observed during the treatment of fibroblasts with oleic, linoleic and γ-linolenic acids. ROS production was dependent on the addition of β-NADH or NADPH to the medium. Diphenyleneiodonium inhibited the effect of oleic, linoleic and γ-linolenic acids on fibroblast superoxide release by 79%, 92% and 82%, respectively. Increased levels of p47phox phosphorylation due to fatty acid treatment were detected by Western blotting analyses of fibroblast proteins. Increased p47phox mRNA expression was observed using real-time PCR. The rank order for the fatty acid stimulation of the fibroblast oxidative burst was as follows: γ-linolenic > linoleic > oleic. In conclusion, oleic, linoleic and γ-linolenic acids stimulated ROS production via activation of the NADPH oxidase enzyme complex in fibroblasts. PMID:23579616

  15. Plant and animal glycolate oxidases have a common eukaryotic ancestor and convergently duplicated to evolve long-chain 2-hydroxy acid oxidases.

    PubMed

    Esser, Christian; Kuhn, Anke; Groth, Georg; Lercher, Martin J; Maurino, Veronica G

    2014-05-01

    Glycolate oxidase (GOX) is a crucial enzyme of plant photorespiration. The encoding gene is thought to have originated from endosymbiotic gene transfer between the eukaryotic host and the cyanobacterial endosymbiont at the base of plantae. However, animals also possess GOX activities. Plant and animal GOX belong to the gene family of (L)-2-hydroxyacid-oxidases ((L)-2-HAOX). We find that all (L)-2-HAOX proteins in animals and archaeplastida go back to one ancestral eukaryotic sequence; the sole exceptions are green algae of the chlorophyta lineage. Chlorophyta replaced the ancestral eukaryotic (L)-2-HAOX with a bacterial ortholog, a lactate oxidase that may have been obtained through the primary endosymbiosis at the base of plantae; independent losses of this gene may explain its absence in other algal lineages (glaucophyta, rhodophyta, and charophyta). We also show that in addition to GOX, plants possess (L)-2-HAOX proteins with different specificities for medium- and long-chain hydroxyacids (lHAOX), likely involved in fatty acid and protein catabolism. Vertebrates possess lHAOX proteins acting on similar substrates as plant lHAOX; however, the existence of GOX and lHAOX subfamilies in both plants and animals is not due to shared ancestry but is the result of convergent evolution in the two most complex eukaryotic lineages. On the basis of targeting sequences and predicted substrate specificities, we conclude that the biological role of plantae (L)-2-HAOX in photorespiration evolved by co-opting an existing peroxisomal protein. PMID:24408912

  16. Plant and animal glycolate oxidases have a common eukaryotic ancestor and convergently duplicated to evolve long-chain 2-hydroxy acid oxidases.

    PubMed

    Esser, Christian; Kuhn, Anke; Groth, Georg; Lercher, Martin J; Maurino, Veronica G

    2014-05-01

    Glycolate oxidase (GOX) is a crucial enzyme of plant photorespiration. The encoding gene is thought to have originated from endosymbiotic gene transfer between the eukaryotic host and the cyanobacterial endosymbiont at the base of plantae. However, animals also possess GOX activities. Plant and animal GOX belong to the gene family of (L)-2-hydroxyacid-oxidases ((L)-2-HAOX). We find that all (L)-2-HAOX proteins in animals and archaeplastida go back to one ancestral eukaryotic sequence; the sole exceptions are green algae of the chlorophyta lineage. Chlorophyta replaced the ancestral eukaryotic (L)-2-HAOX with a bacterial ortholog, a lactate oxidase that may have been obtained through the primary endosymbiosis at the base of plantae; independent losses of this gene may explain its absence in other algal lineages (glaucophyta, rhodophyta, and charophyta). We also show that in addition to GOX, plants possess (L)-2-HAOX proteins with different specificities for medium- and long-chain hydroxyacids (lHAOX), likely involved in fatty acid and protein catabolism. Vertebrates possess lHAOX proteins acting on similar substrates as plant lHAOX; however, the existence of GOX and lHAOX subfamilies in both plants and animals is not due to shared ancestry but is the result of convergent evolution in the two most complex eukaryotic lineages. On the basis of targeting sequences and predicted substrate specificities, we conclude that the biological role of plantae (L)-2-HAOX in photorespiration evolved by co-opting an existing peroxisomal protein.

  17. Nanoparticle strategies for cancer therapeutics: Nucleic acids, polyamines, bovine serum amine oxidase and iron oxide nanoparticles (Review).

    PubMed

    Agostinelli, Enzo; Vianello, Fabio; Magliulo, Giuseppe; Thomas, Thresia; Thomas, T J

    2015-01-01

    Nanotechnology for cancer gene therapy is an emerging field. Nucleic acids, polyamine analogues and cytotoxic products of polyamine oxidation, generated in situ by an enzyme-catalyzed reaction, can be developed for nanotechnology-based cancer therapeutics with reduced systemic toxicity and improved therapeutic efficacy. Nucleic acid-based gene therapy approaches depend on the compaction of DNA/RNA to nanoparticles and polyamine analogues are excellent agents for the condensation of nucleic acids to nanoparticles. Polyamines and amine oxidases are found in higher levels in tumours compared to that of normal tissues. Therefore, the metabolism of polyamines spermidine and spermine, and their diamine precursor, putrescine, can be targets for antineoplastic therapy since these naturally occurring alkylamines are essential for normal mammalian cell growth. Intracellular polyamine concentrations are maintained at a cell type-specific set point through the coordinated and highly regulated interplay between biosynthesis, transport, and catabolism. In particular, polyamine catabolism involves copper-containing amine oxidases. Several studies showed an important role of these enzymes in developmental and disease-related processes in animals through the control of polyamine homeostasis in response to normal cellular signals, drug treatment, and environmental and/or cellular stress. The production of toxic aldehydes and reactive oxygen species (ROS), H2O2 in particular, by these oxidases suggests a mechanism by which amine oxidases can be exploited as antineoplastic drug targets. The combination of bovine serum amine oxidase (BSAO) and polyamines prevents tumour growth, particularly well if the enzyme has been conjugated with a biocompatible hydrogel polymer. The findings described herein suggest that enzymatically formed cytotoxic agents activate stress signal transduction pathways, leading to apoptotic cell death. Consequently, superparamagnetic nanoparticles or other

  18. Uric acid accumulation in an Arabidopsis urate oxidase mutant impairs seedling establishment by blocking peroxisome maintenance.

    PubMed

    Hauck, Oliver K; Scharnberg, Jana; Escobar, Nieves Medina; Wanner, Gerhard; Giavalisco, Patrick; Witte, Claus-Peter

    2014-07-01

    Purine nucleotides can be fully catabolized by plants to recycle nutrients. We have isolated a urate oxidase (uox) mutant of Arabidopsis thaliana that accumulates uric acid in all tissues, especially in the developing embryo. The mutant displays a reduced germination rate and is unable to establish autotrophic growth due to severe inhibition of cotyledon development and nutrient mobilization from the lipid reserves in the cotyledons. The uox mutant phenotype is suppressed in a xanthine dehydrogenase (xdh) uox double mutant, demonstrating that the underlying cause is not the defective purine base catabolism, or the lack of UOX per se, but the elevated uric acid concentration in the embryo. Remarkably, xanthine accumulates to similar levels in the xdh mutant without toxicity. This is paralleled in humans, where hyperuricemia is associated with many diseases whereas xanthinuria is asymptomatic. Searching for the molecular cause of uric acid toxicity, we discovered a local defect of peroxisomes (glyoxysomes) mostly confined to the cotyledons of the mature embryos, which resulted in the accumulation of free fatty acids in dry seeds. The peroxisomal defect explains the developmental phenotypes of the uox mutant, drawing a novel link between uric acid and peroxisome function, which may be relevant beyond plants. PMID:25052714

  19. In vitro oxidation of indoleacetic acid by soluble auxin-oxidases and peroxidases from maize roots. [Zea mays L

    SciTech Connect

    Beffa, R.; Martin, H.V.; Pilet, P.E. )

    1990-10-01

    Soluble auxin-oxidases were extracted from Zea mays L. cv LG11 apical root segments and partially separated from peroxidases (EC 1.11.1.7) by size-exclusion chromatography. Auxin-oxidases were resolved into one main peak corresponding to a molecular mass of 32.5 kilodaltons and a minor peak at 54.5 kilodaltons. Peroxidases were separated into at least four peaks, with molecular masses from 32.5 to 78 kilodaltons. In vitro activity of indoleacetic acid-oxidases was dependent on the presence of MnCl{sub 2} and p-coumaric acid. Compound(s) present in the crude extract and several synthetic auxin transport inhibitors (including 2,3,5-triiodobenzoic acid and N-1-naphthylphthalamic acid) inhibited auxin-oxidase activity, but had no effect on peroxidases. The products resulting from the in vitro enzymatic oxidation of ({sup 3}H)indoleacetic acid were separated by HPLC and the major metabolite was found to cochromatograph with indol-3yl-methanol.

  20. Salmonella Evades d-Amino Acid Oxidase To Promote Infection in Neutrophils

    PubMed Central

    Tuinema, Brian R.; Reid-Yu, Sarah A.

    2014-01-01

    ABSTRACT Neutrophils engulf and kill bacteria using oxidative and nonoxidative mechanisms. Despite robust antimicrobial activity, neutrophils are impaired in directing Salmonella clearance and harbor viable intracellular bacteria during early stages of infection that can subsequently escape to more-permissive cell types. The mechanisms accounting for this immune impairment are not understood. We report that Salmonella limits exposure to oxidative damage elicited by d-amino acid oxidase (DAO) in neutrophils by expressing an ABC importer specific for d-alanine, a DAO substrate found in peptidoglycan stem peptides. A Salmonella dalS mutant defective for d-alanine import was more susceptible to killing by DAO through exposure to greater oxidative stress during infection. This fitness defect was reversed by selective depletion of neutrophils or by inhibition of DAO in vivo with a small-molecule inhibitor. DalS-mediated subversion of neutrophil DAO is a novel host-pathogen interaction that enhances Salmonella survival during systemic infection. PMID:25425233

  1. Interaction of D-Amino Acid Oxidase with Carbon Nanotubes: Implications in the Design of Biosensors

    PubMed Central

    Mora, Maria F.; Giacomelli, Carla E.; Garcia, Carlos D.

    2009-01-01

    We have investigated the interaction of D-amino acid oxidase (DAAO) with single walled carbon nanotubes (CNT) by spectroscopic ellipsometry. Dynamic adsorption experiments were performed at different experimental conditions. In addition, the activity of the enzyme adsorbed at different conditions was studied. Our results indicate that DAAO can be adsorbed to CNT at different pH values and concentrations by a combination of hydrophobic and electrostatic interactions. Considering that the highest enzymatic activity was obtained by adsorbing the protein at pH 5.7 and 0.1 mg.mL−1, our results indicate that DAAO can adopt multiple orientations on the surface; which are ultimately responsible for significant differences in catalytic activity. PMID:19132842

  2. Contributions of spinal D-amino acid oxidase to chronic morphine-induced hyperalgesia.

    PubMed

    Ma, Shuai; Li, Xin-Yan; Gong, Nian; Wang, Yong-Xiang

    2015-12-10

    Spinal D-amino acid oxidase (DAAO) is an FAD-dependent peroxisomal flavoenzyme which mediates the conversion of neutral and polar D-amino acids (including D-serine) to the corresponding α-keto acids, and simultaneously produces hydrogen peroxide and ammonia. This study has aimed to explore the potential contributions of spinal DAAO and its mediated hydrogen peroxide/D-serine metabolism to the development of morphine-induced hyperalgesia. Bi-daily subcutaneous injections of morphine to mice over 7 days induced thermal hyperalgesia as measured by both the hot-plate and tail-immersion tests, and spinal astroglial activation with increased spinal gene expression of DAAO, glial fibrillary acidic protein (GFAP) and pro-inflammatory cytokines (interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α)). Subcutaneous injections of the potent DAAO inhibitor CBIO (5-chloro-benzo[D]isoxazol-3-ol) prevented and reversed the chronic morphine-induced hyperalgesia. CBIO also inhibited both astrocyte activation and the expression of pro-inflammatory cytokines. Intrathecal injection of the hydrogen peroxide scavenger PBN (phenyl-N-tert-butylnitrone) and of catalase completely reversed established morphine hyperalgesia, whereas subcutaneous injections of exogenous D-serine failed to alter chronic morphine-induced hyperalgesia. These results provided evidence that spinal DAAO and its subsequent production of hydrogen peroxide rather than the D-serine metabolism contributed to the development of morphine-induced hyperalgesia.

  3. Use of glucose oxidase in a membrane reactor for gluconic acid production.

    PubMed

    das Neves, Luiz Carlos Martins; Vitolo, Michele

    2007-04-01

    This article aims at the evaluation of the catalytic performance of glucose oxidase (GO) (EC.1.1.3.4) for the glucose/gluconic acid conversion in the ultrafiltration cell type membrane reactor (MB-CSTR). The reactor was coupled with a Millipore ultrafiltration-membrane (cutoff of 100 kDa) and operated for 24 h under agitation of 100 rpm, pH 5.5, and 30 degrees C. The experimental conditions varied were the glucose concentration (2.5, 5.0, 10.0, 20.0, and 40.0 mM), the feeding rate (0.5, 1.0, 3.0, and 6.0/h), dissolved oxygen (8.0 and 16.0 mg/L), GO concentration (2.5, 5.0, 10.0, and 20.0 U(GO)/mL), and the glucose oxidase/catalase activity ratio (U(GO)/U(CAT))(1:0, 1:10, 1:20, and 1:30). A conversion yield of 80% and specific reaction rate of 40 x 10(-4) mmol/h x U(GO) were attained when the process was carried out under the following conditions: D =3.0/h, dissolved oxygen =16.0 mg/L, [G] =40 mM, and (U(GO)/U(CAT)) =1:20. A simplified model for explaining the inhibition of GO activity by hydrogen peroxide, formed during the glucose/gluconic acid conversion, was presented.

  4. Enzymatic production of α-ketoglutaric acid from l-glutamic acid via l-glutamate oxidase.

    PubMed

    Niu, Panqing; Dong, Xiaoxiang; Wang, Yuancai; Liu, Liming

    2014-06-10

    In this study, a novel strategy for α-ketoglutaric acid (α-KG) production from l-glutamic acid using recombinant l-glutamate oxidase (LGOX) was developed. First, by analyzing the molecular structure characteristics of l-glutamic acid and α-KG, LGOX was found to be the best catalyst for oxidizing the amino group of l-glutamic acid to a ketonic group without the need for exogenous cofactor. Then the LGOX gene was expressed in Escherichia coli BL21 (DE3) in a soluble and active form, and the recombinant LGOX activity reached to a maximum value of 0.59U/mL at pH 6.5, 30°C. Finally, the maximum α-KG concentration reached 104.7g/L from 110g/L l-glutamic acid in 24h, under the following optimum conditions: 1.5U/mL LGOX, 250U/mL catalase, 3mM MnCl2, 30°C, and pH 6.5.

  5. New amperometric biosensors based on diamond paste for the assay of L- and D-pipecolic acids in serum samples.

    PubMed

    Stefan, Raluca-Ioana; Nejem, R'afat Mahmoud; van Staden, Jacobus F; Aboul-Enein, Hassan Y

    2004-05-01

    Monocrystalline natural diamond, L-amino acid oxidase (L-AAOD), D-amino acid oxidase (D-AAOD), and paraffin oil were used for the design of the modified diamond paste. The technique used for the direct voltammetric assay was differential pulse voltammetry (DPV) with applied potential pulse amplitude of 25 mV vs. Ag/AgCl. Using the new amperometric biosensors L-pipecolic acid (L-PA) and D-pipecolic acid (D-PA) were determined reliably from serum samples at 700 and 200 mV vs. Ag/AgCl, respectively, with low limits of detection.

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

    PubMed Central

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

    2013-01-01

    Abstract 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. PMID:23873697

  7. Synthesis and evaluation of quinazoline amino acid derivatives as mono amine oxidase (MAO) inhibitors.

    PubMed

    Khattab, Sherine Nabil; Haiba, Nesreen Saied; Asal, Ahmed Mosaad; Bekhit, Adnan A; Amer, Adel; Abdel-Rahman, Hamdy M; El-Faham, Ayman

    2015-07-01

    A series of quinazolinone amino acid ester and quinazolinone amino acid hydrazides were prepared under microwave irradiation as well as conventional condition. The microwave irradiation afforded the product in less reaction time, higher yield and purity. The structures of the synthesized compounds were confirmed by IR, NMR, and elemental analysis. The new synthesized compounds were studied for their monoamine oxidase inhibitory activity. They showed more selective inhibitory activity toward MAO-A than MAO-B. Compounds 7, 10, and 15 showed MAO-A inhibition activity (IC50=3.6×10(-9), 2.8×10(-9), 2.1×10(-9) M, respectively) comparable to that of the standard clorgyline (IC50=2.9×10(-9)M). 2-(2-(Benzo[d][1,3]dioxol-5-yl)-4-oxo-1,2-dihydroquinazolin-3(4H)-yl)acetohydrazide 15 showed selective MAO-A inhibition activity (SI=39524) superior to that of the standard clorgyline (SI=33793). The acute toxicity of the synthesized compounds was determined. In addition, computer-assisted simulated docking experiments were performed to rationalize the biological activity. PMID:25922182

  8. Synthesis and evaluation of quinazoline amino acid derivatives as mono amine oxidase (MAO) inhibitors.

    PubMed

    Khattab, Sherine Nabil; Haiba, Nesreen Saied; Asal, Ahmed Mosaad; Bekhit, Adnan A; Amer, Adel; Abdel-Rahman, Hamdy M; El-Faham, Ayman

    2015-07-01

    A series of quinazolinone amino acid ester and quinazolinone amino acid hydrazides were prepared under microwave irradiation as well as conventional condition. The microwave irradiation afforded the product in less reaction time, higher yield and purity. The structures of the synthesized compounds were confirmed by IR, NMR, and elemental analysis. The new synthesized compounds were studied for their monoamine oxidase inhibitory activity. They showed more selective inhibitory activity toward MAO-A than MAO-B. Compounds 7, 10, and 15 showed MAO-A inhibition activity (IC50=3.6×10(-9), 2.8×10(-9), 2.1×10(-9) M, respectively) comparable to that of the standard clorgyline (IC50=2.9×10(-9)M). 2-(2-(Benzo[d][1,3]dioxol-5-yl)-4-oxo-1,2-dihydroquinazolin-3(4H)-yl)acetohydrazide 15 showed selective MAO-A inhibition activity (SI=39524) superior to that of the standard clorgyline (SI=33793). The acute toxicity of the synthesized compounds was determined. In addition, computer-assisted simulated docking experiments were performed to rationalize the biological activity.

  9. The neurobiology of D-amino acid oxidase (DAO) and its involvement in schizophrenia

    PubMed Central

    Verrall, L; Burnet, PWJ; Betts, JF; Harrison, PJ

    2010-01-01

    D-amino acid oxidase (DAO, DAAO) is a flavoenzyme that metabolises certain D-amino acids, notably the endogenous N-methyl D-aspartate receptor (NMDAR) co-agonist, D-serine. As such, it has the potential to modulate NMDAR function and to contribute to the widely hypothesized involvement of NMDAR signalling in schizophrenia. Three lines of evidence now provide support for this possibility: DAO shows genetic associations to the disorder in several, though not all, studies; the expression and activity of DAO are increased in schizophrenia; and DAO inactivation in rodents produces behavioural and biochemical effects suggestive of potential therapeutic benefits. However, several key issues remain unclear. These include the regional, cellular and subcellular localization of DAO, the physiological importance of DAO and of its substrates other than D-serine, and the causes and consequences of elevated DAO in schizophrenia. Here we critically review the neurobiology of DAO, its involvement in schizophrenia, and the therapeutic value of DAO inhibition. The review also illuminates issues that have a broader relevance beyond DAO itself: how should we weigh up convergent and cumulatively impressive, but individually inconclusive, pieces of evidence regarding the role that a given gene may play in the aetiology, pathophysiology, and pharmacotherapy of schizophrenia? PMID:19786963

  10. D-amino acid oxidase is expressed in the ventral tegmental area and modulates cortical dopamine.

    PubMed

    Betts, Jill F; Schweimer, Judith V; Burnham, Katherine E; Burnet, Philip W J; Sharp, Trevor; Harrison, Paul J

    2014-01-01

    D-amino acid oxidase (DAO, DAAO) degrades the NMDA receptor co-agonist D-serine, modulating D-serine levels and thence NMDA receptor function. DAO inhibitors are under development as a therapy for schizophrenia, a disorder involving both NMDA receptor and dopaminergic dysfunction. However, a direct role for DAO in dopamine regulation has not been demonstrated. Here, we address this question in two ways. First, using in situ hybridization and immunohistochemistry, we show that DAO mRNA and immunoreactivity are present in the ventral tegmental area (VTA) of the rat, in tyrosine hydroxylase (TH)-positive and -negative neurons, and in glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes. Second, we show that injection into the VTA of sodium benzoate, a DAO inhibitor, increases frontal cortex extracellular dopamine, as measured by in vivo microdialysis and high performance liquid chromatography. Combining sodium benzoate and D-serine did not enhance this effect, and injection of D-serine alone affected dopamine metabolites but not dopamine. These data show that DAO is expressed in the VTA, and suggest that it impacts on the mesocortical dopamine system. The mechanism by which the observed effects occur, and the implications of these findings for schizophrenia therapy, require further study. PMID:24822045

  11. Differential expression of two 1-aminocyclopropane-1-carboxylic acid oxidase genes in broccoli after harvest.

    PubMed Central

    Pogson, B J; Downs, C G; Davies, K M

    1995-01-01

    Broccoli (Brassica oleracea L.) floral tissues rapidly differentiate and grow before harvest and then senesce rapidly after harvest. Associated with this postharvest deterioration is an increase in ethylene production by florets. Two cDNA clones having high nucleotide identity to sequences encoding 1-amino-cyclopropane-1-carboxylic acid (ACC) oxidase were isolated from senescing florets. The cDNAs, ACC Ox1 and ACC Ox2, apparently encode mRNAs from different genes. ACC Ox1 transcripts were found at low levels in whole florets at the time of harvest and increased markedly in abundance after harvest. ACC Ox1 transcript abundance also increased in sepals after harvest and in excised yellowing leaves. Transcripts corresponding to ACC Ox2 were found exclusively within the reproductive structures. These ACC Ox2 transcripts were absent at harvest but started to increase in abundance within 2 h of harvest and then accumulated to high levels. Hormone treatment did not alter the abundance of ACC Ox1 transcripts, whereas ACC Ox2 transcripts increased in abundance after treatment with abscisic acid and propylene. Wounding did not affect the levels of ACC Ox1 or Ox2 transcripts after harvest. At harvest, individual broccoli florets were closed and remained unpollinated. We propose a model whereby the rapid increase in ACC Ox1 and Ox2 transcript abundance after harvest contributes to increased ethylene production by florets. This ethylene may regulate aspects of postharvest senescence, in particular chlorophyll loss. PMID:7610162

  12. 1-Aminocyclopropane-1-carboxylic acid oxidase: insight into cofactor binding from experimental and theoretical studies.

    PubMed

    Brisson, Lydie; El Bakkali-Taheri, Nadia; Giorgi, Michel; Fadel, Antoine; Kaizer, József; Réglier, Marius; Tron, Thierry; Ajandouz, El Hassan; Simaan, A Jalila

    2012-08-01

    1-Aminocyclopropane-1-carboxylic acid oxidase (ACCO) is a nonheme Fe(II)-containing enzyme that is related to the 2-oxoglutarate-dependent dioxygenase family. The binding of substrates/cofactors to tomato ACCO was investigated through kinetics, tryptophan fluorescence quenching, and modeling studies. α-Aminophosphonate analogs of the substrate (1-aminocyclopropane-1-carboxylic acid, ACC), 1-aminocyclopropane-1-phosphonic acid (ACP) and (1-amino-1-methyl)ethylphosphonic acid (AMEP), were found to be competitive inhibitors versus both ACC and bicarbonate (HCO(3)(-)) ions. The measured dissociation constants for Fe(II) and ACC clearly indicate that bicarbonate ions improve both Fe(II) and ACC binding, strongly suggesting a stabilization role for this cofactor. A structural model of tomato ACCO was constructed and used for docking experiments, providing a model of possible interactions of ACC, HCO(3)(-), and ascorbate at the active site. In this model, the ACC and bicarbonate binding sites are located close together in the active pocket. HCO(3)(-) is found at hydrogen-bond distance from ACC and interacts (hydrogen bonds or electrostatic interactions) with residues K158, R244, Y162, S246, and R300 of the enzyme. The position of ascorbate is also predicted away from ACC. Individually docked at the active site, the inhibitors ACP and AMEP were found coordinating the metal ion in place of ACC with the phosphonate groups interacting with K158 and R300, thus interlocking with both ACC and bicarbonate binding sites. In conclusion, HCO(3)(-) and ACC together occupy positions similar to the position of 2-oxoglutarate in related enzymes, and through a hydrogen bond HCO(3)(-) likely plays a major role in the stabilization of the substrate in the active pocket. PMID:22711330

  13. Synthesis and bioevaluation of 2-phenyl-4-methyl-1,3-selenazole-5-carboxylic acids as potent xanthine oxidase inhibitors.

    PubMed

    Guan, Qi; Cheng, Zengjin; Ma, Xiaoxue; Wang, Lijie; Feng, Dongjie; Cui, Yuanhang; Bao, Kai; Wu, Lan; Zhang, Weige

    2014-10-01

    A series of 2-phenyl-4-methyl-1,3-selenazole-5-carboxylic acid derivatives (8a-f, 9a-m) were synthesized and evaluated for inhibitory activity against xanthine oxidase in vitro. Structure-activity relationship analyses have also been presented. Most of the target compounds exhibited potency levels in the nanomolar range. Compound 9e emerged as the most potent xanthine oxidase inhibitor (IC50 = 5.5 nM) in comparison to febuxostat (IC50 = 18.6 nM). Steady-state kinetics measurements with the bovine milk enzyme indicated a mixed type inhibition with Ki and Ki' values of 0.9 and 2.3 nM, respectively. A molecular modeling study on compounds 9e was performed to gain an insight into its binding mode with xanthine oxidase, and to provide the basis for further structure-guided design of new non-purine xanthine oxidase inhibitors related with 2-phenyl-4-methyl-1,3-selenazole-5-carboxylic acid scaffold.

  14. Lysyl oxidase-like 4 involvement in retinoic acid epithelial wound healing.

    PubMed

    Comptour, Aurélie; Rouzaire, Marion; Belville, Corinne; Bonnin, Nicolas; Daniel, Estelle; Chiambaretta, Frédéric; Blanchon, Loïc; Sapin, Vincent

    2016-01-01

    Vitamin A and its active forms (retinoic acids/RAs) are known to have pro-healing properties, but their mechanisms of action are still poorly understood. This work aimed to identify the cellular and molecular processes by which atRA (all-trans RA) improves wound healing, using an in vivo model of mouse corneal alkali burns and an in vitro cellular human corneal epithelial injury model. Regulation by atRA has been studied on most of the cellular events that occur in wound healing. We investigated the direct influence of atRA on a specific target gene known to be involved in the extracellular matrix (ECM) dynamics, one of the pathways contributing to epithelial repair. Our results demonstrate that atRA promotes corneal epithelial wound healing by acting preferentially on migration. The induction of lysyl oxidase-like 4 (LOXL4) expression by atRA in the corneal epithelium environment was established as essential in the mechanism of atRA-dependent wound healing. Our study describes for the first time a direct link between a retinoic-induced gene and protein, LOXL4, and its general clinical pro-healing properties in ECM dynamics. PMID:27597564

  15. Manageable cytotoxicity of nanocapsules immobilizing D-amino acid oxidase via exogenous administration of nontoxic prodrug

    NASA Astrophysics Data System (ADS)

    Zhao, Yang; Zhu, Yingchun; Fu, Jingke

    2014-02-01

    D-Amino acid oxidase (DAO), which could catalyze generation of hydrogen peroxide with strong oxidbility and cytotoxicity, has become of interest as a biocatalyst for therapeutic treatments. Herein we report that amino-functional hollow mesoporous silica with large pore size (10.27 nm) and positively charged surface effectively immobilize DAO with negative charge. The adsorption, activity and stability of DAO are demonstrated to depend mainly on the amino-functionalization of surface. Significant cancer cell killing effect is observed when the cells are treated by the nanocapsules entrapping DAO together with D-alanine, showing distinct dose-dependency on concentration of the nanocapsules entrapping DAO or D-alanine. Nevertheless, the toxicity is completely neutralized by the addition of catalase, and anti-tumor effect is not observed when either the nanocapsules entrapping DAO or D-alanine is applied alone. The results indicate that cytotoxicity of the nanocapsules entrapping DAO could be managed by exogenous administration of nontoxic prodrug to tumor tissue, due to the stereoselectivity of DAO and the scarcity of its substrates in mammalian organisms. Thus, the method might be exploited as a potential treatment for cancer therapy.

  16. Lonicera hypoglauca inhibits xanthine oxidase and reduces serum uric acid in mice.

    PubMed

    Chien, Shih-Chang; Yang, Chen-Wei; Tseng, Yen-Hsueh; Tsay, Hsin-Sheng; Kuo, Yueh-Hsiung; Wang, Sheng-Yang

    2009-03-01

    Xanthine oxidase (XOD) catalyzes the oxidation of hypoxanthine to xanthine and then to uric acid, and is a key enzyme in the pathogenesis of hyperuricemia. The ability of extracts of Lonicera hypoglauca (Caprifoliaceae) to inhibit XOD was investigated in this study. An ethanol extract (LH-crude) of the leaves of L. hypoglauca and its derived EtOAc soluble sub-fractions (LH-EA) significantly inhibited XOD activity, with IC50 values for LH-crude and LH-EA of 48.8 and 35.2 microg/mL. Moreover, LH-EA reduced serum urate levels IN VIVO in a potassium oxonate-induced hyperuricemic mouse model, by 70.1% and 93.7% of the hyperuricemic untreated group at doses of 300 and 500 mg/kg of LH-EA, respectively. Finally, we used bioactivity-guided fractionation to isolate a new bisflavonoid, loniceraflavone, which showed significant inhibition of XOD (IC50=0.85 microg/mL). These results suggest that L. hypoglauca and its extracts may have a considerable potential for development as an anti-hyperuricemia agent for clinical application.

  17. Cardiolipin linoleic acid content and mitochondrial cytochrome c oxidase activity are associated in rat skeletal muscle.

    PubMed

    Fajardo, Val Andrew; McMeekin, Lauren; Saint, Caitlin; LeBlanc, Paul J

    2015-04-01

    Cardiolipin (CL) is an inner-mitochondrial membrane phospholipid that is important for optimal mitochondrial function. Specifically, CL and CL linoleic (18:2ω6) content are known to be positively associated with cytochrome c oxidase (COX) activity. However, this association has not been examined in skeletal muscle. In this study, rats were fed high-fat diets with a naturally occurring gradient in linoleic acid (coconut oil [CO], 5.8%; flaxseed oil [FO], 13.2%; safflower oil [SO], 75.1%) in an attempt to alter both mitochondrial CL fatty acyl composition and COX activity in rat mixed hind-limb muscle. In general, mitochondrial membrane lipid composition was fairly resistant to dietary treatments as only modest changes in fatty acyl composition were detected in CL and other major mitochondrial phospholipids such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE). As a result of this resistance, CL 18:2ω6 content was not different between the dietary groups. Consistent with the lack of changes in CL 18:2ω6 content, mitochondrial COX activity was also not different between the dietary groups. However, correlational analysis using data obtained from rats across the dietary groups showed a significant relationship (p = 0.009, R(2) = 0.21). Specifically, our results suggest that CL 18:2ω6 content may positively influence mitochondrial COX activity thereby making this lipid molecule a potential factor related to mitochondrial health and function in skeletal muscle.

  18. Lysyl oxidase-like 4 involvement in retinoic acid epithelial wound healing

    PubMed Central

    Comptour, Aurélie; Rouzaire, Marion; Belville, Corinne; Bonnin, Nicolas; Daniel, Estelle; Chiambaretta, Frédéric; Blanchon, Loïc; Sapin, Vincent

    2016-01-01

    Vitamin A and its active forms (retinoic acids/RAs) are known to have pro-healing properties, but their mechanisms of action are still poorly understood. This work aimed to identify the cellular and molecular processes by which atRA (all-trans RA) improves wound healing, using an in vivo model of mouse corneal alkali burns and an in vitro cellular human corneal epithelial injury model. Regulation by atRA has been studied on most of the cellular events that occur in wound healing. We investigated the direct influence of atRA on a specific target gene known to be involved in the extracellular matrix (ECM) dynamics, one of the pathways contributing to epithelial repair. Our results demonstrate that atRA promotes corneal epithelial wound healing by acting preferentially on migration. The induction of lysyl oxidase-like 4 (LOXL4) expression by atRA in the corneal epithelium environment was established as essential in the mechanism of atRA-dependent wound healing. Our study describes for the first time a direct link between a retinoic-induced gene and protein, LOXL4, and its general clinical pro-healing properties in ECM dynamics. PMID:27597564

  19. The antiviral drug acyclovir is a slow-binding inhibitor of (D)-amino acid oxidase.

    PubMed

    Katane, Masumi; Matsuda, Satsuki; Saitoh, Yasuaki; Sekine, Masae; Furuchi, Takemitsu; Koyama, Nobuhiro; Nakagome, Izumi; Tomoda, Hiroshi; Hirono, Shuichi; Homma, Hiroshi

    2013-08-20

    d-Amino acid oxidase (DAO) is a degradative enzyme that is stereospecific for d-amino acids, including d-serine and d-alanine, which are believed to be coagonists of the N-methyl-d-aspartate (NMDA) receptor. To identify a new class of DAO inhibitor(s) that can be used to elucidate the molecular details of the active site environment of DAO, manifold biologically active compounds of microbial origin and pre-existing drugs were screened for their ability to inhibit DAO activity, and several compounds were identified as candidates. One of these compounds, acyclovir (ACV), a well-known antiviral drug used for the treatment of herpesvirus infections, was characterized and evaluated as a novel DAO inhibitor in vitro. Analysis showed that ACV acts on DAO as a reversible slow-binding inhibitor, and interestingly, the time required to achieve equilibrium between DAO, ACV, and the DAO/ACV complex was highly dependent on temperature. The binding mechanism of ACV to DAO was investigated in detail by several approaches, including kinetic analysis, structural modeling of DAO complexed with ACV, and site-specific mutagenesis of an active site residue postulated to be involved in the binding of ACV. The results confirm that ACV is a novel, active site-directed inhibitor of DAO that can be a valuable tool for investigating the structure-function relationships of DAO, including the molecular details of the active site environment of DAO. In particular, it appears that ACV can serve as an active site probe to study the structural basis of temperature-induced conformational changes of DAO.

  20. Functional Analysis of Fructosyl-Amino Acid Oxidases of Aspergillus oryzae

    PubMed Central

    Akazawa, Shin-ichi; Karino, Tetsuya; Yoshida, Nobuyuki; Katsuragi, Tohoru; Tani, Yoshiki

    2004-01-01

    Three active fractions of fructosyl-amino acid oxidase (FAOD-Ao1, -Ao2a, and -Ao2b) were isolated from Aspergillus oryzae strain RIB40. N-terminal and internal amino acid sequences of FAOD-Ao2a corresponded to those of FAOD-Ao2b, suggesting that these two isozymes were derived from the same protein. FAOD-Ao1 and -Ao2 were different in substrate specificity and subunit assembly; FAOD-Ao2 was active toward Nɛ-fructosyl Nα-Z-lysine and fructosyl valine (Fru-Val), whereas FAOD-Ao1 was not active toward Fru-Val. The genes encoding the FAOD isozymes (i.e., FAOAo1 and FAOAo2) were cloned by PCR with an FAOD-specific primer set. The deduced amino acid sequences revealed that FAOD-Ao1 was 50% identical to FAOD-Ao2, and each isozyme had a peroxisome-targeting signal-1, indicating their localization in peroxisomes. The genes was expressed in Escherichia coli and rFaoAo2 showed the same characteristics as FAOD-Ao2, whereas rFaoAo1 was not active. FAOAo2 disruptant was obtained by using ptrA as a selective marker. Wild-type strain grew on the medium containing Fru-Val as the sole carbon and nitrogen sources, but strain ΔfaoAo2 did not grow. Addition of glucose or (NH4)2SO4 to the Fru-Val medium did not affect the assimilation of Fru-Val by wild-type, indicating glucose and ammonium repressions did not occur in the expression of the FAOAo2 gene. Furthermore, conidia of the wild-type strain did not germinate on the medium containing Fru-Val and NaNO2 as the sole carbon and nitrogen sources, respectively, suggesting that Fru-Val may also repress gene expression of nitrite reductase. These results indicated that FAOD is needed for utilization of fructosyl-amino acids as nitrogen sources in A. oryzae. PMID:15466528

  1. Thermal stability of ascorbic acid and ascorbic acid oxidase in african cowpea leaves ( Vigna unguiculata ) of different maturities.

    PubMed

    Wawire, Michael; Oey, Indrawati; Mathooko, Francis; Njoroge, Charles; Shitanda, Douglas; Hendrickx, Marc

    2011-03-01

    Cowpea, an African leafy vegetable ( Vigna unguiculata ), contains a high level of vitamin C. The leaves harvested at 4-9 weeks are highly prone to vitamin C losses during handling and processing. Therefore, the purpose of this research was to study the effect of thermal treatment on the stability of ascorbic acid oxidase (AAO), total vitamin C content (l-ascorbic acid, l-AA), and dehydroascorbic acid (DHAA) and l-AA/DHAA ratio in cowpea leaves harvested at different maturities (4, 6, and 8 weeks old). The results showed that AAO activity, total vitamin C content, and l-AA/DHAA ratio in cowpea leaves increased with increasing maturity (up to 8 weeks). Eight-week-old leaves were the best source of total vitamin C and showed a high ratio of l-AA/DHAA (4:1). Thermal inactivation of AAO followed first-order reaction kinetics. Heating at temperatures above 90 °C for short times resulted in a complete AAO inactivation, resulting in a protective effect of l-AA toward enzyme-catalyzed oxidation. Total vitamin C in young leaves (harvested at 4 and 6 weeks) was predominantly in the form of DHAA, and therefore temperature treatment at 30-90 °C for 10 min decreased the total vitamin C content, whereas total vitamin C in 8-week-old cowpea leaves was more than 80% in the form of l-AA, so that a high retention of the total vitamin C can be obtained even after heating and/or reheating (30-90 °C for 10 min) before consumption. The results indicated that the stability of total vitamin C in situ was strongly dependent on the plant maturity stage and the processing conditions applied.

  2. Thermal stability of ascorbic acid and ascorbic acid oxidase in african cowpea leaves ( Vigna unguiculata ) of different maturities.

    PubMed

    Wawire, Michael; Oey, Indrawati; Mathooko, Francis; Njoroge, Charles; Shitanda, Douglas; Hendrickx, Marc

    2011-03-01

    Cowpea, an African leafy vegetable ( Vigna unguiculata ), contains a high level of vitamin C. The leaves harvested at 4-9 weeks are highly prone to vitamin C losses during handling and processing. Therefore, the purpose of this research was to study the effect of thermal treatment on the stability of ascorbic acid oxidase (AAO), total vitamin C content (l-ascorbic acid, l-AA), and dehydroascorbic acid (DHAA) and l-AA/DHAA ratio in cowpea leaves harvested at different maturities (4, 6, and 8 weeks old). The results showed that AAO activity, total vitamin C content, and l-AA/DHAA ratio in cowpea leaves increased with increasing maturity (up to 8 weeks). Eight-week-old leaves were the best source of total vitamin C and showed a high ratio of l-AA/DHAA (4:1). Thermal inactivation of AAO followed first-order reaction kinetics. Heating at temperatures above 90 °C for short times resulted in a complete AAO inactivation, resulting in a protective effect of l-AA toward enzyme-catalyzed oxidation. Total vitamin C in young leaves (harvested at 4 and 6 weeks) was predominantly in the form of DHAA, and therefore temperature treatment at 30-90 °C for 10 min decreased the total vitamin C content, whereas total vitamin C in 8-week-old cowpea leaves was more than 80% in the form of l-AA, so that a high retention of the total vitamin C can be obtained even after heating and/or reheating (30-90 °C for 10 min) before consumption. The results indicated that the stability of total vitamin C in situ was strongly dependent on the plant maturity stage and the processing conditions applied. PMID:21309563

  3. Proposed temperature-dependent conformational transition in D-amino acid oxidase: a differential scanning microcalorimetric study.

    PubMed Central

    Sturtevant, J M; Mateo, P L

    1978-01-01

    A number of authors have reported observations on D-amino acid oxidase [D-amino acid: O2 oxidoreductase (deaminating), EC 1.4.3.3.] that they have interpreted in terms of a temperature-dependent conformational transition having a van't Hoff enthalpy amounting to more than 1 cal per g of protein (1 cal = 4.184J). No indication of this transition is obtained by using a differential scanning calorimeter having a sensitivity considerably in excess of that required to detect such a transition. The implications of this discrepancy are discussed. PMID:26913

  4. Modulation of NMDA receptor function by inhibition of D-amino acid oxidase in rodent brain.

    PubMed

    Strick, Christine A; Li, Cheryl; Scott, Liam; Harvey, Brian; Hajós, Mihály; Steyn, Stefanus J; Piotrowski, Mary A; James, Larry C; Downs, James T; Rago, Brian; Becker, Stacey L; El-Kattan, Ayman; Xu, Youfen; Ganong, Alan H; Tingley, F David; Ramirez, Andres D; Seymour, Patricia A; Guanowsky, Victor; Majchrzak, Mark J; Fox, Carol B; Schmidt, Christopher J; Duplantier, Allen J

    2011-01-01

    Observations that N-Methyl-D-Aspartate (NMDA) antagonists produce symptoms in humans that are similar to those seen in schizophrenia have led to the current hypothesis that schizophrenia might result from NMDA receptor hypofunction. Inhibition of D-amino acid oxidase (DAAO), the enzyme responsible for degradation of D-serine, should lead to increased levels of this co-agonist at the NMDA receptor, and thereby provide a therapeutic approach to schizophrenia. We have profiled some of the preclinical biochemical, electrophysiological, and behavioral consequences of administering potent and selective inhibitors of DAAO to rodents to begin to test this hypothesis. Inhibition of DAAO activity resulted in a significant dose and time dependent increase in D-serine only in the cerebellum, although a time delay was observed between peak plasma or brain drug concentration and cerebellum D-serine response. Pharmacokinetic/pharmacodynamic (PK/PD) modeling employing a mechanism-based indirect response model was used to characterize the correlation between free brain drug concentration and D-serine accumulation. DAAO inhibitors had little or no activity in rodent models considered predictive for antipsychotic activity. The inhibitors did, however, affect cortical activity in the Mescaline-Induced Scratching model, produced a modest but significant increase in NMDA receptor-mediated synaptic currents in primary neuronal cultures from rat hippocampus, and resulted in a significant increase in evoked hippocampal theta rhythm, an in vivo electrophysiological model of hippocampal activity. These findings demonstrate that although DAAO inhibition did not cause a measurable increase in D-serine in forebrain, it did affect hippocampal and cortical activity, possibly through augmentation of NMDA receptor-mediated currents.

  5. Revisitation of the βCl-Elimination Reaction of d-Amino Acid Oxidase

    PubMed Central

    Ghisla, Sandro; Pollegioni, Loredano; Molla, Gianluca

    2011-01-01

    d-Amino acid oxidase (DAAO) from pig has been reported to catalyze the β-elimination of Cl− from βCl-d-alanine via abstraction of the substrate α-H as H+ (“carbanion mechanism”) (Walsh, C. T., Schonbrunn, A., and Abeles, R. H. (1971) J. Biol. Chem. 246, 6855–6866). In view of the fundamental mechanistic importance of this reaction and of the recent reinterpretation of the DAAO dehydrogenation step as occurring via a hydride mechanism, we reinvestigated the elimination reaction using yeast DAAO. That enzyme catalyzes the same reactions as the pig enzyme but with a much higher efficiency and a substantially different kinetic behavior. The reaction is initiated by a very rapid and fully reversible dehydrogenation step. This leads to an equilibrium (kon ≈ kreverse) between the complexes of oxidized enzyme-βCl-d-alanine and reduced enzyme-βCl-iminopyruvate. In the presence of O2 the latter complex can partition between an oxidative half-reaction and elimination of Cl−, which proceeds at a rate of ≈50 s−1. This step forms a complex between oxidized enzyme and enamine that is characterized by a charge transfer absorption (which describes its rates of formation and decay). A minimal scheme that lists relevant steps of the reductive and oxidative half-reactions and elimination pathways along with the estimate of the corresponding rate constants is presented. β-Elimination of Cl− is proposed to originate at the locus of the enzyme-βCl-iminopyruvate complex. A chemical mechanism that can account for elimination is discussed in detail. PMID:21949129

  6. A single amino acid change in the plant alternative oxidase alters the specificity of organic acid activation.

    PubMed

    Djajanegara, I; Holtzapffel, R; Finnegan, P M; Hoefnagel, M H; Berthold, D A; Wiskich, J T; Day, D A

    1999-07-01

    The alternative oxidase is a quinol oxidase of the respiratory chain of plants and some fungi and protists. Its activity is regulated by redox-sensitive disulphide bond formation between neighbouring subunits and direct interaction with certain alpha-ketoacids. To investigate these regulatory mechanisms, we undertook site-directed mutagenesis of soybean and Arabidopsis alternative oxidase cDNAs, and expressed them in tobacco plants and Escherichia coli, respectively. The homologous C99 and C127 residues of GmAOX3 and AtAOX1a, respectively, were changed to serine. In the plant system, this substitution prevented oxidative inactivation of alternative oxidase and rendered the protein insensitive to pyruvate activation, in agreement with the recent results from other laboratories [Rhoads et al. (1998) J. Biol. Chem. 273, 30750-30756; Vanlerberghe et al. (1998) Plant Cell 10, 1551-1560]. However, the mutated protein is instead activated specifically by succinate. Measurements of AtAOX1a activity in bacterial membranes lacking succinate dehydrogenase confirmed that the stimulation of the mutant protein's activity by succinate did not involve its metabolism. Examples of alternative oxidase proteins with the C to S substitution occur in nature and these oxidases are expected to be activated under most conditions in vivo, with implications for the efficiency of respiration in the tissues which express them.

  7. Enhanced fatty acid accumulation in Isochrysis galbana by inhibition of the mitochondrial alternative oxidase pathway under nitrogen deprivation.

    PubMed

    Zhang, Litao; Liu, Jianguo

    2016-07-01

    The purpose of this study was to clarify the interrelation between the mitochondrial alternative oxidase (AOX) pathway and fatty acid accumulation in marine microalga Isochrysis galbana. Under normal conditions, the activity of the AOX pathway was maintained at a low level in I. galbana. Compared with the normal condition, nitrogen deprivation significantly increased the AOX pathway activity and fatty acid accumulation. Under nitrogen deprivation, the inhibition of the AOX pathway by salicylhydroxamic acid caused the accumulation of reducing equivalents and the over-reduction of chloroplasts in I. galbana cells, leading to a decrease in the photosynthetic O2 evolution rate. The over-production of reducing equivalents due to the inhibition of the AOX pathway under nitrogen deprivation further enhanced the accumulation of fatty acids in I. galbana cells.

  8. Novel human D-amino acid oxidase inhibitors stabilize an active-site lid-open conformation

    PubMed Central

    Terry-Lorenzo, Ryan T.; Chun, Lawrence E.; Brown, Scott P.; Heffernan, Michele L. R.; Fang, Q. Kevin; Orsini, Michael A.; Pollegioni, Loredano; Hardy, Larry W.; Spear, Kerry L.; Large, Thomas H.

    2014-01-01

    The NMDAR (N-methyl-D-aspartate receptor) is a central regulator of synaptic plasticity and learning and memory. hDAAO (human D-amino acid oxidase) indirectly reduces NMDAR activity by degrading the NMDAR co-agonist D-serine. Since NMDAR hypofunction is thought to be a foundational defect in schizophrenia, hDAAO inhibitors have potential as treatments for schizophrenia and other nervous system disorders. Here, we sought to identify novel chemicals that inhibit hDAAO activity. We used computational tools to design a focused, purchasable library of compounds. After screening this library for hDAAO inhibition, we identified the structurally novel compound, ‘compound 2’ [3-(7-hydroxy-2-oxo-4-phenyl-2H-chromen-6-yl)propanoic acid], which displayed low nM hDAAO inhibitory potency (Ki=7 nM). Although the library was expected to enrich for compounds that were competitive for both D-serine and FAD, compound 2 actually was FAD uncompetitive, much like canonical hDAAO inhibitors such as benzoic acid. Compound 2 and an analog were independently co-crystalized with hDAAO. These compounds stabilized a novel conformation of hDAAO in which the active-site lid was in an open position. These results confirm previous hypotheses regarding active-site lid flexibility of mammalian D-amino acid oxidases and could assist in the design of the next generation of hDAAO inhibitors. PMID:25001371

  9. Lithospermic acid as a novel xanthine oxidase inhibitor has anti-inflammatory and hypouricemic effects in rats.

    PubMed

    Liu, Xiaoyu; Chen, Ruohua; Shang, Yanjun; Jiao, Binghua; Huang, Caiguo

    2008-11-25

    Lithospermic acid (LSA) was originally isolated from the roots of Salvia mitiorrhiza, a common herb of oriental medicine. Previous studies demonstrated that LSA has antioxidant effects. In this study, we investigated the in vitro xanthine oxidase (XO) inhibitory activity, and in vivo hypouricemic and anti-inflammatory effects of rats. XO activity was detected by measuring the formation of uric acid or superoxide radicals in the xanthine/xanthine oxidase system. The results showed that LSA inhibited the formation of uric acid and superoxide radicals significantly with an IC50 5.2 and 1.08 microg/ml, respectively, and exhibited competitive inhibition. It was also found that LSA scavenged superoxide radicals directly in the system beta-NADH/PMS and inhibited the production of superoxide in human neutrophils stimulated by PMA and fMLP. LSA was also found to have hypouricemic activity on oxonate-pretreated rats in vivo and have anti-inflammatory effects in a model of gouty arthritis. These results suggested that LSA is a competitive inhibitor of XO, able to directly scavenge superoxide and inhibit superoxide production in vitro, and presents hypouricemic and anti-inflammatory actions in vivo.

  10. Fabricating an Amperometric Cholesterol Biosensor by a Covalent Linkage between Poly(3-thiopheneacetic acid) and Cholesterol Oxidase

    PubMed Central

    Nien, Po-Chin; Chen, Po-Yen; Ho, Kuo-Chuan

    2009-01-01

    In this study, use of the covalent enzyme immobilization method was proposed to attach cholesterol oxidase (ChO) on a conducting polymer, poly(3-thiopheneacetic acid), [poly(3-TPAA)]. Three red-orange poly(3-TPAA) films, named electrodes A, B and C, were electropolymerized on a platinum electrode by applying a constant current of 1.5 mA, for 5, 20 and 100 s, respectively. Further, 1-ethyl-3-(3-dimethylamiopropyl)carbodiimide hydrochloride (EDC · HCl) and N-hydroxysuccinimide (NHS) were used to activate the free carboxylic groups of the conducting polymer. Afterwards, the amino groups of the cholesterol oxidase were linked on the activated groups to form peptide bonds. The best sensitivity obtained for electrode B is 4.49 mA M−1 cm−2, with a linear concentration ranging from 0 to 8 mM, which is suitable for the analysis of cholesterol in humans. The response time (t95) is between 70 and 90 s and the limit of detection is 0.42 mM, based on the signal to noise ratio equal to 3. The interference of species such as ascorbic acid and uric acid increased to 5.2 and 10.3% of the original current response, respectively, based on the current response of cholesterol (100%). With respect to the long-term stability, the sensing response retains 88% of the original current after 13 days. PMID:22573987

  11. Co-localization of glutamic acid decarboxylase and vesicular GABA transporter in cytochrome oxidase patches of macaque striate cortex.

    PubMed

    Adams, Daniel L; Economides, John R; Horton, Jonathan C

    2015-01-01

    The patches in primary visual cortex constitute hot spots of metabolic activity, manifested by enhanced levels of cytochrome oxidase (CO) activity. They are also labeled preferentially by immunostaining for glutamic acid decarboxylase (GAD), γ-aminobutyric acid (GABA), and parvalbumin. However, calbindin shows stronger immunoreactivity outside patches. In light of this discrepancy, the distribution of the vesicular GABA transporter (VGAT) was examined in striate cortex of two normal macaques. VGAT immunoreactivity was strongest in layers 4B, 4Cα, and 5. In tangential sections, the distribution of CO, GAD, and VGAT was compared in layer 2/3. There was a close match between all three labels. This finding indicates that GABA synthesis is enriched in patches, and that inhibitory synapses are more active in patches than interpatches. PMID:26579566

  12. Surface modification of polyvinyl alcohol/malonic acid nanofibers by gaseous dielectric barrier discharge plasma for glucose oxidase immobilization

    NASA Astrophysics Data System (ADS)

    Afshari, Esmail; Mazinani, Saeedeh; Ranaei-Siadat, Seyed-Omid; Ghomi, Hamid

    2016-11-01

    Polymeric nanofiber prepares a suitable situation for enzyme immobilization for variety of applications. In this research, we have fabricated polyvinyl alcohol (PVA)/malonic acid nanofibers using electrospinning. After fabrication of nanofibers, the effect of air, nitrogen, CO2, and argon DBD (dielectric barrier discharge) plasmas on PVA/malonic acid nanofibers were analysed. Among them, air plasma had the most significant effect on glucose oxidase (GOx) immobilization. Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectrum analysis and X-ray photoelectron spectroscopy (XPS) results revealed that in case of air plasma modified nanofibers, the carboxyl groups on the surface are increased. The scanning electron microscopy (SEM) images showed that, after GOx immobilization, the modified nanofibers with plasma has retained its nanofiber structure. Finally, we analysed reusability and storage stability of GOx immobilized on plasma modified and unmodified nanofibers. The results were more satisfactory for modified nanofibers with respect to unmodified ones.

  13. Positive feedback regulation of maize NADPH oxidase by mitogen-activated protein kinase cascade in abscisic acid signalling

    PubMed Central

    Lin, Fan; Ding, Haidong; Wang, Jinxiang; Zhang, Hong; Zhang, Aying; Zhang, Yun; Tan, Mingpu; Dong, Wen; Jiang, Mingyi

    2009-01-01

    In maize (Zea mays), abscisic acid (ABA)-induced H2O2 production activates a 46 kDa mitogen-activated protein kinase (p46MAPK), and the activation of p46MAPK also regulates the production of H2O2. However, the mechanism for the regulation of H2O2 production by MAPK in ABA signalling remains to be elucidated. In this study, four reactive oxygen species (ROS)-producing NADPH oxidase (rboh) genes (ZmrbohA–D) were isolated and characterized in maize leaves. ABA treatment induced a biphasic response (phase I and phase II) in the expression of ZmrbohA–D and the activity of NADPH oxidase. Phase II induced by ABA was blocked by pretreatments with two MAPK kinase (MPKKK) inhibitors and two H2O2 scavengers, but phase I was not affected by these inhibitors or scavengers. Treatment with H2O2 alone also only induced phase II, and the induction was arrested by the MAPKK inhibitors. Furthermore, the ABA-activated p46MAPK was partially purified. Using primers corresponding to the sequences of internal tryptic peptides, the p46MAPK gene was cloned. Analysis of the tryptic peptides and the p46MAPK sequence indicate it is the known ZmMPK5. Treatments with ABA and H2O2 led to a significant increase in the activity of ZmMPK5, although ABA treatment only induced a slight increase in the expression of ZmMPK5. The data indicate that H2O2-activated ZmMPK5 is involved in the activation of phase II in ABA signalling, but not in phase I. The results suggest that there is a positive feedback loop involving NADPH oxidase, H2O2, and ZmMPK5 in ABA signalling. PMID:19592501

  14. Interfacial electron transfer of glucose oxidase on poly(glutamic acid)-modified glassy carbon electrode and glucose sensing.

    PubMed

    Zhou, Xuechou; Tan, Bingcan; Zheng, Xinyu; Kong, Dexian; Li, Qinglu

    2015-11-15

    The interfacial electron transfer of glucose oxidase (GOx) on a poly(glutamic acid)-modified glassy carbon electrode (PGA/GCE) was investigated. The redox peaks measured for GOx and flavin adenine dinucleotide (FAD) are similar, and the anodic peak of GOx does not increase in the presence of glucose in a mediator-free solution. These indicate that the electroactivity of GOx is not the direct electron transfer (DET) between GOx and PGA/GCE and that the observed electroactivity of GOx is ascribed to free FAD that is released from GOx. However, efficient electron transfer occurred if an appropriate mediator was placed in solution, suggesting that GOx is active. The PGA/GCE-based biosensor showed wide linear response in the range of 0.5-5.5 mM with a low detection limit of 0.12 mM and high sensitivity and selectivity for measuring glucose. PMID:26278169

  15. Salicylic acid inhibits enzymatic browning of fresh-cut Chinese chestnut (Castanea mollissima) by competitively inhibiting polyphenol oxidase.

    PubMed

    Zhou, Dan; Li, Lin; Wu, Yanwen; Fan, Junfeng; Ouyang, Jie

    2015-03-15

    The inhibitory effect and associated mechanisms of salicylic acid (SA) on the browning of fresh-cut Chinese chestnut were investigated. Shelled and sliced chestnuts were immersed in different concentrations of an SA solution, and the browning of the chestnut surface and interior were inhibited. The activities of polyphenol oxidase (PPO) and peroxidase (POD) extracted from chestnuts were measured in the presence and absence of SA. SA at concentrations higher than 0.3g/L delayed chestnut browning by significantly inhibiting the PPO activity (P<0.01), and the POD activity was not significantly affected (P>0.05). The binding and inhibition modes of SA with PPO and POD, determined by AUTODOCK 4.2 and Lineweaver-Burk plots, respectively, established SA as a competitive inhibitor of PPO. PMID:25308637

  16. Interfacial electron transfer of glucose oxidase on poly(glutamic acid)-modified glassy carbon electrode and glucose sensing.

    PubMed

    Zhou, Xuechou; Tan, Bingcan; Zheng, Xinyu; Kong, Dexian; Li, Qinglu

    2015-11-15

    The interfacial electron transfer of glucose oxidase (GOx) on a poly(glutamic acid)-modified glassy carbon electrode (PGA/GCE) was investigated. The redox peaks measured for GOx and flavin adenine dinucleotide (FAD) are similar, and the anodic peak of GOx does not increase in the presence of glucose in a mediator-free solution. These indicate that the electroactivity of GOx is not the direct electron transfer (DET) between GOx and PGA/GCE and that the observed electroactivity of GOx is ascribed to free FAD that is released from GOx. However, efficient electron transfer occurred if an appropriate mediator was placed in solution, suggesting that GOx is active. The PGA/GCE-based biosensor showed wide linear response in the range of 0.5-5.5 mM with a low detection limit of 0.12 mM and high sensitivity and selectivity for measuring glucose.

  17. Salicylic acid inhibits enzymatic browning of fresh-cut Chinese chestnut (Castanea mollissima) by competitively inhibiting polyphenol oxidase.

    PubMed

    Zhou, Dan; Li, Lin; Wu, Yanwen; Fan, Junfeng; Ouyang, Jie

    2015-03-15

    The inhibitory effect and associated mechanisms of salicylic acid (SA) on the browning of fresh-cut Chinese chestnut were investigated. Shelled and sliced chestnuts were immersed in different concentrations of an SA solution, and the browning of the chestnut surface and interior were inhibited. The activities of polyphenol oxidase (PPO) and peroxidase (POD) extracted from chestnuts were measured in the presence and absence of SA. SA at concentrations higher than 0.3g/L delayed chestnut browning by significantly inhibiting the PPO activity (P<0.01), and the POD activity was not significantly affected (P>0.05). The binding and inhibition modes of SA with PPO and POD, determined by AUTODOCK 4.2 and Lineweaver-Burk plots, respectively, established SA as a competitive inhibitor of PPO.

  18. Substrate Orientation and Catalytic Specificity in the Action of Xanthine Oxidase: The Sequential Hydroxylation of Hypoxanthine to Uric Acid

    SciTech Connect

    Cao, Hongnan; Pauff, James M.; Hille, Russ

    2010-11-29

    Xanthine oxidase is a molybdenum-containing enzyme catalyzing the hydroxylation of a sp{sup 2}-hybridized carbon in a broad range of aromatic heterocycles and aldehydes. Crystal structures of the bovine enzyme in complex with the physiological substrate hypoxanthine at 1.8 {angstrom} resolution and the chemotherapeutic agent 6-mercaptopurine at 2.6 {angstrom} resolution have been determined, showing in each case two alternate orientations of substrate in the two active sites of the crystallographic asymmetric unit. One orientation is such that it is expected to yield hydroxylation at C-2 of substrate, yielding xanthine. The other suggests hydroxylation at C-8 to give 6,8-dihydroxypurine, a putative product not previously thought to be generated by the enzyme. Kinetic experiments demonstrate that >98% of hypoxanthine is hydroxylated at C-2 rather than C-8, indicating that the second crystallographically observed orientation is significantly less catalytically effective than the former. Theoretical calculations suggest that enzyme selectivity for the C-2 over C-8 of hypoxanthine is largely due to differences in the intrinsic reactivity of the two sites. For the orientation of hypoxanthine with C-2 proximal to the molybdenum center, the disposition of substrate in the active site is such that Arg880 and Glu802, previous shown to be catalytically important for the conversion of xanthine to uric acid, play similar roles in hydroxylation at C-2 as at C-8. Contrary to the literature, we find that 6,8-dihydroxypurine is effectively converted to uric acid by xanthine oxidase.

  19. A single amino acid substitution confers high cinchonidine oxidation activity comparable with that of rabbit to monkey aldehyde oxidase 1.

    PubMed

    Fukiya, Kensuke; Itoh, Kunio; Yamaguchi, Satoshi; Kishiba, Akiko; Adachi, Mayuko; Watanabe, Nobuaki; Tanaka, Yorihisa

    2010-02-01

    Aldehyde oxidase 1 (AOX1) is a major member of the xanthine oxidase family belonging to the class of complex molybdo-flavoenzymes and plays an important role in the nucleophilic oxidation of N-heterocyclic aromatic compounds and various aldehydes. The enzyme has been well known to show remarkable species differences. Comparing the rabbit and monkey enzymes, the former showed extremely high activity toward cinchonidine and methotrexate, but the latter exhibited only marginal activities. In contrast, monkey had several times greater activity than did rabbit toward zonisamide and (+)-4-(4-cyanoanilino)-5,6-dihydro-7-hydroxy-7H-cyclopenta[d]-pyrimidine [(S)-RS-8359]. In this report, we tried to confer high cinchonidine oxidation activity comparable with that of rabbit AOX1 to monkey AOX1. The chimera proteins prepared by restriction enzyme digestion and recombination methods between monkey and rabbit AOX1s indicated that the sequences from Asn993 to Ala1088 of rabbit AOX1 are essential for the activity. The kinetic parameters were then measured using monkey AOX1 mutants prepared by site-directed mutagenesis. The monkey V1085A mutant acquired the high cinchonidine oxidation activity. Inversely, the reciprocal rabbit A1081V mutant lost the activity entirely: amino acid 1081 of rabbit AOX1 corresponding to amino acid 1085 of monkey AOX1. Thus, cinchonidine oxidation activity was drastically changed by mutation of a single residue in AOX1. However, this might be true for bulky substrates such as cinchonidine but not for small substrates. The mechanism of substrate-dependent species differences in AOX1 activity toward bulky substrates is discussed.

  20. 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).

  1. Polyacrylic acid-coated cerium oxide nanoparticles: An oxidase mimic applied for colorimetric assay to organophosphorus pesticides.

    PubMed

    Zhang, Shi-Xiang; Xue, Shi-Fan; Deng, Jingjing; Zhang, Min; Shi, Guoyue; Zhou, Tianshu

    2016-11-15

    It is important and urgent to develop reliable and highly sensitive methods that can provide on-site and rapid detection of extensively used organophosphorus pesticides (OPs) for their neurotoxicity. In this study, we developed a novel colorimetric assay for the detection of OPs based on polyacrylic acid-coated cerium oxide nanoparticles (PAA-CeO2) as an oxidase mimic and OPs as inhibitors to suppress the activity of acetylcholinesterase (AChE). Firstly, highly dispersed PAA-CeO2 was prepared in aqueous solution, which could catalyze the oxidation of TMB to produce a color reaction from colorless to blue. And the enzyme of AChE was used to catalyze the substrate of acetylthiocholine (ATCh) to produce thiocholine (TCh). As a thiol-containing compound with reducibility, TCh can decrease the oxidation of TMB catalyzed by PAA-CeO2. Upon incubated with OPs, the enzymatic activity of AChE was inhibited to produce less TCh, resulting in more TMB catalytically oxidized by PAA-CeO2 to show an increasing blue color. The two representative OPs, dichlorvos and methyl-paraoxon, were tested using our proposed assay. The novel assay showed notable color change in a concentration-dependent manner, and as low as 8.62 ppb dichlorvos and 26.73 ppb methyl-paraoxon can be readily detected. Therefore, taking advantage of such oxidase-like activity of PAA-CeO2, our proposed colorimetric assay can potentially be a screening tool for the precise and rapid evaluation of the neurotoxicity of a wealth of OPs.

  2. Polyacrylic acid-coated cerium oxide nanoparticles: An oxidase mimic applied for colorimetric assay to organophosphorus pesticides.

    PubMed

    Zhang, Shi-Xiang; Xue, Shi-Fan; Deng, Jingjing; Zhang, Min; Shi, Guoyue; Zhou, Tianshu

    2016-11-15

    It is important and urgent to develop reliable and highly sensitive methods that can provide on-site and rapid detection of extensively used organophosphorus pesticides (OPs) for their neurotoxicity. In this study, we developed a novel colorimetric assay for the detection of OPs based on polyacrylic acid-coated cerium oxide nanoparticles (PAA-CeO2) as an oxidase mimic and OPs as inhibitors to suppress the activity of acetylcholinesterase (AChE). Firstly, highly dispersed PAA-CeO2 was prepared in aqueous solution, which could catalyze the oxidation of TMB to produce a color reaction from colorless to blue. And the enzyme of AChE was used to catalyze the substrate of acetylthiocholine (ATCh) to produce thiocholine (TCh). As a thiol-containing compound with reducibility, TCh can decrease the oxidation of TMB catalyzed by PAA-CeO2. Upon incubated with OPs, the enzymatic activity of AChE was inhibited to produce less TCh, resulting in more TMB catalytically oxidized by PAA-CeO2 to show an increasing blue color. The two representative OPs, dichlorvos and methyl-paraoxon, were tested using our proposed assay. The novel assay showed notable color change in a concentration-dependent manner, and as low as 8.62 ppb dichlorvos and 26.73 ppb methyl-paraoxon can be readily detected. Therefore, taking advantage of such oxidase-like activity of PAA-CeO2, our proposed colorimetric assay can potentially be a screening tool for the precise and rapid evaluation of the neurotoxicity of a wealth of OPs. PMID:27208478

  3. Two New Alleles of the abscisic aldehyde oxidase 3 Gene Reveal Its Role in Abscisic Acid Biosynthesis in Seeds1

    PubMed Central

    González-Guzmán, Miguel; Abia, David; Salinas, Julio; Serrano, Ramón; Rodríguez, Pedro L.

    2004-01-01

    The abscisic aldehyde oxidase 3 (AAO3) gene product of Arabidopsis catalyzes the final step in abscisic acid (ABA) biosynthesis. An aao3-1 mutant in a Landsberg erecta genetic background exhibited a wilty phenotype in rosette leaves, whereas seed dormancy was not affected (Seo et al., 2000a). Therefore, it was speculated that a different aldehyde oxidase would be the major contributor to ABA biosynthesis in seeds (Seo et al., 2000a). Through a screening based on germination under high-salt concentration, we isolated two mutants in a Columbia genetic background, initially named sre2-1 and sre2-2 (for salt resistant). Complementation tests with different ABA-deficient mutants indicated that sre2-1 and sre2-2 mutants were allelic to aao3-1, and therefore they were renamed as aao3-2 and aao3-3, respectively. Indeed, molecular characterization of the aao3-2 mutant revealed a T-DNA insertional mutation that abolished the transcription of AAO3 gene, while sequence analysis of AAO3 in aao3-3 mutant revealed a deletion of three nucleotides and several missense mutations. Physiological characterization of aao3-2 and aao3-3 mutants revealed a wilty phenotype and osmotolerance in germination assays. In contrast to aao3-1, both aao3-2 and aao3-3 mutants showed a reduced dormancy. Accordingly, ABA levels were reduced in dry seeds and rosette leaves of both aao3-2 and aao3-3. Taken together, these results indicate that AAO3 gene product plays a major role in seed ABA biosynthesis. PMID:15122034

  4. Gluconic acid production in bioreactor with immobilized glucose oxidase plus catalase on polymer membrane adjacent to anion-exchange membrane.

    PubMed

    Godjevargova, Tzonka; Dayal, Rajeshwar; Turmanova, Sevdalina

    2004-10-20

    Gluconic acid was obtained in the permeate side of the bioreactor with glucose oxidase (GOD) immobilized onto anion-exchange membrane (AEM) of low-density polyethylene grafted with 4-vinylpiridine. The electric resistance of the anion-exchange membranes was increased after the enzyme immobilization on the membrane. The gluconic acid productions were relatively low with the GOD immobilized by any method on the AEM. To increase the enzyme reaction efficiency, GOD was immobilized on membrane of AN copolymer (PAN) adjacent to an anion-exchange membrane in bioreactor. Uses of anion-exchange membrane led to selective removal of the gluconic acid from the glucose solution and reduce the gluconic acid inhibition. The amount of gluconic acid obtained in the permeate side of the bioreactor with the GOD immobilized on the PAN membrane adjacent to the AEM under electrodialysis was about 30 times higher than that obtained with enzyme directly bound to the AEM. The optimal substrate concentration in the feed side was found to be about 1 g/l. Further experiments were carried out with the co-immobilized GOD plus Catalase (CAT) on the PAN membrane adjacent to the AEM to improve the efficiency of the immobilize system. The yield of this process was at least 95%. The storage stability of the co-immobilized GOD and CAT was studied (lost 20% of initial activity for 90 d). The results obtained clearly showed the higher potential of the dual membrane bioreactor with GOD plus CAT bound to ultrafiltration polymer membrane adjacent to the AEM. Storage stability of GOD activity in GOD plus CAT immobilized on PAN//AEM membranes and on AEM.

  5. Control of skin colour and polyphenol oxidase activity in santol fruit by dipping in organic acid solution.

    PubMed

    Benjawan, Chutichudet; Chutichudet, P

    2009-06-01

    This laboratory experiment was carried out at the Department of Agricultural Technology, Mahasarakham University, Northeast Thailand during July and August 2008. The experiment aimed to determine an effective natural organic acid that would delay the unattractive skin browning of santol fruit, while at the same time not damaging the quality of the fruit. The experiment included a study of the fruit's polyphenol oxidase (PPO) activity, phenolic content and quinone content, as they relate to colour and a study of total soluble solid content, pH, titratable acidity and vitamin C content as they relate to fruit quality. A Completely Randomized Design (CRD) with four replications was used. Each replication consisted of 10 fruits. Santol fruit was harvested at 145 days after full bloom and dipped for 30 min in aqueous solutions of two organic acids that were used as treatments, i.e., 0% for T1 (control), 5% citric acid for T2, 5% ascorbic acid for T3, 10% citric acid for T4 and 10% ascorbic acid for T5 and stored at room temperature (28 degrees C, 90% R.H.) to investigate the effect of the acid on fruit weight, skin colour, PPO activity and other internal parameters. The results showed that the most appropriate anti-browning agent for santol fruit was found with T2. It gave the highest mean values, 57.37 and 55.95, of brightness (L*) at 4 and 10 Days After Storage (DAS), respectively. In addition, PPO activity of flesh tissue was lowest for T2 with mean values of 0.0078 to 0.0092 by 0 and 300 S, respectively. The phenolic content in the flesh tissue significantly increased with an increase in numbers ofDAS, whereas the reverse was found with the pH level in the fruits. They were lowest for T2, with mean values of 6.00, by 10 DAS. There were no significant differences among the treatments in any of the measured Total Soluble Solids (TSS), Titratable Acidity (TA) and vitamin C content.

  6. The conformational state of polyphenol oxidase from field bean (Dolichos lablab) upon SDS and acid-pH activation.

    PubMed

    Kanade, Santosh R; Paul, Beena; Rao, A G Appu; Gowda, Lalitha R

    2006-05-01

    Field bean (Dolichos lablab) contains a single isoform of PPO (polyphenol oxidase)--a type III copper protein that catalyses the o-hydroxylation of monophenols and oxidation of o-diphenols using molecular oxygen--and is a homotetramer with a molecular mass of 120 kDa. The enzyme is activated manyfold either in the presence of the anionic detergent SDS below its critical micellar concentration or on exposure to acid-pH. The enhancement of kcat upon activation is accompanied by a marked shift in the pH optimum for the oxidation of t-butyl catechol from 4.5 to 6.0, an increased sensitivity to tropolone, altered susceptibility to proteolytic degradation and decreased thermostability. The Stokes radius of the native enzyme is found to increase from 49.1+/-2 to 75.9+/-0.6 A (1 A=0.1 nm). The activation by SDS and acid-pH results in a localized conformational change that is anchored around the catalytic site of PPO that alters the microenvironment of an essential glutamic residue. Chemical modification of field bean and sweet potato PPO with 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide followed by kinetic analysis leads to the conclusion that both the enzymes possess a core carboxylate essential to activity. This enhanced catalytic efficiency of PPO, considered as an inducible defence oxidative enzyme, is vital to the physiological defence strategy adapted by plants to insect herbivory and pathogen attack. PMID:16393141

  7. Structural characterization of CalO2: A putative orsellinic acid P450 oxidase in the calicheamicin biosynthetic

    SciTech Connect

    McCoy, Jason G.; Johnson, Heather D.; Singh, Shanteri; Bingman, Craig A.; Lei, In-Kyoung; Thorson, Jon S.; Phillips, Jr., George N.

    2009-08-13

    Although bacterial iterative Type I polyketide synthases are now known to participate in the biosynthesis of a small set of diverse natural products, the subsequent downstream modification of the resulting polyketide products remains poorly understood. Toward this goal, we report the X-ray structure determination at 2.5 A resolution and preliminary characterization of the putative orsellenic acid P450 oxidase (CalO2) involved in calicheamicin biosynthesis. These studies represent the first crystal structure for a P450 involved in modifying a bacterial iterative Type I polyketide product and suggest the CalO2-catalyzed step may occur after CalO3-catalyzed iodination and may also require a coenzyme A- (CoA) or acyl carrier protein- (ACP) bound substrate. Docking studies also reveal a putative docking site within CalO2 for the CLM orsellinic acid synthase (CalO5) ACP domain which involves a well-ordered helix along the CalO2 active site cavity that is unique compared with other P450 structures.

  8. 4-Hydroxypyridazin-3(2H)-one derivatives as novel D-amino acid oxidase inhibitors.

    PubMed

    Hondo, Takeshi; Warizaya, Masaichi; Niimi, Tatsuya; Namatame, Ichiji; Yamaguchi, Tomohiko; Nakanishi, Keita; Hamajima, Toshihiro; Harada, Katsuya; Sakashita, Hitoshi; Matsumoto, Yuzo; Orita, Masaya; Takeuchi, Makoto

    2013-05-01

    D-Amino acid oxidase (DAAO) catalyzes the oxidation of d-amino acids including d-serine, a coagonist of the N-methyl-d-aspartate receptor. We identified a series of 4-hydroxypyridazin-3(2H)-one derivatives as novel DAAO inhibitors with high potency and substantial cell permeability using fragment-based drug design. Comparisons of complex structures deposited in the Protein Data Bank as well as those determined with in-house fragment hits revealed that a hydrophobic subpocket was formed perpendicular to the flavin ring by flipping Tyr224 in a ligand-dependent manner. We investigated the ability of the initial fragment hit, 3-hydroxy-pyridine-2(1H)-one, to fill this subpocket with the aid of complex structure information. 3-Hydroxy-5-(2-phenylethyl)pyridine-2(1H)-one exhibited the predicted binding mode and demonstrated high inhibitory activity for human DAAO in enzyme- and cell-based assays. We further designed and synthesized 4-hydroxypyridazin-3(2H)-one derivatives, which are equivalent to the 3-hydroxy-pyridine-2(1H)-one series but lack cell toxicity. 6-[2-(3,5-Difluorophenyl)ethyl]-4-hydroxypyridazin-3(2H)-one was found to be effective against MK-801-induced cognitive deficit in the Y-maze.

  9. Spinal D-amino acid oxidase contributes to mechanical pain hypersensitivity induced by sleep deprivation in the rat.

    PubMed

    Wei, Hong; Gong, Nian; Huang, Jin-Lu; Fan, Hui; Ma, Ai-Niu; Li, Xin-Yan; Wang, Yong-Xiang; Pertovaara, Antti

    2013-10-01

    We studied the hypothesis that spinal d-amino acid oxidase (DAAO) that is expressed in astrocytes and that has been reported to promote tonic pain in various pathophysiological conditions plays a role in 'physiological' pain hypersensitivity induced by rapid eye movement sleep deprivation (REMSD). The experiments were performed in healthy rats with a chronic intrathecal (i.t.) catheter. Pain behavior was assessed by determining limb withdrawal response to repetitive stimulation of the hind paw with a calibrated series of monofilaments. REMSD of 48 h duration produced a significant mechanical hypersensitivity. At 48 h of REMSD, the animals were treated i.t. with a DAAO inhibitor or vehicle. Three structurally different DAAO inhibitors were tested in this study: 6-chlorobenzo[d]isoxazol-3-ol (CBIO), sodium benzoate, and 5-methylpyrazole-3-carboxylic acid (AS-057278). CBIO (1-3 μg), sodium benzoate (30-100 μg) and AS-057278 (3-10 μg) produced dose-related antihypersensitivity effects in sleep-deprived animals. In control animals (with no sleep deprivation), the currently used doses of DAAO inhibitors failed to produce significant changes in mechanically evoked pain behavior. The results indicate that among spinal pain facilitatory mechanisms that contribute to the sleep deprivation-induced mechanical pain hypersensitivity is DAAO, presumably due to production of reactive oxygen species, such as hydrogen peroxide, an endogenous agonist of the pronociceptive TRPA1 ion channel.

  10. Changes in D-aspartic acid and D-glutamic acid levels in the tissues and physiological fluids of mice with various D-aspartate oxidase activities.

    PubMed

    Han, Hai; Miyoshi, Yurika; Koga, Reiko; Mita, Masashi; Konno, Ryuichi; Hamase, Kenji

    2015-12-10

    D-Aspartic acid (D-Asp) and D-glutamic acid (D-Glu) are currently paid attention as modulators of neuronal transmission and hormonal secretion. These two D-amino acids are metabolized only by D-aspartate oxidase (DDO) in mammals. Therefore, in order to design and develop new drugs controlling the D-Asp and D-Glu amounts via regulation of the DDO activities, changes in these acidic D-amino acid amounts in various tissues are expected to be clarified in model animals having various DDO activities. In the present study, the amounts of Asp and Glu enantiomers in 6 brain tissues, 11 peripheral tissues and 2 physiological fluids of DDO(+/+), DDO(+/-) and DDO(-/-) mice were determined using a sensitive and selective two-dimensional HPLC system. As a result, the amounts of D-Asp were drastically increased with the decrease in the DDO activity in all the tested tissues and physiological fluids. On the other hand, the amounts of D-Glu were almost the same among the 3 strains of mice. The present results are useful for designing new drug candidates, such as DDO inhibitors, and further studies are expected.

  11. Esters of 3,4-dihydroxybenzoic acid, highly effective inhibitors of the sn-glycerol-3-phosphate oxidase of Trypanosoma brucei brucei.

    PubMed

    Grady, R W; Bienen, E J; Clarkson, A B

    1986-10-01

    Alkyl esters of 3,4-dihydroxybenzoic acid are inhibitors of the sn-glycerol-3-phosphate oxidase system of Trypanosoma brucei brucei in vitro and have significant trypanocidal activity in vivo when combined with glycerol. While the parent acid has little inhibitory activity in vitro, the esters are highly active with activity increasing as the chain length of the esterifying alcohol increases. The n-dodecyl ester was more than 400 times as active as salicylhydroxamic acid and 15 times more active than the corresponding p-n-alkyloxybenzhydroxamic acid, one of the most active sn-glycerol-3-phosphate oxidase inhibitors previously reported. When combined with glycerol (to block an alternative pathway of glycolysis) and tested in vitro against intact parasites, this ester was 100 times more effective than salicylhydroxamic acid and 10 times more effective than p-n-dodecyloxybenzhydroxamic acid. It was also active against T. b. brucei in mice when combined with glycerol whereas the latter compound was not. Esters of 3,4,5-trihydroxybenzoic acid (gallic acid) were also highly active while those of 2,3-dihydroxybenzoic acid were much less inhibitory and those of 2,5-dihydroxybenzoic acid were inactive. A related compound, 2',4',5'-trihydroxybutyrophenone, was also active as predicted by its structure but was too toxic to be of interest as a drug candidate.

  12. Indispensable but insufficient role of renal D-amino acid oxidase in chiral inversion of NG-nitro-D-arginine.

    PubMed

    Xin, Yan-Fei; Li, Xin; Hao, Bin; Gong, Nian; Sun, Wen-Qiang; Konno, Ryuichi; Wang, Yong-Xiang

    2010-06-01

    Unidirectionally chiral inversion of N(G)-nitro-D-arginine (D-NNA) to its L-enantiomer (L-NNA) occurred in rats, and it was blocked markedly (ca. 80%) by renal vascular ligation, and entirely (100%) by the D-amino acid oxidase (DAO) inhibitor sodium benzoate, suggesting that renal DAO is essential for the inversion. However, the doses of sodium benzoate administrated were extremely high (e.g., 400 mg/kg) due to its low potency. It is thus possible that sodium benzoate-mediated blockade of D-NNA inversion might be due to its nonspecific (or non-DAO-related) effects. In addition, after D-NNA was incubated with the pure enzyme of DAO in vitro without tissue homogenates, L-NNA was not produced, even though D-NNA was disposed. We propose that this occurred because D-NNA was first converted to its corresponding alpha-keto acid by DAO and then to L-NNA by transaminase(s); however, there was no direct evidence for this process. The goal of this study is to further elucidate the process of D-NNA chiral inversion both in vivo and in in vitro tissue homogenates by comparing mutant ddY/DAO(-/-) mice that lack DAO activity entirely compared to normal ddY/DAO(+/+) mice and Swiss mice. Furthermore, the ability to produce L-NNA from D-NNA-corresponding alpha-keto acids (N(G)-nitroguanidino-2-oxopentanoic acid) produced by porcine kidney-derived DAO (pkDAO) was also studied in the DAO inhibitor-pretreated rats. We found that D-NNA chiral inversion occurred in Swiss mice and ddY/DAO(+/+) mice both in vivo and in in vitro kidney homogenates, but not in ddY/DAO(-/-) mice, correlated to their DAO activities. The alpha-keto acid (N(G)-nitro-guanidino-2-oxopentanoic acid) from D-NNA was able to produce L-NNA, and subsequent vasoconstriction and pressor responses. These results indicate that the role of renal DAO is indispensible but insufficient for chiral inversion of D-NNA and other neutral and polar D-amino acids, and unidentified aminotransferase(s) are involved in a subsequent

  13. Indispensable but insufficient role of renal D-amino acid oxidase in chiral inversion of NG-nitro-D-arginine.

    PubMed

    Xin, Yan-Fei; Li, Xin; Hao, Bin; Gong, Nian; Sun, Wen-Qiang; Konno, Ryuichi; Wang, Yong-Xiang

    2010-06-01

    Unidirectionally chiral inversion of N(G)-nitro-D-arginine (D-NNA) to its L-enantiomer (L-NNA) occurred in rats, and it was blocked markedly (ca. 80%) by renal vascular ligation, and entirely (100%) by the D-amino acid oxidase (DAO) inhibitor sodium benzoate, suggesting that renal DAO is essential for the inversion. However, the doses of sodium benzoate administrated were extremely high (e.g., 400 mg/kg) due to its low potency. It is thus possible that sodium benzoate-mediated blockade of D-NNA inversion might be due to its nonspecific (or non-DAO-related) effects. In addition, after D-NNA was incubated with the pure enzyme of DAO in vitro without tissue homogenates, L-NNA was not produced, even though D-NNA was disposed. We propose that this occurred because D-NNA was first converted to its corresponding alpha-keto acid by DAO and then to L-NNA by transaminase(s); however, there was no direct evidence for this process. The goal of this study is to further elucidate the process of D-NNA chiral inversion both in vivo and in in vitro tissue homogenates by comparing mutant ddY/DAO(-/-) mice that lack DAO activity entirely compared to normal ddY/DAO(+/+) mice and Swiss mice. Furthermore, the ability to produce L-NNA from D-NNA-corresponding alpha-keto acids (N(G)-nitroguanidino-2-oxopentanoic acid) produced by porcine kidney-derived DAO (pkDAO) was also studied in the DAO inhibitor-pretreated rats. We found that D-NNA chiral inversion occurred in Swiss mice and ddY/DAO(+/+) mice both in vivo and in in vitro kidney homogenates, but not in ddY/DAO(-/-) mice, correlated to their DAO activities. The alpha-keto acid (N(G)-nitro-guanidino-2-oxopentanoic acid) from D-NNA was able to produce L-NNA, and subsequent vasoconstriction and pressor responses. These results indicate that the role of renal DAO is indispensible but insufficient for chiral inversion of D-NNA and other neutral and polar D-amino acids, and unidentified aminotransferase(s) are involved in a subsequent

  14. Synthesis and characterization of microparticles based on poly-methacrylic acid with glucose oxidase for biosensor applications.

    PubMed

    Hervás Pérez, J P; López-Ruiz, B; López-Cabarcos, E

    2016-03-01

    In the line of the applicability of biocompatible monomers pH and temperature dependent, we assayed poly-methacrylic acid (p-MAA) microparticles as immobilization system in the design of enzymatic biosensors. Glucose oxidase was used as enzyme model for the study of microparticles as immobilization matrices and as biological material in the performance of glucose biosensors. The enzyme immobilization method was optimized by investigating the influence of monomer concentration and cross-linker content (N',N'-methylenebisacrylamide), used in the preparation of the microparticles in the response of the biosensors. The kinetics of the polymerization and the effects of the temperature were studied, also the conversion of the polymerization was determinates by a weight method. The structure of the obtained p-MAA microparticles were studied through scanning electron microscopy (SEM) and differential scanning microscopy (DSC). The particle size measurements were performed with a Galai-Cis 1 particle analyzer system. Furthermore, the influence of the swelling behavior of hydrogel matrix as a function of pH and temperature were studied. Analytical properties such as sensitivity, linear range, response time and detection limit were studied for the glucose biosensors. The sensitivity for glucose detection obtained with poly-methacrylic acid (p-MAA) microparticles was 11.98mAM(-1)cm(-2) and 10μM of detection limit. A Nafion® layer was used to eliminate common interferents of the human serum such as uric and ascorbic acids. The biosensors were used to determine glucose in human serum samples with satisfactory results. When stored in a frozen phosphate buffer solution (pH 6.0) at -4°C, the useful lifetime of all biosensors was at least 550 days. PMID:26717846

  15. Synthesis and characterization of microparticles based on poly-methacrylic acid with glucose oxidase for biosensor applications.

    PubMed

    Hervás Pérez, J P; López-Ruiz, B; López-Cabarcos, E

    2016-01-01

    In the line of the applicability of biocompatible monomers pH and temperature dependent, we assayed poly-methacrylic acid (p-MAA) microparticles as immobilization system in the design of enzymatic biosensors. Glucose oxidase was used as enzyme model for the study of microparticles as immobilization matrices and as biological material in the performance of glucose biosensors. The enzyme immobilization method was optimized by investigating the influence of monomer concentration and cross-linker content (N',N'-methylenebisacrylamide), used in the preparation of the microparticles in the response of the biosensors. The kinetics of the polymerization and the effects of the temperature were studied, also the conversion of the polymerization was determinates by a weight method. The structure of the obtained p-MAA microparticles were studied through scanning electron microscopy (SEM) and differential scanning microscopy (DSC). The particle size measurements were performed with a Galai-Cis 1 particle analyzer system. Furthermore, the influence of the swelling behavior of hydrogel matrix as a function of pH and temperature were studied. Analytical properties such as sensitivity, linear range, response time and detection limit were studied for the glucose biosensors. The sensitivity for glucose detection obtained with poly-methacrylic acid (p-MAA) microparticles was 11.98mAM(-1)cm(-2) and 10μM of detection limit. A Nafion® layer was used to eliminate common interferents of the human serum such as uric and ascorbic acids. The biosensors were used to determine glucose in human serum samples with satisfactory results. When stored in a frozen phosphate buffer solution (pH 6.0) at -4°C, the useful lifetime of all biosensors was at least 550 days.

  16. Optimization of media composition for D-amino acid oxidase production by Trigonopsis variabilis using biostatistical analysis.

    PubMed

    Gupta, Neeraj; Gundampati, Ravi Kumar; Debnath, M

    2012-08-01

    D-amino acid oxidase (DAAO) is biotechnologically relevant enzyme that is used in various food and pharmaceutical industries. DAAO from the yeast Trigonopsis variabilis is an important agent for use in commercial applications because of its high activity with cephalosporin C and is reasonable resistant to the oxidants O2 and H2O2 byproducts of reaction. In this study, response surface methodology (RSM) in shake flask culture was used to enhance the production of DAAO from T. variabilis by optimization of fermentation media composition. The effects of six factors (DL-alanine, glucose, pH, ZnSO4, (NH4)2SO4 and temperature) were evaluated on DAAO production. Results of Placket-Burman design showed that DL-alanine, pH, glucose and ZnSO4 were significant factors for DAAO production (P<0.05). The optimum values of media components as predicted by the central composite design were inducer (DL-alanine) concentration 3 g/L, pH 7.7, glucose 17 g/L and ZnSO4 34 mg/L. At these optimum values of media composition, maximum production of DAAO was 153 U/g yeast dry weight. Two-fold increase in DAAO production was achieved after optimization of the physical parameters by RSM.

  17. Factors influencing diamine oxidase activity and γ-aminobutyric acid content of fava bean (Vicia faba L.) during germination.

    PubMed

    Yang, Runqiang; Chen, Hui; Gu, Zhenxin

    2011-11-01

    Factors (germination time, spectra, temperature, pH, and chemical inhibitors) influencing diamine oxidase (DAO, EC 1.4.3.6) activity and γ-aminobutyric acid (GABA) content of fava bean (Vicia faba L.) during germination were investigated in this study. DAO activity significantly increased in germinating seeds but varied with different organs. The enzyme activity was higher in shoot than that in cotyledon, hypocotyl, and radicle. When seeds were germinated in the dark, DAO activity was 2.35-, 2.00-, 2.36-, 4.40-, and 1.67-fold of that under white, red, blue, green, and yellow spectra, respectively. The optimum germination temperature and pH value for increasing DAO activity were 30 °C and 3.0, respectively. The DAO activity was inhibited significantly by aminoguanidine and sodium ethylenediamine tetracetate, while it was activated by CuCl(2) and CaCl(2). Germinating at an appropriate temperature and pH, 30% of GABA formation was supplied by DAO. Calcium was related to the regulation of DAO activity and GABA accumulation. PMID:21942768

  18. Factors influencing diamine oxidase activity and γ-aminobutyric acid content of fava bean (Vicia faba L.) during germination.

    PubMed

    Yang, Runqiang; Chen, Hui; Gu, Zhenxin

    2011-11-01

    Factors (germination time, spectra, temperature, pH, and chemical inhibitors) influencing diamine oxidase (DAO, EC 1.4.3.6) activity and γ-aminobutyric acid (GABA) content of fava bean (Vicia faba L.) during germination were investigated in this study. DAO activity significantly increased in germinating seeds but varied with different organs. The enzyme activity was higher in shoot than that in cotyledon, hypocotyl, and radicle. When seeds were germinated in the dark, DAO activity was 2.35-, 2.00-, 2.36-, 4.40-, and 1.67-fold of that under white, red, blue, green, and yellow spectra, respectively. The optimum germination temperature and pH value for increasing DAO activity were 30 °C and 3.0, respectively. The DAO activity was inhibited significantly by aminoguanidine and sodium ethylenediamine tetracetate, while it was activated by CuCl(2) and CaCl(2). Germinating at an appropriate temperature and pH, 30% of GABA formation was supplied by DAO. Calcium was related to the regulation of DAO activity and GABA accumulation.

  19. The conformational state of polyphenol oxidase from field bean (Dolichos lablab) upon SDS and acid-pH activation

    PubMed Central

    Kanade, Santosh R.; Paul, Beena; Rao, A. G. Appu; Gowda, Lalitha R.

    2006-01-01

    Field bean (Dolichos lablab) contains a single isoform of PPO (polyphenol oxidase) – a type III copper protein that catalyses the o-hydroxylation of monophenols and oxidation of o-diphenols using molecular oxygen – and is a homotetramer with a molecular mass of 120 kDa. The enzyme is activated manyfold either in the presence of the anionic detergent SDS below its critical micellar concentration or on exposure to acid-pH. The enhancement of kcat upon activation is accompanied by a marked shift in the pH optimum for the oxidation of t-butyl catechol from 4.5 to 6.0, an increased sensitivity to tropolone, altered susceptibility to proteolytic degradation and decreased thermostability. The Stokes radius of the native enzyme is found to increase from 49.1±2 to 75.9±0.6 Å (1 Å=0.1 nm). The activation by SDS and acid-pH results in a localized conformational change that is anchored around the catalytic site of PPO that alters the microenvironment of an essential glutamic residue. Chemical modification of field bean and sweet potato PPO with 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide followed by kinetic analysis leads to the conclusion that both the enzymes possess a core carboxylate essential to activity. This enhanced catalytic efficiency of PPO, considered as an inducible defence oxidative enzyme, is vital to the physiological defence strategy adapted by plants to insect herbivory and pathogen attack. PMID:16393141

  20. CHARACTERISTICS OF POLYPHENOL OXIDASES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyphenol oxidase (PPO, EC 1.14.18.1 or EC 1.10.3.1) catalyzes the oxidation of o-diphenols to o-quinones. Highly reactive o-quinones couple with phenolics and specific amino acids on proteins to form the characteristic browning products in many wounded fruits, vegetables, and leaf tissues of plant...

  1. Hypohalous acid-modified human serum albumin induces neutrophil NADPH oxidase activation, degranulation, and shape change.

    PubMed

    Gorudko, Irina V; Grigorieva, Daria V; Shamova, Ekaterina V; Kostevich, Valeria A; Sokolov, Alexey V; Mikhalchik, Elena V; Cherenkevich, Sergey N; Arnhold, Jürgen; Panasenko, Oleg M

    2014-03-01

    Halogenated lipids, proteins, and lipoproteins formed in reactions with myeloperoxidase (MPO)-derived hypochlorous acid (HOCl) and hypobromous acid (HOBr) can contribute to the regulation of functional activity of cells and serve as mediators of inflammation. Human serum albumin (HSA) is the major plasma protein target of hypohalous acids. This study was performed to assess the potency of HSA modified by HOCl (HSA-Cl) and HOBr (HSA-Br) to elicit selected neutrophil responses. HSA-Cl/Br were found to induce neutrophil degranulation, generation of reactive oxygen intermediates, shape change, and actin cytoskeleton reorganization. Thus HSA-Cl/Br can initially act as a switch and then as a feeder of the "inflammatory loop" under oxidative stress. In HSA-Cl/Br-treated neutrophils, monoclonal antibodies against CD18, the β subunit of β2 integrins, reduced the production of superoxide anion radicals and hydrogen peroxide as well as MPO exocytosis, suggesting that CD18 contributed to neutrophil activation. HSA-Cl/Br-induced neutrophil responses were also inhibited by genistein, a broad-specificity tyrosine kinase inhibitor, and wortmannin, a phosphoinositide 3-kinase (PI3K) inhibitor, supporting the notion that activation of both tyrosine kinase and PI3K may play a role in neutrophil activation by HSA modified in MPO-dependent reactions. These results confirm the hypothesis that halogenated molecules formed in vivo via MPO-dependent reactions can be considered as a new class of biologically active substances potentially able to contribute to activation of myeloid cells in sites of inflammation and serve as inflammatory response modulators. PMID:24384524

  2. Distribution, industrial applications, and enzymatic synthesis of D-amino acids.

    PubMed

    Gao, Xiuzhen; Ma, Qinyuan; Zhu, Hailiang

    2015-04-01

    D-Amino acids exist widely in microbes, plants, animals, and food and can be applied in pharmaceutical, food, and cosmetics. Because of their widespread applications in industry, D-amino acids have recently received more and more attention. Enzymes including D-hydantoinase, N-acyl-D-amino acid amidohydrolase, D-amino acid amidase, D-aminopeptidase, D-peptidase, L-amino acid oxidase, D-amino acid aminotransferase, and D-amino acid dehydrogenase can be used for D-amino acids synthesis by kinetic resolution or asymmetric amination. In this review, the distribution, industrial applications, and enzymatic synthesis methods are summarized. And, among all the current enzymatic methods, D-amino acid dehydrogenase method not only produces D-amino acid by a one-step reaction but also takes environment and atom economics into consideration; therefore, it is deserved to be paid more attention.

  3. Bile acid receptor TGR5, NADPH Oxidase NOX5-S and CREB Mediate Bile Acid-Induced DNA Damage In Barrett’s Esophageal Adenocarcinoma Cells

    PubMed Central

    Li, Dan; Cao, Weibiao

    2016-01-01

    The mechanisms whereby bile acid reflux may accelerate the progression from Barrett’s esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. In this study we found that bile acid taurodeoxycholic acid (TDCA) significantly increased the tail moment (TM) and histone H2AX phosphorylation in FLO-1 EA cells, an increase which was significantly decreased by knockdown of TGR5. Overexpression of TGR5 significantly increased TDCA-induced TM increase and H2AX phosphorylation. In addition, NADPH oxidase inhibitor diphenylene iodonium significantly inhibited the TDCA-induced increase in TM and H2AX phosphorylation. TDCA-induced increase in TM and H2AX phosphorylation was significantly decreased by knockdown of NOX5-S and overexpression of NOX5-S significantly increased TDCA-induced increase in the tail moment and H2AX phosphorylation. Furthermore, TDCA significantly increased cAMP response element binding protein (CREB) phosphorylation in FLO-1 cells. Knockdown of CREB significantly decreased TDCA-induced increase in NOX5-S mRNA and the tail moment. Conversely, overexpression of CREB significantly increased TDCA-induced TM increase. We conclude that TDCA-induced DNA damage may depend on the activation of TGR5, CREB and NOX5-S. It is possible that in Barrett’s patients bile acids may activate NOX5-S and increase reactive oxygen species (ROS) production via activation of TGR5 and CREB. NOX5-S-derived ROS may cause DNA damage, thereby contributing to the progression from BE to EA. PMID:27511066

  4. Bile acid receptor TGR5, NADPH Oxidase NOX5-S and CREB Mediate Bile Acid-Induced DNA Damage In Barrett's Esophageal Adenocarcinoma Cells.

    PubMed

    Li, Dan; Cao, Weibiao

    2016-01-01

    The mechanisms whereby bile acid reflux may accelerate the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. In this study we found that bile acid taurodeoxycholic acid (TDCA) significantly increased the tail moment (TM) and histone H2AX phosphorylation in FLO-1 EA cells, an increase which was significantly decreased by knockdown of TGR5. Overexpression of TGR5 significantly increased TDCA-induced TM increase and H2AX phosphorylation. In addition, NADPH oxidase inhibitor diphenylene iodonium significantly inhibited the TDCA-induced increase in TM and H2AX phosphorylation. TDCA-induced increase in TM and H2AX phosphorylation was significantly decreased by knockdown of NOX5-S and overexpression of NOX5-S significantly increased TDCA-induced increase in the tail moment and H2AX phosphorylation. Furthermore, TDCA significantly increased cAMP response element binding protein (CREB) phosphorylation in FLO-1 cells. Knockdown of CREB significantly decreased TDCA-induced increase in NOX5-S mRNA and the tail moment. Conversely, overexpression of CREB significantly increased TDCA-induced TM increase. We conclude that TDCA-induced DNA damage may depend on the activation of TGR5, CREB and NOX5-S. It is possible that in Barrett's patients bile acids may activate NOX5-S and increase reactive oxygen species (ROS) production via activation of TGR5 and CREB. NOX5-S-derived ROS may cause DNA damage, thereby contributing to the progression from BE to EA. PMID:27511066

  5. Retinoic acid modulation of thyroid dual oxidase activity in rats and its impact on thyroid iodine organification.

    PubMed

    Mühlbauer, Mônica; da Silva, Alba Cenélia Matos; Marassi, Michelle Porto; Lourenço, Alexandre Lopes; Ferreira, Andrea Claudia Freitas; de Carvalho, Denise Pires

    2010-06-01

    The sodium-iodide symporter (NIS) mediates iodide uptake into the thyrocytes, which is important for the diagnosis and therapy of thyroid disorders. Decreased ability to uptake iodide in thyroid carcinomas reduces the efficacy of radioiodine therapy, and retinoic acid (RA) treatment reinduces iodide uptake. The effectiveness of treatment depends not only on iodide uptake but also on the ability of thyrocytes to organify iodine, which is catalyzed by thyroperoxidase (TPO) in the presence of H(2)O(2). Our goal was to determine the influence of RA on thyroid iodide uptake, iodine organification, and TPO and dual oxidase (DuOx) activities. Normal rats were treated with all-trans-RA or 13-cis-RA (100 or 1500 microg/100 g body weight (b.w.), s.c.) for 14 and 28 days. The 2 h thyroid radioiodine content significantly decreased in rats treated with all-trans-RA (100 microg/100 g b.w.) for 14 days. In this group, NIS function and TPO activity were unchanged, whereas DuOx activity was significantly decreased, which might have contributed to the decrease in iodine organification. Both doses of 13-cis-RA for 28 days increased the 15 min thyroid radioiodine uptake, while the 2 h radioiodide uptake increased only in rats treated with the highest dose of 13-cis-RA. While TPO activity did not change, H(2)O(2) generation was increased in this group, and serum thyroxine levels were normal. Since radioiodine half-life in the thyroid gland is important for treatment efficacy, our results highlight the importance of correctly choosing the RA isomer, the time and the dose of treatment, in order to improve the efficacy of radioiodine therapy.

  6. D-amino acid oxidase activity is inhibited by an interaction with bassoon protein at the presynaptic active zone.

    PubMed

    Popiolek, Michael; Ross, John F; Charych, Erik; Chanda, Pranab; Gundelfinger, Eckart D; Moss, Stephen J; Brandon, Nicholas J; Pausch, Mark H

    2011-08-19

    Schizophrenia is a highly heritable neuropsychiatric disorder affecting ∼1% of the world's population. Linkage and association studies have identified multiple candidate schizophrenia susceptibility genes whose functions converge on the glutamatergic neurotransmitter system. One such susceptibility gene encoding D-amino acid oxidase (DAO), an enzyme that metabolizes the NMDA receptor (NMDAR) co-agonist D-serine, has the potential to modulate NMDAR function in the context of schizophrenia. To further investigate its cellular regulation, we sought to identify DAO-interacting proteins that participate in its functional regulation in rat cerebellum, where DAO expression is especially high. Immunoprecipitation with DAO-specific antibodies and subsequent mass spectrometric analysis of co-precipitated proteins yielded 24 putative DAO-interacting proteins. The most robust interactions occurred with known components of the presynaptic active zone, such as bassoon (BSN) and piccolo (PCLO). The interaction of DAO with BSN was confirmed through co-immunoprecipitation assays using DAO- and BSN-specific antibodies. Moreover, DAO and BSN colocalized with one another in cultured cerebellar granule cells and in synaptic junction membrane protein fractions derived from rat cerebellum. The functional consequences of this interaction were studied through enzyme assay experiments, where DAO enzymatic activity was significantly inhibited as a result of its interaction with BSN. Taking these results together, we hypothesize that synaptic D-serine concentrations may be under tight regulation by a BSN-DAO complex. We therefore predict that this mechanism plays a role in the modulation of glutamatergic signaling through NMDARs. It also furthers our understanding of the biology underlying this potential therapeutic entry point for schizophrenia and other psychiatric disorders.

  7. Haplotypes of the D-Amino Acid Oxidase Gene Are Significantly Associated with Schizophrenia and Its Neurocognitive Deficits

    PubMed Central

    Hwu, Hai-Gwo; Fann, Cathy Shen-Jang; Yang, Ueng-Cheng; Yang, Wei-Chih; Hsu, Pei-Chun; Chang, Chien-Ching; Wen, Chun-Chiang; Tsai-Wu, Jyy-Jih; Hwang, Tzung-Jeng; Hsieh, Ming H.; Liu, Chen-Chung; Chien, Yi-Ling; Fang, Chiu-Ping; Faraone, Stephen V.; Tsuang, Ming T.; Chen, Wei J.; Liu, Chih-Min

    2016-01-01

    D-amino acid oxidase (DAO) has been reported to be associated with schizophrenia. This study aimed to search for genetic variants associated with this gene. The genomic regions of all exons, highly conserved regions of introns, and promoters of this gene were sequenced. Potentially meaningful single-nucleotide polymorphisms (SNPs) obtained from direct sequencing were selected for genotyping in 600 controls and 912 patients with schizophrenia and in a replicated sample consisting of 388 patients with schizophrenia. Genetic associations were examined using single-locus and haplotype association analyses. In single-locus analyses, the frequency of the C allele of a novel SNP rs55944529 located at intron 8 was found to be significantly higher in the original large patient sample (p = 0.016). This allele was associated with a higher level of DAO mRNA expression in the Epstein-Barr virus-transformed lymphocytes. The haplotype distribution of a haplotype block composed of rs11114083-rs2070586-rs2070587-rs55944529 across intron 1 and intron 8 was significantly different between the patients and controls and the haplotype frequencies of AAGC were significantly higher in patients, in both the original (corrected p < 0.0001) and replicated samples (corrected p = 0.0003). The CGTC haplotype was specifically associated with the subgroup with deficits in sustained attention and executive function and the AAGC haplotype was associated with the subgroup without such deficits. The DAO gene was a susceptibility gene for schizophrenia and the genomic region between intron 1 and intron 8 may harbor functional genetic variants, which may influence the mRNA expression of DAO and neurocognitive functions in schizophrenia. PMID:26986737

  8. The xanthine oxidase inhibitor Febuxostat reduces tissue uric acid content and inhibits injury-induced inflammation in the liver and lung.

    PubMed

    Kataoka, Hiroshi; Yang, Ke; Rock, Kenneth L

    2015-01-01

    Necrotic cell death in vivo induces a robust neutrophilic inflammatory response and the resulting inflammation can cause further tissue damage and disease. Dying cells induce this inflammation by releasing pro-inflammatory intracellular components, one of which is uric acid. Cells contain high levels of intracellular uric acid, which is produced when purines are oxidized by the enzyme xanthine oxidase. Here we test whether a non-nucleoside xanthine oxidase inhibitor, Febuxostat (FBX), can reduce intracellular uric acid levels and inhibit cell death-induced inflammation in two different murine tissue injury models; acid-induced acute lung injury and acetaminophen liver injury. Infiltration of inflammatory cells induced by acid injection into lungs or peritoneal administration of acetaminophen was evaluated by quantification with flow cytometry and tissue myeloperoxidase activity in the presence or absence of FBX treatment. Uric acid levels in serum and tissue were measured before giving the stimuli and during inflammation. The impact of FBX treatment on the peritoneal inflammation caused by the microbial stimulus, zymosan, was also analyzed to see whether FBX had a broad anti-inflammatory effect. We found that FBX reduced uric acid levels in acid-injured lung tissue and inhibited acute pulmonary inflammation triggered by lung injury. Similarly, FBX reduced uric acid levels in the liver and inhibited inflammation in response to acetaminophen-induced hepatic injury. In contrast, FBX did not reduce inflammation to zymosan, and therefore is not acting as a general anti-inflammatory agent. These results point to the potential of using agents like FBX to treat cell death-induced inflammation.

  9. Role of the Molybdoflavoenzyme Aldehyde Oxidase Homolog 2 in the Biosynthesis of Retinoic Acid: Generation and Characterization of a Knockout Mouse▿ †

    PubMed Central

    Terao, Mineko; Kurosaki, Mami; Barzago, Maria Monica; Fratelli, Maddalena; Bagnati, Renzo; Bastone, Antonio; Giudice, Chiara; Scanziani, Eugenio; Mancuso, Alessandra; Tiveron, Cecilia; Garattini, Enrico

    2009-01-01

    The mouse aldehyde oxidase AOH2 (aldehyde oxidase homolog 2) is a molybdoflavoenzyme. Harderian glands are the richest source of AOH2, although the protein is detectable also in sebaceous glands, epidermis, and other keratinized epithelia. The levels of AOH2 in the Harderian gland and skin are controlled by genetic background, being maximal in CD1 and C57BL/6 and minimal in DBA/2, CBA, and 129/Sv strains. Testosterone is a negative regulator of AOH2 in Harderian glands. Purified AOH2 oxidizes retinaldehyde into retinoic acid, while it is devoid of pyridoxal-oxidizing activity. Aoh2−/− mice, the first aldehyde oxidase knockout animals ever generated, are viable and fertile. The data obtained for this knockout model indicate a significant role of AOH2 in the local synthesis and biodisposition of endogenous retinoids in the Harderian gland and skin. The Harderian gland's transcriptome of knockout mice demonstrates overall downregulation of direct retinoid-dependent genes as well as perturbations in pathways controlling lipid homeostasis and cellular secretion, particularly in sexually immature animals. The skin of knockout mice is characterized by thickening of the epidermis in basal conditions and after UV light exposure. This has correlates in the corresponding transcriptome, which shows enrichment and overall upregulation of genes involved in hypertrophic responses. PMID:18981221

  10. Identification and Structural Analysis of Amino Acid Substitutions that Increase the Stability and Activity of Aspergillus niger Glucose Oxidase

    PubMed Central

    Marín-Navarro, Julia; Roupain, Nicole; Talens-Perales, David; Polaina, Julio

    2015-01-01

    Glucose oxidase is one of the most conspicuous commercial enzymes due to its many different applications in diverse industries such as food, chemical, energy and textile. Among these applications, the most remarkable is the manufacture of glucose biosensors and in particular sensor strips used to measure glucose levels in serum. The generation of ameliorated versions of glucose oxidase is therefore a significant biotechnological objective. We have used a strategy that combined random and rational approaches to isolate uncharacterized mutations of Aspergillus niger glucose oxidase with improved properties. As a result, we have identified two changes that increase significantly the enzyme's thermal stability. One (T554M) generates a sulfur-pi interaction and the other (Q90R/Y509E) introduces a new salt bridge near the interphase of the dimeric protein structure. An additional double substitution (Q124R/L569E) has no significant effect on stability but causes a twofold increase of the enzyme's specific activity. Our results disclose structural motifs of the protein which are critical for its stability. The combination of mutations in the Q90R/Y509E/T554M triple mutant yielded a version of A. niger glucose oxidase with higher stability than those previously described. PMID:26642312

  11. Glutathione and cinnamic acid: natural dietary components used in preventing the process of browning by inhibition of Polyphenol Oxidase in apple juice.

    PubMed

    Gacche, R N; Warangkar, S C; Ghole, V S

    2004-04-01

    Consumer demands for 'freshness' in processed foods has been given increasing attention by food processing industries by searching for minimally processed products. Polyphenol Oxidase (PPO) mediated browning is a major cause of undesirable flavors and nutritional losses in fruit juices. Here the anti-browning efficiency of glutathione (GSH, reduced form) and cinnamic acid (CA) in apple juice is evaluated. It was observed that the rate of the browning reaction could be efficiently delayed using GSH and CA, which act as inhibitors of PPO. Kinetic studies confirm that GSH and CA are non-competitive and competitive inhibitors of PPO respectively.

  12. A fifth member of the tomato 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase gene family harbours a leucine zipper and is anaerobically induced.

    PubMed

    Sell, Simone; Hehl, Reinhard

    2005-02-01

    Using the leucine zipper domain of a small anaerobically induced bZIP transcription factor in a yeast two hybrid screen, anaerobically induced genes were identified. One peptide corresponds to an anaerobically induced IDS4-like protein that maybe involved in G-protein signaling. Surprisingly, another interacting peptide corresponds to a novel anaerobically induced 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase, designated ACO5. ACO5 harbours a leucine zipper and transcription is mainly induced in fruits and to a lesser extend in leaves. The role of ACO5 in the low oxygen response of tomato is discussed. PMID:16040352

  13. 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.

  14. Cr(VI) reduction by gluconolactone and hydrogen peroxide, the reaction products of fungal glucose oxidase: Cooperative interaction with organic acids in the biotransformation of Cr(VI).

    PubMed

    Romo-Rodríguez, Pamela; Acevedo-Aguilar, Francisco Javier; Lopez-Torres, Adolfo; Wrobel, Kazimierz; Wrobel, Katarzyna; Gutiérrez-Corona, J Félix

    2015-09-01

    The Cr(VI) reducing capability of growing cells of the environmental A. tubingensis Ed8 strain is remarkably efficient compared to reference strains A. niger FGSC322 and A. tubingensis NRRL593. Extracellular glucose oxidase (GOX) activity levels were clearly higher in colonies developed in solid medium and in concentrated extracts of the spent medium of liquid cultures of the Ed8 strain in comparison with the reference strains. In addition, concentrated extracts of the spent medium of A. tubingensis Ed8, but not those of the reference strains, exhibited the ability to reduce Cr(VI). In line with this observation, it was found that A. niger purified GOX is capable of mediating the conversion of Cr(VI) to Cr(III) in a reaction dependent on the presence of glucose that is stimulated by organic acids. Furthermore, it was found that a decrease in Cr(VI) may occur in the absence of the GOX enzyme, as long as the reaction products gluconolactone and hydrogen peroxide are present; this conversion of Cr(VI) is stimulated by organic acids in a reaction that generates hydroxyl radicals, which may involve the formation of an intermediate peroxichromate(V) complex. These findings indicated that fungal glucose oxidase acts an indirect chromate reductase through the formation of Cr(VI) reducing molecules, which interact cooperatively with other fungal metabolites in the biotransformation of Cr(VI).

  15. Spatio-Temporal Detection of the Thiomonas Population and the Thiomonas Arsenite Oxidase Involved in Natural Arsenite Attenuation Processes in the Carnoulès Acid Mine Drainage

    PubMed Central

    Hovasse, Agnès; Bruneel, Odile; Casiot, Corinne; Desoeuvre, Angélique; Farasin, Julien; Hery, Marina; Van Dorsselaer, Alain; Carapito, Christine; Arsène-Ploetze, Florence

    2016-01-01

    The acid mine drainage (AMD) impacted creek of the Carnoulès mine (Southern France) is characterized by acid waters with a high heavy metal content. The microbial community inhabiting this AMD was extensively studied using isolation, metagenomic and metaproteomic methods, and the results showed that a natural arsenic (and iron) attenuation process involving the arsenite oxidase activity of several Thiomonas strains occurs at this site. A sensitive quantitative Selected Reaction Monitoring (SRM)-based proteomic approach was developed for detecting and quantifying the two subunits of the arsenite oxidase and RpoA of two different Thiomonas groups. Using this approach combined with FISH and pyrosequencing-based 16S rRNA gene sequence analysis, it was established here for the first time that these Thiomonas strains are ubiquitously present in minor proportions in this AMD and that they express the key enzymes involved in natural remediation processes at various locations and time points. In addition to these findings, this study also confirms that targeted proteomics applied at the community level can be used to detect weakly abundant proteins in situ. PMID:26870729

  16. Organochlorine insecticides induce NADPH oxidase-dependent reactive oxygen species in human monocytic cells via phospholipase A2/arachidonic acid.

    PubMed

    Mangum, Lee C; Borazjani, Abdolsamad; Stokes, John V; Matthews, Anberitha T; Lee, Jung Hwa; Chambers, Janice E; Ross, Matthew K

    2015-04-20

    Bioaccumulative organohalogen chemicals, such as organochlorine (OC) insecticides, have been increasingly associated with disease etiology; however, the mechanistic link between chemical exposure and diseases, such as atherosclerosis, cancer, and diabetes, is complex and poorly defined. Systemic oxidative stress stemming from OC exposure might play a vital role in the development of these pathologies. Monocytes are important surveillance cells of the innate immune system that respond to extracellular signals possessing danger-associated molecular patterns by synthesizing oxyradicals, such as superoxide, for the purpose of combating infectious pathogens. We hypothesized that OC chemicals can be toxic to monocytes because of an inappropriate elevation in superoxide-derived reactive oxygen species (ROS) capable of causing cellular oxidative damage. Reactive oxyradicals are generated in monocytes in large part by NADPH oxidase (Nox). The present study was conducted to examine the ability of two chlorinated cyclodiene compounds, trans-nonachlor and dieldrin, as well as p,p'-DDE, a chlorinated alicyclic metabolite of DDT, to stimulate Nox activity in a human monocytic cell line and to elucidate the mechanisms for this activation. Human THP-1 monocytes treated with either trans-nonachlor or dieldrin (0.1-10 μM in the culture medium) exhibited elevated levels of intracellular ROS, as evidenced by complementary methods, including flow cytometry analysis using the probe DCFH-DA and hydroethidine-based fluorometric and UPLC-MS assays. In addition, the induced reactive oxygen flux caused by trans-nonachlor was also observed in two other cell lines, murine J774 macrophages and human HL-60 cells. The central role of Nox in OC-mediated oxidative stress was demonstrated by the attenuated superoxide production in OC-exposed monocytes treated with the Nox inhibitors diphenyleneiodonium and VAS-2870. Moreover, monocytes challenged with OCs exhibited increased phospho-p47(phox

  17. The Apoplastic Copper AMINE OXIDASE1 Mediates Jasmonic Acid-Induced Protoxylem Differentiation in Arabidopsis Roots1

    PubMed Central

    Ghuge, Sandip A.; Carucci, Andrea; Rodrigues-Pousada, Renato A.; Tisi, Alessandra; Franchi, Stefano; Tavladoraki, Paraskevi; Cona, Alessandra

    2015-01-01

    Polyamines are involved in key developmental processes and stress responses. Copper amine oxidases oxidize the polyamine putrescine (Put), producing an aldehyde, ammonia, and hydrogen peroxide (H2O2). The Arabidopsis (Arabidopsis thaliana) amine oxidase gene At4g14940 (AtAO1) encodes an apoplastic copper amine oxidase expressed at the early stages of vascular tissue differentiation in roots. Here, its role in root development and xylem differentiation was explored by pharmacological and forward/reverse genetic approaches. Analysis of the AtAO1 expression pattern in roots by a promoter::green fluorescent protein-β-glucuronidase fusion revealed strong gene expression in the protoxylem at the transition, elongation, and maturation zones. Methyl jasmonate (MeJA) induced AtAO1 gene expression in vascular tissues, especially at the transition and elongation zones. Early protoxylem differentiation was observed upon MeJA treatment along with Put level decrease and H2O2 accumulation in wild-type roots, whereas Atao1 loss-of-function mutants were unresponsive to the hormone. The H2O2 scavenger N,N1-dimethylthiourea reversed the MeJA-induced early protoxylem differentiation in wild-type seedlings. Likewise, Put, which had no effect on Atao1 mutants, induced early protoxylem differentiation in the wild type, this event being counteracted by N,N1-dimethylthiourea treatment. Consistently, AtAO1-overexpressing plants showed lower Put levels and early protoxylem differentiation concurrent with H2O2 accumulation in the root zone where the first protoxylem cells with fully developed secondary wall thickenings are found. These results show that the H2O2 produced via AtAO1-driven Put oxidation plays a role in MeJA signaling leading to early protoxylem differentiation in root. PMID:25883242

  18. Effect of Soy Sauce on Serum Uric Acid Levels in Hyperuricemic Rats and Identification of Flazin as a Potent Xanthine Oxidase Inhibitor.

    PubMed

    Li, Huipin; Zhao, Mouming; Su, Guowan; Lin, Lianzhu; Wang, Yong

    2016-06-15

    This is the first report on the ability of soy sauce to effectively reduce the serum uric acid levels and xanthine oxidase (XOD) activities of hyperuricemic rats. Soy sauce was partitioned sequentially into ethyl acetate and water fractions. The ethyl acetate fraction with strong XOD inhibition effect was purified further. On the basis of xanthine oxidase inhibitory (XOI) activity-guided purification, nine compounds including 3,4-dihydroxy ethyl cinnamate, diisobutyl terephthalate, harman, daidzein, flazin, catechol, thymine, genistein, and uracil were obtained. It was the first time that 3,4-dihydroxy ethyl cinnamate and diisobutyl terephthalate had been identified from soy sauce. Flazin with hydroxymethyl furan ketone group at C-1 and carboxyl at C-3 exhibited the strongest XOI activity (IC50 = 0.51 ± 0.05 mM). According to fluorescence quenching and molecular docking experiments, flazin could enter into the catalytic center of XOD to interact with Lys1045, Gln1194, and Arg912 mainly by hydrophobic forces and hydrogen bonds. Flazin, catechol, and genistein not only were potent XOD inhibitors but also held certain antioxidant activities. According to ADME (absorption, distribution, metabolism, and excretion) simulation in silico, flazin had good oral bioavailability in vivo. PMID:27181598

  19. In vitro Xanthine Oxidase Inhibitory Studies of Lippia nodiflora and Isolated Flavonoids and Phenylethanoid Glycosides as Potential Uric Acid-lowering Agents.

    PubMed

    Cheng, Lee-Chuen; Murugaiyah, Vikneswaran; Chan, Kit-Lam

    2015-06-01

    Lippia nodiflora has been traditionally used for treatment of knee joint pain. Hitherto, no studies have been reported on the effective use of L. nodiflora against hyperuricemia, gout or other metabolic disorders. In this present study, L. nodiflora was examined for its ability to lower uric acid levels using an in vitro xanthine oxidase inhibitory assay. The whole plant methanolic extract was subjected to bioactivity-guided fractionation to yield 4 fractions (F1-F4). F3 displayed the highest potency and was further purified by column chromatography to afford two phenylethanoid glycosides, arenarioside (1) and verbascoside (2), and three flavonoids, 6-hydroxyluteolin (3), 6-hydroxyluteolin-7-O-glycoside (4), and nodifloretin (5). These compounds inhibited xanthine oxidase activity, with IC50 values between 7.52 ± 0.01 and 130.00 ± 2.25 μM, of which 3 was the most potent. In contrast, allopurinol, serving as a positive control, was 0.22 ± 0.00 μM. Thus, L. nodiflora, and its chemical constituents are worthy of further studies as potential anti-hyperuricemic agents.

  20. Expression of Mitochondrial Cytochrome C Oxidase Chaperone Gene (COX20) Improves Tolerance to Weak Acid and Oxidative Stress during Yeast Fermentation

    PubMed Central

    Kumar, Vinod; Hart, Andrew J.; Keerthiraju, Ethiraju R.; Waldron, Paul R.; Tucker, Gregory A.; Greetham, Darren

    2015-01-01

    Introduction Saccharomyces cerevisiae is the micro-organism of choice for the conversion of fermentable sugars released by the pre-treatment of lignocellulosic material into bioethanol. Pre-treatment of lignocellulosic material releases acetic acid and previous work identified a cytochrome oxidase chaperone gene (COX20) which was significantly up-regulated in yeast cells in the presence of acetic acid. Results A Δcox20 strain was sensitive to the presence of acetic acid compared with the background strain. Overexpressing COX20 using a tetracycline-regulatable expression vector system in a Δcox20 strain, resulted in tolerance to the presence of acetic acid and tolerance could be ablated with addition of tetracycline. Assays also revealed that overexpression improved tolerance to the presence of hydrogen peroxide-induced oxidative stress. Conclusion This is a study which has utilised tetracycline-regulated protein expression in a fermentation system, which was characterised by improved (or enhanced) tolerance to acetic acid and oxidative stress. PMID:26427054

  1. A study of ozone-induced edema in the isolated rat lung in relation to arachidonic acid metabolism, mixed-function oxidases and angiotensin converting enzyme activities.

    PubMed

    Dutta, S; Chatterjee, M; Teknos, T N; Carlson, R W

    1990-01-01

    In order to elucidate the role of arachidonic acid in the pathogenesis of ozone-induced pulmonary edema, isolated rat lungs were exposed to 14C-arachidonic acid in the presence or absence of ozone and the incorporation of radiolabelled arachidonate into pulmonary cell lipids was studied. The perfusates from these studies were also subjected to differential extraction and thin layer chromatography (t.l.c.) to determine synthesis of both cyclo-oxygenase and lipoxygenase products. In the presence of an edemagenic concentration of ozone, isolated lungs incorporated significantly less exogenous arachidonic acid into phosphatidyl choline and phosphatidyl ethanolamine, whereas incorporation into phosphatidyl inositol or serine was not affected. The edemagenic concentration of ozone also increased production of a variety of arachidonic acid metabolites via cyclo-oxygenase and lipoxygenase pathways. In separate studies, a similar ozone exposure did not affect 14CO2 production, resulting from the metabolism of 14C-antipyrine by mixed function oxidases (MFO). Similarly, an edemagenic concentration of ozone did not affect pulmonary angiotensin converting enzyme activity (ACE) as determined by the rate of formation of 14C-hippuric acid from 14C-hippuryl-histidyl-leucine (14C-HHL). Thus, acute ozone exposure is specifically associated with a reduced incorporation of arachidonate into phospholipids and with an increased conversion of arachidonate into bio-active metabolites.

  2. Recombinant expression, molecular characterization and crystal structure of antitumor enzyme, L-lysine α-oxidase from Trichoderma viride.

    PubMed

    Amano, Marie; Mizuguchi, Haruka; Sano, Tadahisa; Kondo, Hiroki; Shinyashiki, Kengo; Inagaki, Junko; Tamura, Takashi; Kawaguchi, Tatsuya; Kusakabe, Hitoshi; Imada, Katsumi; Inagaki, Kenji

    2015-06-01

    L-Lysine α-oxidase (LysOX) from Trichoderma viride is a homodimeric 112 kDa flavoenzyme that catalyzes the oxidative deamination of L-lysine to form α-keto-ε-aminocaproate. LysOX severely inhibited growth of cancer cells but showed relatively low cytotoxicity for normal cells. We have determined the cDNA nucleotide sequence encoding LysOX from T. viride. The full-length cDNA consists of 2,119 bp and encodes a possible signal peptide (Met1-Arg77) and the mature protein (Ala78-Ile617). The LysOX gene have been cloned and heterologously expressed in Streptomyces lividans TK24 with the enzyme activity up to 9.8 U/ml. The enzymatic properties of the purified recombinant LysOX, such as substrate specificity and thermal stability, are same as those of native LysOX. The crystal structure of LysOX at 1.9 Å resolution revealed that the overall structure is similar to that of snake venom L-amino acid oxidase (LAAO), and the residues involved in the interaction with the amino or carboxy group of the substrate are structurally conserved. However, the entrance and the inner surface structures of the funnel to the active site, as well as the residues involved in the substrate side-chain recognition, are distinct from LAAOs. These structural differences well explain the unique substrate specificity of LysOX. PMID:25648943

  3. Selected biochemical properties of polyphenol oxidase in butter lettuce leaves (Lactuca sativa L. var. capitata) elicited with dl-β-amino-n-butyric acid.

    PubMed

    Złotek, Urszula; Gawlik-Dziki, Urszula

    2015-02-01

    The study concentrated on changes in certain biochemical parameters of polyphenol oxidase (PPO) from lettuce leaves caused by dl-β-amino-n-butyric acid (BABA) elicitation. PPO from control plants demonstrated the highest affinity toward catechol, whereas PPO from BABA-elicited lettuce showed the highest affinity to 4-methylcatechol. The optimum temperature for enzymes from control plants was 35°C, whereas from plants elicited with 1mM BABA this was 25°C. PPO from plants elicited with BABA was also more sensitive to the tested inhibitors than PPO from control plants. l-Cysteine was the most effective inhibitor. Native gel stained for PPO activity in control samples showed two isoforms. However, in BABA-treated lettuce three bands visualising PPO activity were observed. The information obtained in this study will be valuable for the development of treatment technology and storage conditions to control undesirable browning reactions in elicited lettuce.

  4. Selected biochemical properties of polyphenol oxidase in butter lettuce leaves (Lactuca sativa L. var. capitata) elicited with dl-β-amino-n-butyric acid.

    PubMed

    Złotek, Urszula; Gawlik-Dziki, Urszula

    2015-02-01

    The study concentrated on changes in certain biochemical parameters of polyphenol oxidase (PPO) from lettuce leaves caused by dl-β-amino-n-butyric acid (BABA) elicitation. PPO from control plants demonstrated the highest affinity toward catechol, whereas PPO from BABA-elicited lettuce showed the highest affinity to 4-methylcatechol. The optimum temperature for enzymes from control plants was 35°C, whereas from plants elicited with 1mM BABA this was 25°C. PPO from plants elicited with BABA was also more sensitive to the tested inhibitors than PPO from control plants. l-Cysteine was the most effective inhibitor. Native gel stained for PPO activity in control samples showed two isoforms. However, in BABA-treated lettuce three bands visualising PPO activity were observed. The information obtained in this study will be valuable for the development of treatment technology and storage conditions to control undesirable browning reactions in elicited lettuce. PMID:25172730

  5. Potent and Selective Inhibitors of Long Chain l-2-Hydroxy Acid Oxidase Reduced Blood Pressure in DOCA Salt-Treated Rats

    PubMed Central

    2011-01-01

    l-2-Hydroxy acid oxidase (Hao2) is a peroxisomal enzyme with predominant expression in the liver and kidney. Hao2 was recently identified as a candidate gene for blood pressure quantitative trait locus in rats. To investigate a pharmacological role of Hao2 in the management of blood pressure, selective Hao2 inhibitors were developed. Optimization of screening hits 1 and 2 led to the discovery of compounds 3 and 4 as potent and selective rat Hao2 inhibitors with pharmacokinetic properties suitable for in vivo studies in rats. Treatment with compound 3 or 4 resulted in a significant reduction or attenuation of blood pressure in an established or developing model of hypertension, deoxycorticosterone acetate-treated rats. This is the first report demonstrating a pharmacological benefit of selective Hao2 inhibitors in a relevant model of hypertension. PMID:24900281

  6. Growth of transplastomic cells expressing D-amino acid oxidase in chloroplasts is tolerant to D-alanine and inhibited by D-valine.

    PubMed

    Gisby, Martin F; Mudd, Elisabeth A; Day, Anil

    2012-12-01

    Dual-conditional positive/negative selection markers are versatile genetic tools for manipulating genomes. Plastid genomes are relatively small and conserved DNA molecules that can be manipulated precisely by homologous recombination. High-yield expression of recombinant products and maternal inheritance of plastid-encoded traits make plastids attractive sites for modification. Here, we describe the cloning and expression of a dao gene encoding D-amino acid oxidase from Schizosaccharomyces pombe in tobacco (Nicotiana tabacum) plastids. The results provide genetic evidence for the uptake of D-amino acids into plastids, which contain a target that is inhibited by D-alanine. Importantly, this nonantibiotic-based selection system allows the use of cheap and widely available D-amino acids, which are relatively nontoxic to animals and microbes, to either select against (D-valine) or for (D-alanine) cells containing transgenic plastids. Positive/negative selection with d-amino acids was effective in vitro and against transplastomic seedlings grown in soil. The dual functionality of dao is highly suited to the polyploid plastid compartment, where it can be used to provide tolerance against potential D-alanine-based herbicides, control the timing of recombination events such as marker excision, influence the segregation of transgenic plastid genomes, identify loci affecting dao function in mutant screens, and develop D-valine-based methods to manage the spread of transgenic plastids tagged with dao.

  7. Synergistic effect of Aspergillus tubingensis CTM 507 glucose oxidase in presence of ascorbic acid and alpha amylase on dough properties, baking quality and shelf life of bread.

    PubMed

    Kriaa, Mouna; Ouhibi, Rabeb; Graba, Héla; Besbes, Souhail; Jardak, Mohamed; Kammoun, Radhouane

    2016-02-01

    The impact of Aspergillus tubingensis glucose oxidase (GOD) in combination with α-amylase and ascorbic acid on dough properties, qualities and shelf life of bread was investigated. Regression models of alveograph and texture parameters of dough and bread were adjusted. Indeed, the mixture of GOD (44 %) and ascorbic acid (56 %) on flour containing basal improver showed its potential as a corrective action to get better functional and rheological properties of dough and bread texture. Furthermore, wheat flour containing basal additives and enriched with GOD (63.8 %), ascorbic acid (32 %) and α- amylase (4.2 %) led to high technological bread making parameters, to decrease the crumb firmness and chewiness and to improve elasticity, adhesion, cohesion and specific volume of bread. In addition to that, the optimized formulation addition significantly reduced water activity and therefore decreased bread susceptibility to microbial spoilage. These findings demonstrated that GOD could partially substitute not only ascorbic acid but also α-amylase. The generated models allowed to predict the behavior of wheat flour containing additives in the range of values tested and to define the additives formula that led to desired rheological and baking qualities of dough. This fact provides new perspectives to compensate flour quality deficiencies at the moment of selecting raw materials and technological parameters reducing the production costs and facilitating gluten free products development. Graphical abstractᅟ.

  8. Synergistic effect of Aspergillus tubingensis CTM 507 glucose oxidase in presence of ascorbic acid and alpha amylase on dough properties, baking quality and shelf life of bread.

    PubMed

    Kriaa, Mouna; Ouhibi, Rabeb; Graba, Héla; Besbes, Souhail; Jardak, Mohamed; Kammoun, Radhouane

    2016-02-01

    The impact of Aspergillus tubingensis glucose oxidase (GOD) in combination with α-amylase and ascorbic acid on dough properties, qualities and shelf life of bread was investigated. Regression models of alveograph and texture parameters of dough and bread were adjusted. Indeed, the mixture of GOD (44 %) and ascorbic acid (56 %) on flour containing basal improver showed its potential as a corrective action to get better functional and rheological properties of dough and bread texture. Furthermore, wheat flour containing basal additives and enriched with GOD (63.8 %), ascorbic acid (32 %) and α- amylase (4.2 %) led to high technological bread making parameters, to decrease the crumb firmness and chewiness and to improve elasticity, adhesion, cohesion and specific volume of bread. In addition to that, the optimized formulation addition significantly reduced water activity and therefore decreased bread susceptibility to microbial spoilage. These findings demonstrated that GOD could partially substitute not only ascorbic acid but also α-amylase. The generated models allowed to predict the behavior of wheat flour containing additives in the range of values tested and to define the additives formula that led to desired rheological and baking qualities of dough. This fact provides new perspectives to compensate flour quality deficiencies at the moment of selecting raw materials and technological parameters reducing the production costs and facilitating gluten free products development. Graphical abstractᅟ. PMID:27162406

  9. [Alternative oxidase in industrial fungi].

    PubMed

    Gu, Shuai; Liu, Qiang; He, Hao; Li, Shuang

    2015-01-01

    Filamentous fungi have been used in industrial fermentation extensively. Based on non-phosphorylating electron transport process, alternative respiration pathway (ARP) acts as an energy overflow, which can balance carbon metabolism and electron transport, allow the continuance of tricarboxylic acid cycle without the formation of ATP, and permit the turnover of carbon skeletons. Alternative respiration pathway also plays an important role in the stress response of fungi and the physiological function of conditioned pathogen. Alternative oxidase (AOX) is the terminal oxidase responsible for the activity of alternative respiration pathway, which exists widely in higher plants, parts of fungi and algae. Owing to the property that alternative oxidase (AOX) is sensitive to salicylhydroxamic acid (SHAM) and insensitive to conventional inhibitors of cytochrome respiration, alternative respiration pathway by AOX is also named as cyanide-resistant respiration (CRR). In recent years, the study of the alternative respiration pathway and alternative oxidase has been a hot topic in the area involving cellular respiration metabolism. In this review we summarized the latest research advances about the functions of alternative respiration pathway and alternative oxidase in industrial fungi.

  10. Construction of a D-amino acid oxidase reactor based on magnetic nanoparticles modified by a reactive polymer and its application in screening enzyme inhibitors.

    PubMed

    Mu, Xiaoyu; Qiao, Juan; Qi, Li; Liu, Ying; Ma, Huimin

    2014-08-13

    Developing facile and high-throughput methods for exploring pharmacological inhibitors of D-amino acid oxidase (DAAO) has triggered increasing interest. In this work, DAAO was immobilized on the magnetic nanoparticles, which were modified by a biocompatible reactive polymer, poly(glycidyl methacrylate) (PGMA) via an atom transfer radical polymerization technique. Interestingly, the enzyme immobilization process was greatly promoted with the assistance of a lithium perchlorate catalyst. Meanwhile, a new amino acid ionic liquid (AAIL) was successfully synthesized and employed as the efficient chiral ligand in a chiral ligand exchange capillary electrophoresis (CLE-CE) system for chiral separation of amino acids (AAs) and quantitation of methionine, which was selected as the substrate of DAAO. Then, the apparent Michaelis-Menten constants in the enzyme system were determined with the proposed CLE-CE method. The prepared DAAO-PGMA-Fe3O4 nanoparticles exhibited excellent reusability and good stability. Moreover, the enzyme reactor was successfully applied in screening DAAO inhibitors. These results demonstrated that the enzyme could be efficiently immobilized on the polymer-grafted magnetic nanoparticles and that the obtained enzyme reactor has great potential in screening enzyme inhibitors, further offering new insight into monitoring the relevant diseases.

  11. A high-performance liquid chromatography assay with a triazole-bonded column for evaluation of d-amino acid oxidase activity.

    PubMed

    Iwasaki, Megumi; Kashiwaguma, Yoshiyuki; Nagashima, Chihiro; Izumi, Mao; Uekusa, Ayano; Iwasa, Sumiko; Onozato, Mayu; Ichiba, Hideaki; Fukushima, Takeshi

    2016-03-01

    Elution profiles of kynurenic acid (KYNA) and 7-chlorokynurenic acid (Cl-KYNA) were examined by high-performance liquid chromatography (HPLC) using a triazole-bonded stationary phase column (Cosmosil® HILIC) under isocratic elution of a mobile phase consisting of CH3 CN-aqueous 10 mm ammonium formate between pH 3.0 and 6.0. The capacity factors of KYNA and Cl-KYNA varied with both the CH3 CN content and the pH of the mobile phase. The elution order of KYNA and Cl-KYNA was reversed between the CH3 CN- and H2 O-rich mobile phases, suggesting that hydrophilic interactions and anion-exchange interactions caused retention of KYNA and Cl-KYNA in the CH3 CN- and H2 O-rich mobile phases, respectively. The present HPLC method using a triazole-bonded column and fluorescence detection (excitation 250 nm, emission 398 nm) was applied to monitor in vitro production of KYNA from d-kynurenine (d-KYN) by d-amino acid oxidase (DAO) using Cl-KYNA as an internal standard. A single KYNA peak was clearly observed after enzymatic reaction of d-KYN with DAO. Production of KYNA from d-KYN was suppressed by the addition of commercial DAO inhibitors. The present HPLC method can be used to evaluate DAO activity and DAO inhibitory effects in candidate drugs for the treatment of schizophrenia.

  12. Essential oil from leaves of Cinnamomum osmophloeum acts as a xanthine oxidase inhibitor and reduces the serum uric acid levels in oxonate-induced mice.

    PubMed

    Wang, S Y; Yang, C W; Liao, J W; Zhen, W W; Chu, F H; Chang, S T

    2008-11-01

    The xanthine oxidase (XOD) inhibitory activity and anti-hyperuricemia effect in mice of Cinnamomum osmophloeum, which is an endemic tree in Taiwan, were evaluated in this study. The results demonstrated that the essential oil of C. osmophloeum leaves presented the strongest XOD inhibition activity (IC(50)=16.3 μg/ml); however, no significant XOD inhibition activities were found in ethanolic and hot water extracts. Furthermore, among the main compounds of essential oil, the cinnamaldehyde exhibited the potent XOD inhibition activity with an IC(50)=8.4 μg/ml. Besides, the reducing serum uric acid levels in oxonate-induced mice by cinnamaldehyde were further investigated. The hyperuricemic mice were oral administrated cinnamaldehyde at a dosage of 150 mg/kg, the uric acid value in serum was reduced from 5.25±0.63 to 2.10±0.04 mg/dl, the levels of serum uric acid in mice was lowered down by 84.48% as compared to the hyperuricemic control group. Based on the results obtained in this study, cinnamaldehyde may be a potential lead compound for developing the pharmaceutic for anti-hyperuricemia agent. PMID:18693097

  13. Inhibition of aconitase by nitric oxide leads to induction of the alternative oxidase and to a shift of metabolism towards biosynthesis of amino acids.

    PubMed

    Gupta, Kapuganti J; Shah, Jay K; Brotman, Yariv; Jahnke, Kathrin; Willmitzer, Lothar; Kaiser, Werner M; Bauwe, Hermann; Igamberdiev, Abir U

    2012-02-01

    Nitric oxide (NO) is a free radical molecule involved in signalling and in hypoxic metabolism. This work used the nitrate reductase double mutant of Arabidopsis thaliana (nia) and studied metabolic profiles, aconitase activity, and alternative oxidase (AOX) capacity and expression under normoxia and hypoxia (1% oxygen) in wild-type and nia plants. The roots of nia plants accumulated very little NO as compared to wild-type plants which exhibited ∼20-fold increase in NO emission under low oxygen conditions. These data suggest that nitrate reductase is involved in NO production either directly or by supplying nitrite to other sites of NO production (e.g. mitochondria). Various studies revealed that NO can induce AOX in mitochondria, but the mechanism has not been established yet. This study demonstrates that the NO produced in roots of wild-type plants inhibits aconitase which in turn leads to a marked increase in citrate levels. The accumulating citrate enhances AOX capacity, expression, and protein abundance. In contrast to wild-type plants, the nia double mutant failed to show AOX induction. The overall induction of AOX in wild-type roots correlated with accumulation of glycine, serine, leucine, lysine, and other amino acids. The findings show that NO inhibits aconitase under hypoxia which results in accumulation of citrate, the latter in turn inducing AOX and causing a shift of metabolism towards amino acid biosynthesis.

  14. Fabrication of a chitosan/glucose oxidase-poly(anilineboronic acid)-Au(nano)/Au-plated Au electrode for biosensor and biofuel cell.

    PubMed

    Huang, Yi; Qin, Xiaoli; Li, Zou; Fu, Yingchun; Qin, Cong; Wu, Feng; Su, Zhaohong; Ma, Ming; Xie, Qingji; Yao, Shouzhuo; Hu, Jiming

    2012-01-15

    Enzyme immobilization is one of the key factors in constructing high-performance enzyme biosensors and biofuel cells (BFCs). Herein, we propose a new protocol for efficient immobilization of a glycoprotein enzyme based on the interaction of the 1, 2- or 1, 3-diols in the glycoprotein with a boronic acid functionalized monomer. Briefly, casting a mixture of glucose oxidase (GOx) and anilineboronic acid (ABA) followed by a NaAuCl(4) solution to an Au-plated Au electrode surface yielded a GOx-poly(ABA) (PABA)-gold nanoparticle (Au(nano)) bionanocomposite, and chitosan (CS) was then cast and air-dried. In the present protocol, the small-sized Au(nano) or Au subnanostructures can form near/on the enzyme molecule, which greatly promotes the electron transfer of enzymatic reaction and enhances the amperometric responses. The thus-prepared CS/GOx-PABA-Au(nano)/Au-plated Au electrode worked well in the first-/second generation biosensing modes and as a bioanode in a monopolar biofuel cell, with analytical or cell-power performance superior to those of most analogues hitherto reported. PMID:22099959

  15. Complementary DNA cloning of the pear 1-aminocyclopropane-1-carboxylic acid oxidase gene and agrobacterium-mediated anti-sense genetic transformation.

    PubMed

    Qi, Jing; Dong, Zhen; Zhang, Yu-Xing

    2015-12-01

    The aim of the present study was to genetically modify plantlets of the Chinese yali pear to reduce their expression of ripening-associated 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) and therefore increase the shelf-life of the fruit. Primers were designed with selectivity for the conserved regions of published ACO gene sequences, and yali complementary DNA (cDNA) cloning was performed by reverse transcription quantitative polymerase chain reaction (PCR). The obtained cDNA fragment contained 831 base pairs, encoding 276 amino acid residues, and shared no less than 94% nucleotide sequence identity with other published ACO genes. The cDNA fragment was inversely inserted into a pBI121 expression vector, between the cauliflower mosaic virus 35S promoter and the nopaline synthase terminator, in order to construct the anti‑sense expression vector of the ACO gene; it was transfected into cultured yali plants using Agrobacterium LBA4404. Four independent transgenic lines of pear plantlets were obtained and validated by PCR analysis. A Southern blot assay revealed that there were three transgenic lines containing a single copy of exogenous gene and one line with double copies. The present study provided germplasm resources for the cultivation of novel storage varieties of pears, therefore providing a reference for further applications of anti‑sense RNA technology in the genetic improvement of pears and other fruit.

  16. Aldehyde oxidase 1 is highly abundant in hepatic steatosis and is downregulated by adiponectin and fenofibric acid in hepatocytes in vitro

    SciTech Connect

    Neumeier, Markus; Weigert, Johanna; Schaeffler, Andreas; Weiss, Thomas S.; Schmidl, Christian; Buettner, Roland; Bollheimer, Cornelius; Aslanidis, Charalampos; Schoelmerich, Juergen; Buechler, Christa . E-mail: christa.buechler@klinik.uni-regensburg.de

    2006-11-24

    Adiponectin protects the liver from steatosis caused by obesity or alcohol and therefore the influence of adiponectin on human hepatocytes was analyzed. GeneChip experiments indicated that recombinant adiponectin downregulates aldehyde oxidase 1 (AOX1) expression and this was confirmed by real-time RT-PCR and immunoblot. AOX1 is a xenobiotic metabolizing protein and produces reactive oxygen species (ROS), that promote cell damage and fibrogenesis. Adiponectin and fenofibric acid activate peroxisome proliferator-activated receptor-{alpha} (PPAR-{alpha}) and both suppress AOX1 protein and this is blocked by the PPAR-{alpha} antagonist RU486. Obesity is associated with low adiponectin, reduced hepatic PPAR-{alpha} activity and fatty liver, and AOX1 was found induced in the liver of rats on a high-fat diet when compared to controls. Free fatty acids and leptin, that are elevated in obesity, failed to upregulate AOX1 in vitro. The current data indicate that adiponectin reduces AOX1 by activating PPAR-{alpha} whereas fatty liver disease is associated with elevated hepatic AOX1. High AOX1 may be associated with higher ROS well described to induce fibrogenesis in liver tissue but may also influence drug metabolism and activity.

  17. Rho Kinase ROCK2 Mediates Acid-Induced NADPH Oxidase NOX5-S Expression in Human Esophageal Adenocarcinoma Cells

    PubMed Central

    Cao, Weibiao

    2016-01-01

    Mechanisms of the progression from Barrett’s esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. We have shown that NOX5-S may be involved in this progression. However, how acid upregulates NOX5-S is not well known. We found that acid-induced increase in NOX5-S expression was significantly decreased by the Rho kinase (ROCK) inhibitor Y27632 in BE mucosal biopsies and FLO-1 EA cells. In addition, acid treatment significantly increased the Rho kinase activity in FLO-1 cells. The acid-induced increase in NOX5-S expression and H2O2 production was significantly decreased by knockdown of Rho kinase ROCK2, but not by knockdown of ROCK1. Conversely, the overexpression of the constitutively active ROCK2, but not the constitutively active ROCK1, significantly enhanced the NOX5-S expression and H2O2 production. Moreover, the acid-induced increase in Rho kinase activity and in NOX5-S mRNA expression was blocked by the removal of calcium in both FLO-1 and OE33 cells. The calcium ionophore A23187 significantly increased the Rho kinase activity and NOX5-S mRNA expression. We conclude that acid-induced increase in NOX5-S expression and H2O2 production may depend on the activation of ROCK2, but not ROCK1, in EA cells. The acid-induced activation of Rho kinase may be mediated by the intracellular calcium increase. It is possible that persistent acid reflux present in BE patients may increase the intracellular calcium, activate ROCK2 and thereby upregulate NOX5-S. High levels of reactive oxygen species derived from NOX5-S may cause DNA damage and thereby contribute to the progression from BE to EA. PMID:26901778

  18. Prokaryotic orthologues of mitochondrial alternative oxidase and plastid terminal oxidase.

    PubMed

    McDonald, Allison E; Amirsadeghi, Sasan; Vanlerberghe, Greg C

    2003-12-01

    The mitochondrial alternative oxidase (AOX) and the plastid terminal oxidase (PTOX) are two similar members of the membrane-bound diiron carboxylate group of proteins. AOX is a ubiquinol oxidase present in all higher plants, as well as some algae, fungi, and protists. It may serve to dampen reactive oxygen species generation by the respiratory electron transport chain. PTOX is a plastoquinol oxidase in plants and some algae. It is required in carotenoid biosynthesis and may represent the elusive oxidase in chlororespiration. Recently, prokaryotic orthologues of both AOX and PTOX proteins have appeared in sequence databases. These include PTOX orthologues present in four different cyanobacteria as well as an AOX orthologue in an alpha-proteobacterium. We used PCR, RT-PCR and northern analyses to confirm the presence and expression of the PTOX gene in Anabaena variabilis PCC 7120. An extensive phylogeny of newly found prokaryotic and eukaryotic AOX and PTOX proteins supports the idea that AOX and PTOX represent two distinct groups of proteins that diverged prior to the endosymbiotic events that gave rise to the eukaryotic organelles. Using multiple sequence alignment, we identified residues conserved in all AOX and PTOX proteins. We also provide a scheme to readily distinguish PTOX from AOX proteins based upon differences in amino acid sequence in motifs around the conserved iron-binding residues. Given the presence of PTOX in cyanobacteria, we suggest that this acronym now stand for plastoquinol terminal oxidase. Our results have implications for the photosynthetic and respiratory metabolism of these prokaryotes, as well as for the origin and evolution of eukaryotic AOX and PTOX proteins.

  19. Role of arginine 285 in the active site of Rhodotorula gracilis D-amino acid oxidase. A site-directed mutagenesis study.

    PubMed

    Molla, G; Porrini, D; Job, V; Motteran, L; Vegezzi, C; Campaner, S; Pilone, M S; Pollegioni, L

    2000-08-11

    Arg(285), one of the very few conserved residues in the active site of d-amino acid oxidases, has been mutated to lysine, glutamine, aspartate, and alanine in the enzyme from the yeast Rhodotorula gracilis (RgDAAO). The mutated proteins are all catalytically competent. Mutations of Arg(285) result in an increase ( approximately 300-fold) of K(m) for the d-amino acid and in a large decrease ( approximately 500-fold) of turnover number. Stopped-flow analysis shows that the decrease in turnover is paralleled by a similar decrease in the rate of flavin reduction (k(2)), the latter still being the rate-limiting step of the reaction. In agreement with data from the protein crystal structure, loss of the guanidinium group of Arg(285) in the mutated DAAOs drastically reduces the binding of several carboxylic acids (e.g. benzoate). These results highlight the importance of this active site residue in the precise substrate orientation, a main factor in this redox reaction. Furthermore, Arg(285) DAAO mutants have spectral properties similar to those of the wild-type enzyme, but show a low degree of stabilization of the flavin semiquinone and a change in the redox properties of the free enzyme. From this, we can unexpectedly conclude that Arg(285) in the free enzyme form is involved in the stabilization of the negative charge on the N(1)-C(2)=O locus of the isoalloxazine ring of the flavin. We also suggest that the residue undergoes a conformational change in order to bind the carboxylate portion of the substrate/ligand in the complexed enzyme. PMID:10821840

  20. Gold electrode modified with a self-assembled glucose oxidase and 2,6-pyridinedicarboxylic acid as novel glucose bioanode for biofuel cells

    NASA Astrophysics Data System (ADS)

    Ammam, Malika; Fransaer, Jan

    2014-07-01

    In this study, we have constructed a gold electrode modified with (3-aminopropyl)trimethoxysilane/2,6-pyridinedicarboxylic acid/glucose oxidase (abbreviated as, Au/ATS/PDA/GOx) by sequential chemical adsorption. Au/ATS/PDA/GOx electrode was characterized by Fourier Transform Infrared Spectroscopy (FT-IR) and Electrochemical Impedance Spectroscopy (EIS). The data from FT-IR illustrated deposition of ATS, PDA and GOx on the surface of gold electrode. The latter has been confirmed by EIS which showed that the electron transfer resistance of the electrode increases after adsorption of each supplementary layer. Linear sweep voltammetry (LSV) in phosphate buffer solution containing 5 mM glucose displayed that compared to Au/ATS/GOx, oxidation of glucose at Au/ATS/PDA/GOx electrode starts 461 mV earlier. This gain in potential is attributed to presence of PDA in the constructed Au/ATS/PDA/GOx electrode, which plays some sort of electron mediator for glucose oxidation. The Au/ATS/PDA/GOx electrode was stabilized by an outer layer of polystyrene sulfonate (PSS) and was connected to a Pt electrode as cathode and the non-compartmentalized cell was studied under air in phosphate buffer solution pH 7.4 containing 10 mM glucose. Under these conditions, the maximum power density reaches 0.25 μW mm-2 (25 μW cm-2) for the deposited GOx layer that has an estimated surface coverage of ∼70% of a monolayer.

  1. Browning prevention by ascorbic acid and 4-hexylresorcinol: different mechanisms of action on polyphenol oxidase in the presence and in the absence of substrates.

    PubMed

    Arias, E; González, J; Peiró, J M; Oria, R; Lopez-Buesa, P

    2007-11-01

    We have investigated the mechanism of action of 4-hexylresorcinol (4-HR) and ascorbic acid (AA) on the polyphenol oxidase (PPO) catalyzed oxidation of phenolic substrates. Incubation of PPO with 4-HR diminishes strongly PPO activity. This effect can be erroneously interpreted, due to the high affinity of 4-HR for PPO, as irreversible inactivation of PPO. However, PPO activity can be recovered by dialysis after incubation with 4-HR. 4-hexylresorcinol is a canonical enzyme inhibitor that binds preferentially to the oxy form of PPO. It is a mixed-type inhibitor, because it influences both apparent V(max) (1.26 compared with 0.4 units in the absence and presence of 4-HR, respectively) and K(m) values (0.28 mM compared with 0.97 mM in the absence and in the presence of 4-HR, respectively) of PPO. AA can prevent browning by 2 different mechanisms: In the absence of PPO substrates it inactivates PPO irreversibly, probably through binding to its active site, preferentially in its oxy form. In the presence of PPO substrates, AA reduces PPO oxidized reaction products, which results in a lag phase when measuring PPO activity by monitoring dark product formation but not when monitoring O(2) consumption. The simultaneous use of both 4-HR and AA on PPO results in additive prevention of browning.

  2. d-amino acid oxidase knockout (Dao(-/-) ) mice show enhanced short-term memory performance and heightened anxiety, but no sleep or circadian rhythm disruption.

    PubMed

    Pritchett, David; Hasan, Sibah; Tam, Shu K E; Engle, Sandra J; Brandon, Nicholas J; Sharp, Trevor; Foster, Russell G; Harrison, Paul J; Bannerman, David M; Peirson, Stuart N

    2015-05-01

    d-amino acid oxidase (DAO, DAAO) is an enzyme that degrades d-serine, the primary endogenous co-agonist of the synaptic N-methyl-d-aspartate receptor. Convergent evidence implicates DAO in the pathophysiology and potential treatment of schizophrenia. To better understand the functional role of DAO, we characterized the behaviour of the first genetically engineered Dao knockout (Dao(-/-) ) mouse. Our primary objective was to assess both spatial and non-spatial short-term memory performance. Relative to wildtype (Dao(+/+) ) littermate controls, Dao(-/-) mice demonstrated enhanced spatial recognition memory performance, improved odour recognition memory performance, and enhanced spontaneous alternation in the T-maze. In addition, Dao(-/-) mice displayed increased anxiety-like behaviour in five tests of approach/avoidance conflict: the open field test, elevated plus maze, successive alleys, light/dark box and novelty-suppressed feeding. Despite evidence of a reciprocal relationship between anxiety and sleep and circadian function in rodents, we found no evidence of sleep or circadian rhythm disruption in Dao(-/-) mice. Overall, our observations are consistent with, and extend, findings in the natural mutant ddY/Dao(-) line. These data add to a growing body of preclinical evidence linking the inhibition, inactivation or deletion of DAO with enhanced cognitive performance. Our results have implications for the development of DAO inhibitors as therapeutic agents.

  3. Overexpression of Arabidopsis thaliana gibberellic acid 20 oxidase (AtGA20ox) gene enhance the vegetative growth and fiber quality in kenaf (Hibiscus cannabinus L.) plants.

    PubMed

    Withanage, Samanthi Priyanka; Hossain, Md Aktar; Kumar M, Sures; Roslan, Hairul Azman B; Abdullah, Mohammad Puad; Napis, Suhaimi B; Shukor, Nor Aini Ab

    2015-06-01

    Kenaf (Hibiscus cannabinus L.; Family: Malvaceae), is multipurpose crop, one of the potential alternatives of natural fiber for biocomposite materials. Longer fiber and higher cellulose contents are required for good quality biocomposite materials. However, average length of kenaf fiber (2.6 mm in bast and 1.28 mm in whole plant) is below the critical length (4 mm) for biocomposite production. Present study describes whether fiber length and cellulose content of kenaf plants could be enhanced by increasing GA biosynthesis in plants by overexpressing Arabidopsis thaliana Gibberellic Acid 20 oxidase (AtGA20ox) gene. AtGA20ox gene with intron was overexpressed in kenaf plants under the control of double CaMV 35S promoter, followed by in planta transformation into V36 and G4 varieties of kenaf. The lines with higher levels of bioactive GA (0.3-1.52 ng g(-1) fresh weight) were further characterized for their morphological and biochemical traits including vegetative and reproductive growth, fiber dimension and chemical composition. Positive impact of increased gibberellins on biochemical composition, fiber dimension and their derivative values were demonstrated in some lines of transgenic kenaf including increased cellulose content (91%), fiber length and quality but it still requires further study to confirm the critical level of this particular bioactive GA in transgenic plants.

  4. 6-Hydroxy-1,2,4-triazine-3,5(2H,4H)-dione Derivatives as Novel d-Amino Acid Oxidase Inhibitors

    PubMed Central

    2015-01-01

    A series of 2-substituted 6-hydroxy-1,2,4-triazine-3,5(2H,4H)-dione derivatives were synthesized as inhibitors of d-amino acid oxidase (DAAO). Many compounds in this series were found to be potent DAAO inhibitors, with IC50 values in the double-digit nanomolar range. The 6-hydroxy-1,2,4-triazine-3,5(2H,4H)-dione pharmacophore appears metabolically resistant to O-glucuronidation unlike other structurally related DAAO inhibitors. Among them, 6-hydroxy-2-(naphthalen-1-ylmethyl)-1,2,4-triazine-3,5(2H,4H)-dione 11h was found to be selective over a number of targets and orally available in mice. Furthermore, oral coadministration of d-serine with 11h enhanced the plasma levels of d-serine in mice compared to the oral administration of d-serine alone, demonstrating its ability to serve as a pharmacoenhancer of d-serine. PMID:26309148

  5. Overexpression of Arabidopsis thaliana gibberellic acid 20 oxidase (AtGA20ox) gene enhance the vegetative growth and fiber quality in kenaf (Hibiscus cannabinus L.) plants

    PubMed Central

    Withanage, Samanthi Priyanka; Hossain, Md Aktar; Kumar M., Sures; Roslan, Hairul Azman B; Abdullah, Mohammad Puad; Napis, Suhaimi B.; Shukor, Nor Aini Ab.

    2015-01-01

    Kenaf (Hibiscus cannabinus L.; Family: Malvaceae), is multipurpose crop, one of the potential alternatives of natural fiber for biocomposite materials. Longer fiber and higher cellulose contents are required for good quality biocomposite materials. However, average length of kenaf fiber (2.6 mm in bast and 1.28 mm in whole plant) is below the critical length (4 mm) for biocomposite production. Present study describes whether fiber length and cellulose content of kenaf plants could be enhanced by increasing GA biosynthesis in plants by overexpressing Arabidopsis thaliana Gibberellic Acid 20 oxidase (AtGA20ox) gene. AtGA20ox gene with intron was overexpressed in kenaf plants under the control of double CaMV 35S promoter, followed by in planta transformation into V36 and G4 varieties of kenaf. The lines with higher levels of bioactive GA (0.3–1.52 ng g−1 fresh weight) were further characterized for their morphological and biochemical traits including vegetative and reproductive growth, fiber dimension and chemical composition. Positive impact of increased gibberellins on biochemical composition, fiber dimension and their derivative values were demonstrated in some lines of transgenic kenaf including increased cellulose content (91%), fiber length and quality but it still requires further study to confirm the critical level of this particular bioactive GA in transgenic plants. PMID:26175614

  6. NADPH Oxidase and Neurodegeneration

    PubMed Central

    Hernandes, Marina S; Britto, Luiz R G

    2012-01-01

    NADPH oxidase (Nox) is a unique, multi-protein, electron transport system that produces large amounts of superoxide via the reduction of molecular oxygen. Nox-derived reactive oxygen species (ROS) are known to be involved in a variety of physiological processes, including host defense and signal transduction. However, over the past decade, the involvement of (Nox)-dependent oxidative stress in the pathophysiology of several neurodegenerative diseases has been increasingly recognized. ROS produced by Nox proteins contribute to neurodegenerative diseases through distinct mechanisms, such as oxidation of DNA, proteins, lipids, amino acids and metals, in addition to activation of redox-sensitive signaling pathways. In this review, we discuss the recent literature on Nox involvement in neurodegeneration, focusing on Parkinson and Alzheimer diseases. PMID:23730256

  7. Indole-3-ethanol Oxidase

    PubMed Central

    Percival, Frank W.; Purves, William K.; Vickery, Larry E.

    1973-01-01

    We report the further characterization of indole-3-ethanol oxidase from cucumber seedlings. The effects of various inhibitors suggest that the enzyme may be a flavoprotein with a metal ion and sulfhydryl groups required for full activity. Indole-3-acetaldehyde, a product of the reaction, inhibits the enzyme. This inhibition is overcome by O2 but not by indole-3-ethanol, indicating that the kinetic mechanism of the enzyme is a ping-pong Bi-Bi. The enzyme undergoes cooperative interactions with indoleethanol, yielding Hill coefficients as high as 2.96. Gibberellins are without effect on the enzyme, but it is inhibited by several acidic indoles possessing growth-promoting activity and by two synthetic auxins, 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid. Increasing concentrations of indoleacetic acid (IAA) brought about a slight reduction in the indoleethanol concentration producing halfmaximal velocity. Increasing levels of indoleethanol decreased the concentration of IAA required for half-maximal inhibition. At low concentrations of indoleethanol, low levels of IAA activated rather than inhibited. The effect of IAA was not overcome at higher levels of indoleethanol. These results may be interpreted as showing that IAA is a noncompetitive inhibitor which binds to that conformation of the enzyme which also binds indoleethanol. The significance of these interactions for the regulation of IAA biosynthesis is discussed. PMID:16658401

  8. An amperometric enzyme electrode and its biofuel cell based on a glucose oxidase-poly(3-anilineboronic acid)-Pd nanoparticles bionanocomposite for glucose biosensing.

    PubMed

    Sun, Lingen; Ma, Yixuan; Zhang, Pei; Chao, Long; Huang, Ting; Xie, Qingji; Chen, Chao; Yao, Shouzhuo

    2015-06-01

    A new amperometric enzyme electrode and its biofuel cell were fabricated based on a glucose oxidase (GOx)-poly(3-anilineboronic acid) (PABA)-Pd nanoparticles (PdNPs) bionanocomposite for biosensing of glucose. Briefly, Pd was electroplated on a multiwalled carbon nanotubes (MWCNTs)-modified Au electrode, and the GOx-PABA-PdNPs bionanocomposite was prepared on the Pd(plate)/MWCNTs/Au electrode through the chemical oxidation of a GOx-3-anilineboronic acid adduct by Na2PdCl4, followed by electrode-modification with an outer-layer chitosan (CS) film. The thus-prepared CS/GOx-PABA-PdNPs/Pd(plate)/MWCNTs/Au electrode exhibited a linear amperometric response to glucose concentration from 2.0 μM to 4.5 mM with a sensitivity of 160 μA/mM/cm(2), sub-μM detection limit, and excellent operation/storage stability in the first-generation biosensing mode, as well as excellent analytical performance in the second-generation biosensing mode. The good recoveries of glucose obtained from spiked urine samples revealed the application potential of our amperometric enzyme electrode. In addition, a glucose/O2 biofuel cell was constructed using this enzyme electrode as the anode and a Pt/MWCNTs/Au electrode as the cathode, and this biofuel cell as a self-powered biosensing device showed a linear voltage response to glucose concentration from 100 μM to 13.5 mM with a sensitivity of 43.5 mV/mM/cm(2) and excellent operation/storage stability. PMID:25863377

  9. Liver microsomal mixed-function oxidases in response to polyunsaturated/saturated and n-6/n-3 fatty acid ratios of dietary lipids in rats.

    PubMed

    Saito, M; Yamaguchi, M

    1994-01-01

    The effects of various polyunsaturated/saturated (P/S; 0.29-2.74) and n-6/n-3 (0.49-11.21) ratios of dietary fatty acids and of phenobarbital (PB) stimulation on the activity of liver microsomal mixed-function oxidase (MFO) system were studied in rats using a combination of palm oil, safflower oil and fish oil concentrate. When the n-6/n-3 ratio was kept constant (4.6-4.9) without induction by PB, the highest P/S ratio (2.74) tended to increase the basal PB-uninduced MFO activities; while the PB-induced MFO activities were elevated as the dietary P/S ratio increased. This definitely indicates the role of both n-6 and n-3 polyunsaturated fatty acids (PUFAs) in the elevation of MFO activities with and without PB stimulation. Whereas the n-6/n-3 ratio was varied while maintaining a constant P/S ratio (1.3), the PB-induced MFO activities were significantly elevated by decreasing the n-6/n-3 ratio although the MFO activities were not affected without PB stimulation. The MFO activities induced by PB were directly related to the n-3 PUFA content and inversely related to the n-6 PUFA content of the diets with n-6/n-3 ratios less than 4.55. These results obtained herein suggest that there are significant differences in the effects of n-6 and n-3 PUFAs on liver microsomal MFO activities, especially in relation to the induction of MFOs by PB.

  10. Searching for cognitive enhancement in the Morris water maze: better and worse performance in D-amino acid oxidase knockout (Dao(-/-)) mice.

    PubMed

    Pritchett, David; Taylor, Amy M; Barkus, Christopher; Engle, Sandra J; Brandon, Nicholas J; Sharp, Trevor; Foster, Russell G; Harrison, Paul J; Peirson, Stuart N; Bannerman, David M

    2016-04-01

    A common strategy when searching for cognitive-enhancing drugs has been to target the N-methyl-d-aspartate receptor (NMDAR), given its putative role in synaptic plasticity and learning. Evidence in favour of this approach has come primarily from studies with rodents using behavioural assays like the Morris water maze. D-amino acid oxidase (DAO) degrades neutral D-amino acids such as D-serine, the primary endogenous co-agonist acting at the glycine site of the synaptic NMDAR. Inhibiting DAO could therefore provide an effective and viable means of enhancing cognition, particularly in disorders like schizophrenia, in which NMDAR hypofunction is implicated. Indirect support for this notion comes from the enhanced hippocampal long-term potentiation and facilitated water maze acquisition of ddY/Dao(-) mice, which lack DAO activity due to a point mutation in the gene. Here, in Dao knockout (Dao(-/-) ) mice, we report both better and worse water maze performance, depending on the radial distance of the hidden platform from the side wall of the pool. Dao(-/-) mice displayed an increased innate preference for swimming in the periphery of the maze (possibly due to heightened anxiety), which facilitated the discovery of a peripherally located platform, but delayed the discovery of a centrally located platform. By contrast, Dao(-/-) mice exhibited normal performance in two alternative assays of long-term spatial memory: the appetitive and aversive Y-maze reference memory tasks. Taken together, these results question the proposed relationship between DAO inactivation and enhanced long-term associative spatial memory. They also have generic implications for how Morris water maze studies are performed and interpreted. PMID:26833794

  11. Searching for cognitive enhancement in the Morris water maze: better and worse performance in D-amino acid oxidase knockout (Dao(-/-)) mice.

    PubMed

    Pritchett, David; Taylor, Amy M; Barkus, Christopher; Engle, Sandra J; Brandon, Nicholas J; Sharp, Trevor; Foster, Russell G; Harrison, Paul J; Peirson, Stuart N; Bannerman, David M

    2016-04-01

    A common strategy when searching for cognitive-enhancing drugs has been to target the N-methyl-d-aspartate receptor (NMDAR), given its putative role in synaptic plasticity and learning. Evidence in favour of this approach has come primarily from studies with rodents using behavioural assays like the Morris water maze. D-amino acid oxidase (DAO) degrades neutral D-amino acids such as D-serine, the primary endogenous co-agonist acting at the glycine site of the synaptic NMDAR. Inhibiting DAO could therefore provide an effective and viable means of enhancing cognition, particularly in disorders like schizophrenia, in which NMDAR hypofunction is implicated. Indirect support for this notion comes from the enhanced hippocampal long-term potentiation and facilitated water maze acquisition of ddY/Dao(-) mice, which lack DAO activity due to a point mutation in the gene. Here, in Dao knockout (Dao(-/-) ) mice, we report both better and worse water maze performance, depending on the radial distance of the hidden platform from the side wall of the pool. Dao(-/-) mice displayed an increased innate preference for swimming in the periphery of the maze (possibly due to heightened anxiety), which facilitated the discovery of a peripherally located platform, but delayed the discovery of a centrally located platform. By contrast, Dao(-/-) mice exhibited normal performance in two alternative assays of long-term spatial memory: the appetitive and aversive Y-maze reference memory tasks. Taken together, these results question the proposed relationship between DAO inactivation and enhanced long-term associative spatial memory. They also have generic implications for how Morris water maze studies are performed and interpreted.

  12. Isomerization of delta 1-piperideine-2-carboxylate to delta 2-piperideine-2-carboxylate on complexation with flavoprotein D-amino acid oxidase.

    PubMed

    Nishina, Y; Sato, K; Shiga, K

    1991-05-01

    The 1,646 cm-1 band in a resonance Raman spectrum obtained with excitation in the charge-transfer band of the complex of oxidized D-amino acid oxidase (DAO) with the oxidation product of D-lysine catalyzed by DAO shifted to 1,617 cm-1 upon 2-13C substitution of lysine. Thus, the band is assigned to a C(2) = C(3) stretching mode of the enamine, delta 2-piperideine-2-carboxylate (En). In the enzyme-free solution, the product is preferentially in the cyclic imine form, delta 1-piperideine-2-carboxylate (Im). Thus, DAO has a higher affinity for the enamine form than for the imine form. The pH effects on the affinity of DAO for the product and on the molar absorption coefficient at 630 nm in the charge-transfer band, suggest that the enzyme-bound product is En in the neutral form at the N atom. As the value of observed rate constant between DAO and the product was constant at high product concentrations, the binding mechanism can be explained as follows; E + Im in equilibrium with EIm in equilibrium with EEN: rapid bimolecular and slow unimolecular processes. The isomerization of the imine form to the enamine form proceeds in the slow process. The low affinity of Im for DAO may be due to a steric repulsion of the hydrogen atoms of Im at C(3) in the active site. The hydrogen atoms of a substrate D-amino acid at C(3), which correspond to the C(3) hydrogens of Im, may act repulsively in the active site and the repulsive energy may induce strain or distortion of the substrate and the enzyme, accelerating the catalytic reaction.

  13. Oral administration of D-alanine in monkeys robustly increases plasma and cerebrospinal fluid levels but experimental D-amino acid oxidase inhibitors had minimal effect.

    PubMed

    Rojas, Camilo; Alt, Jesse; Ator, Nancy A; Wilmoth, Heather; Rais, Rana; Hin, Niyada; DeVivo, Michael; Popiolek, Michael; Tsukamoto, Takashi; Slusher, Barbara S

    2016-09-01

    Hypofunction of the N-methyl-d-aspartate (NMDA) receptor is thought to exacerbate psychosis in patients diagnosed with schizophrenia. Consistent with this hypothesis, D-alanine, a co-agonist at the glycine site of the NMDA receptor, was shown to improve positive and cognitive symptoms when used as add-on therapy for schizophrenia treatment. However, D-alanine had to be administered at high doses (~7 g) to observe clinical effects. One possible reason for the high dose is that D-alanine could be undergoing oxidation by D-amino acid oxidase (DAAO) before it reaches the brain. If this is the case, the dose could be reduced by co-administration of D-alanine with a DAAO inhibitor (DAAOi). Early studies with rodents showed that co-administration of D-alanine with 5-chloro-benzo[d]isoxazol-3-ol (CBIO), a prototype DAAOi, significantly enhanced the levels of extracellular D-alanine in the frontal cortex compared with D-alanine alone. Further, the use of CBIO reduced the dose of D-alanine needed to attenuate prepulse inhibition deficits induced by dizocilpine. The objective of the work reported herein was to confirm the hypothesis that DAAO inhibition can enhance D-alanine exposure in a species closer to humans: non-human primates. We report that while oral D-alanine administration to baboons (10 mg/kg) enhanced D-alanine plasma and CSF levels over 20-fold versus endogenous levels, addition of experimental DAAOi to the regimen exhibited a 2.2-fold enhancement in plasma and no measurable effect on CSF levels. The results provide caution regarding the utility of DAAO inhibition to increase D-amino acid levels as treatment for patients with schizophrenia. PMID:27287825

  14. The effect of high polyphenol oxidase grass silage on metabolism of polyunsaturated fatty acids and nitrogen across the rumen of beef steers.

    PubMed

    Lee, M R F; Theobald, V J; Gordon, N; Leyland, M; Tweed, J K S; Fychan, R; Scollan, N D

    2014-11-01

    Polyphenol oxidase (PPO) activity in red clover (Trifolium pratense) has been reported to reduce both proteolysis and lipolysis, resulting in greater N use efficiency and protection of PUFA across the rumen. Although high levels of PPO have been reported in grasses such as cocksfoot (orchard grass; Dactylis glomerata), no in vivo research has determined whether grass PPO elicits the same response as red clover PPO. To test the hypothesis that silage ensiled from grass with high levels of PPO protects N and PUFA across the rumen, 6 steers with ruminal and duodenal cannulas were offered cocksfoot silage (CO; high-PPO grass), perennial ryegrass silage (PR; Lolium perenne; low-PPO grass), or red clover silage (RC; high-PPO control) at 16 g DM/kg BW daily with the experiment consisting of two 3 × 3 Latin squares with 21-d periods, consisting of 12 d of diet adaptation, 6 d of duodenal marker infusion, 2 d of duodenal sampling, and 1 d of ruminal sampling. All silages were well preserved, with DM of 34.4, 55.3, and 45.4% for CO, PR, and RC. Activity of PPO in silages was low due to deactivation but was greater in CO than either PR or RC (0.15 vs. 0.05 and 0.08 μkatal/g DM). Protein-bound phenol (mg/g DM) as a measure of the degree of oxidation and an indication of PPO protection was greatest for RC (15.9) but comparable for PR (10.1) and CO (12.2). Biohydrogenation of C18 PUFA was significantly lower on RC compared to the 2 grass silages with CO greater than PR. Despite lower levels of total fatty acid intake and subsequent duodenal flow, CO resulted in greater levels of phytanic acid and total branched and odd chain fatty acids in duodenal digesta than RC or PR. Ruminal ammonia concentration was greatest for RC, with no difference between the grasses. Duodenal flow of microbial N and efficiency of microbial protein synthesis were lowest for CO and comparable for RC and PR. The CO (high-grass PPO) did not result in elevated levels of C18 PUFA escaping the rumen or

  15. The effect of high polyphenol oxidase grass silage on metabolism of polyunsaturated fatty acids and nitrogen across the rumen of beef steers.

    PubMed

    Lee, M R F; Theobald, V J; Gordon, N; Leyland, M; Tweed, J K S; Fychan, R; Scollan, N D

    2014-11-01

    Polyphenol oxidase (PPO) activity in red clover (Trifolium pratense) has been reported to reduce both proteolysis and lipolysis, resulting in greater N use efficiency and protection of PUFA across the rumen. Although high levels of PPO have been reported in grasses such as cocksfoot (orchard grass; Dactylis glomerata), no in vivo research has determined whether grass PPO elicits the same response as red clover PPO. To test the hypothesis that silage ensiled from grass with high levels of PPO protects N and PUFA across the rumen, 6 steers with ruminal and duodenal cannulas were offered cocksfoot silage (CO; high-PPO grass), perennial ryegrass silage (PR; Lolium perenne; low-PPO grass), or red clover silage (RC; high-PPO control) at 16 g DM/kg BW daily with the experiment consisting of two 3 × 3 Latin squares with 21-d periods, consisting of 12 d of diet adaptation, 6 d of duodenal marker infusion, 2 d of duodenal sampling, and 1 d of ruminal sampling. All silages were well preserved, with DM of 34.4, 55.3, and 45.4% for CO, PR, and RC. Activity of PPO in silages was low due to deactivation but was greater in CO than either PR or RC (0.15 vs. 0.05 and 0.08 μkatal/g DM). Protein-bound phenol (mg/g DM) as a measure of the degree of oxidation and an indication of PPO protection was greatest for RC (15.9) but comparable for PR (10.1) and CO (12.2). Biohydrogenation of C18 PUFA was significantly lower on RC compared to the 2 grass silages with CO greater than PR. Despite lower levels of total fatty acid intake and subsequent duodenal flow, CO resulted in greater levels of phytanic acid and total branched and odd chain fatty acids in duodenal digesta than RC or PR. Ruminal ammonia concentration was greatest for RC, with no difference between the grasses. Duodenal flow of microbial N and efficiency of microbial protein synthesis were lowest for CO and comparable for RC and PR. The CO (high-grass PPO) did not result in elevated levels of C18 PUFA escaping the rumen or

  16. Regulating levels of the neuromodulator d-serine in human brain: structural insight into pLG72 and d-amino acid oxidase interaction.

    PubMed

    Birolo, Leila; Sacchi, Silvia; Smaldone, Giovanni; Molla, Gianluca; Leo, Gabriella; Caldinelli, Laura; Pirone, Luciano; Eliometri, Patrick; Di Gaetano, Sonia; Orefice, Ida; Pedone, Emilia; Pucci, Piero; Pollegioni, Loredano

    2016-09-01

    The human flavoenzyme d-amino acid oxidase (hDAAO) degrades the NMDA-receptor modulator d-serine in the brain. Although hDAAO has been extensively characterized, little is known about its main modulator pLG72, a small protein encoded by the primate-specific gene G72 that has been associated with schizophrenia susceptibility. pLG72 interacts with neosynthesized hDAAO, promoting its inactivation and degradation. In this work, we used low-resolution techniques to characterize the surface topology of the hDAAO-pLG72 complex. By using limited proteolysis coupled to mass spectrometry, we could map the exposed regions in the two proteins after complex formation and highlighted an increased sensitivity to proteolysis of hDAAO in complex with pLG72. Cross-linking experiments by using bis(sulfosuccinimidyl)suberate identified the single covalent bond between T182 in hDAAO and K62 in pLG72. In order to validate the designed mode of interaction, three pLG72 variants incrementally truncated at the C terminus, in addition to a form lacking the 71 N-terminal residues, were produced. All variants were dimeric, folded, and interacted with hDAAO. The strongest decrease in affinity for hDAAO (as well as for the hydrophobic drug chlorpromazine) was apparent for the N-terminally deleted pLG72(72-153) form, which lacked K62. On the other hand, eliminating the disordered C-terminal tail yielded a more stable pLG72 protein, improved the binding to hDAAO, although giving lower enzyme inhibition. Elucidation of the mode of hDAAO-pLG72 interaction now makes it possible to design novel molecules that, by targeting the protein complex, can be therapeutically advantageous for diseases related to impairment in d-serine metabolism.

  17. An integrated bienzyme glucose oxidase-fructose dehydrogenase-tetrathiafulvalene-3-mercaptopropionic acid-gold electrode for the simultaneous determination of glucose and fructose.

    PubMed

    Campuzano, Susana; Loaiza, Oscar A; Pedrero, María; de Villena, F Javier Manuel; Pingarrón, José M

    2004-06-01

    A bienzyme biosensor for the simultaneous determination of glucose and fructose was developed by coimmobilising glucose oxidase (GOD), fructose dehydrogenase (FDH), and the mediator, tetrathiafulvalene (TTF), by cross-linking with glutaraldehyde atop a 3-mercaptopropionic acid (MPA) self-assembled monolayer (SAM) on a gold disk electrode (AuE). The performance of this bienzyme electrode under batch and flow injection (FI) conditions, as well as an amperometric detection in high-performance liquid chromatography (HPLC), are reported. The order of enzyme immobilisation atop the MPA-SAM affected the biosensor amperometric response in terms of sensitivity, with the immobilisation order GOD, FDH, TTF being selected. Similar analytical characteristics to those obtained with single GOD or FDH SAM-based biosensors for glucose and fructose were achieved with the bienzyme electrode, indicating that no noticeable changes in the biosensor responses to the analytes occurred as a consequence of the coimmobilisation of both enzymes on the same MPA-AuE. The suitability of the bienzyme biosensor for the analysis of real samples under flow injection conditions was tested by determining glucose in two certified serum samples. The simultaneous determination of glucose and fructose in the same sample cannot be performed without a separation step because at the detection potential used (+0.10 V), both sugars show amperometric response. Consequently, HPLC with amperometric detection at the TTF-FDH-GOD-MPA-AuE was accomplished. Glucose and fructose were simultaneously determined in honey, cola softdrink, and commercial apple juice, and the results were compared with those obtained by using other reference methods.

  18. A Penicillium expansum glucose oxidase-encoding gene, GOX2, is essential for gluconic acid production and acidification during colonization of deciduous fruit.

    PubMed

    Barad, Shiri; Horowitz, Sigal Brown; Moscovitz, Oren; Lichter, Amnon; Sherman, Amir; Prusky, Dov

    2012-06-01

    Penicillium expansum, the causal agent of blue mold rot, causes severe postharvest maceration of fruit through secretion of total, d-gluconic acid (GLA). Two P. expansum glucose oxidase (GOX)-encoding genes, GOX1 and GOX2, were analyzed. GOX activity and GLA accumulation were strongly related to GOX2 expression, which increased with pH to a maximum at pH 7.0, whereas GOX1 was expressed at pH 4.0, where no GOX activity or extracellular GLA were detected. This differential expression was also observed at the leading edge of the decaying tissue, where GOX2 expression was dominant. The roles of the GOX genes in pathogenicity were further studied through i) development of P. expansum goxRNAi mutants exhibiting differential downregulation of GOX2, ii) heterologous expression of the P. expansum GOX2 gene in the nondeciduous fruit-pathogen P. chrysogenum, and iii) modulation of GLA production by FeSO(4) chelation. Interestingly, in P. expansum, pH and GLA production elicited opposite effects on germination and biomass accumulation: 26% of spores germinated at pH 7.0 when GOX activity and GLA were highest whereas, in P. chrysogenum at the same pH, when GLA did not accumulate, 72% of spores germinated. Moreover, heterologous expression of P. expansum GOX2 in P. chrysogenum resulted in enhanced GLA production and reduced germination, suggesting negative regulation of spore germination and GLA production. These results demonstrate that pH modulation, mediated by GLA accumulation, is an important factor in generating the initial signal or signals for fungal development leading to host-tissue colonization by P. expansum.

  19. Coencapsulation of oxygen carriers and glucose oxidase in polyelectrolyte complex capsules for the enhancement of D-gluconic acid and delta-gluconolactone production.

    PubMed

    Bucko, Marek; Gemeiner, Peter; Vikartovská, Alica; Mislovicová, Danica; Lacík, Igor; Tkác, Ján

    2010-04-01

    A novel encapsulated oxidative biocatalyst comprising glucose oxidase (GOD) coencapsulated with oxygen carriers within polyelectrolyte complex capsules was developed for the production of D-gluconic acid and delta-gluconolactone. The capsules containing immobilized GOD were produced by polyelectrolyte complexation with sodium alginate (SA) and cellulose sulfate (CS) as polyanions, poly(methylene-co-guanidine) (PMCG) as the polycation, CaCl(2) as the gelling agent and NaCl as the antigelling agent (GOD-SA-CS/PMCG capsules). Poly(dimethylsiloxane) (PDMS) and an emulsion of n-dodecane (DOD) or perfluorodecaline (PFD) with PDMS were used as the oxygen carriers and MnO(2) was used as a hydrogen peroxide decomposition catalyst. Water-soluble PDMS was found to act as both an oxygen carrier and an emulsifier of water-insoluble DOD and PFD. Stable microcapsules could be produced with concentrations of up to 4% (w/w) of PDMS, 10% (w/w) of DOD and PFD, and 25% (w/w) of MnO(2) in the polyanion solution of SA and CS. Roughly a two-fold increase in the GOD activity from 21.0+/-1.1 to 38.4+/-2.0 U*g(-1) and product space-time yields (STY) from 44.3+/-2.0 to 83.4+/-3.4 g*H*day(-1) could be achieved utilizing coencapsulated oxygen carriers compared to GOD encapsulated in the absence of oxygen carriers. This enhanced production does not significantly depend on the selected oxygen carrier under the conditions used in this study.

  20. Effect of endogenous ascorbic acid oxidase activity and stability on vitamin C in carrots (Daucus carota subsp. sativus) during thermal treatment.

    PubMed

    Leong, Sze Ying; Oey, Indrawati

    2012-10-15

    The purpose of this research was to study the effect of endogenous ascorbic acid oxidase (AAO) on vitamin C in carrots (Daucus carota subsp. sativus), namely Nantes, Egmont Gold and baby carrots during thermal treatment. Enzyme-substrate reaction kinetics of AAO were described using Michaelis-Menten equation. The estimated K(m) and V(max) values of AAO ranged from 50.34 to 63.54 μM and 23.70 to 26.82 μmol/min, respectively. Nantes carrots had the lowest AAO activity. On the other hand, Egmont Gold had the highest V(max). AAO activity in all carrot cultivars was stable up to 50 °C and inactivated above 50 °C. Irreversible thermal inactivation of AAO followed first order kinetics (55-70 °C) and the estimated activation energy of the three carrot cultivars situated between 114.33 and 191.45 kJ/mol. Regarding vitamin C stability, thermal treatment at 60-70 °C has resulted in total conversion of l-AA to DHAA due to residual AAO activity; a complete AAO inactivation was found in 80 °C-treated carrots with high vitamin C retention predominantly in l-AA form, up to 90%. On average, the carrots had a total vitamin C content amounting from 368.24 to 379.87 μg/g dry matter and the Nantes carrots had the highest vitamin C content. The effectiveness of rapid inactivation of endogenous AAO via heating (>80 °C, 10 min) prior to matrix disruption gave protection to l-AA towards enzymatic oxidation, thus resulted in a higher vitamin C content and stability in carrots.

  1. Revisitation of the βCl-elimination reaction of D-amino acid oxidase: new interpretation of the reaction that sparked flavoprotein dehydrogenation mechanisms.

    PubMed

    Ghisla, Sandro; Pollegioni, Loredano; Molla, Gianluca

    2011-11-25

    D-amino acid oxidase (DAAO) from pig has been reported to catalyze the β-elimination of Cl(-) from βCl-D-alanine via abstraction of the substrate α-H as H(+) ("carbanion mechanism") (Walsh, C. T., Schonbrunn, A., and Abeles, R. H. (1971) J. Biol. Chem. 246, 6855-6866). In view of the fundamental mechanistic importance of this reaction and of the recent reinterpretation of the DAAO dehydrogenation step as occurring via a hydride mechanism, we reinvestigated the elimination reaction using yeast DAAO. That enzyme catalyzes the same reactions as the pig enzyme but with a much higher efficiency and a substantially different kinetic behavior. The reaction is initiated by a very rapid and fully reversible dehydrogenation step. This leads to an equilibrium (k(on) ≈ k(reverse)) between the complexes of oxidized enzyme-βCl-D-alanine and reduced enzyme-βCl-iminopyruvate. In the presence of O(2) the latter complex can partition between an oxidative half-reaction and elimination of Cl(-), which proceeds at a rate of ≈50 s(-1). This step forms a complex between oxidized enzyme and enamine that is characterized by a charge transfer absorption (which describes its rates of formation and decay). A minimal scheme that lists relevant steps of the reductive and oxidative half-reactions and elimination pathways along with the estimate of the corresponding rate constants is presented. β-Elimination of Cl(-) is proposed to originate at the locus of the enzyme-βCl-iminopyruvate complex. A chemical mechanism that can account for elimination is discussed in detail. PMID:21949129

  2. Binding geometry, stoichiometry, and thermodynamics of cyclomalto-oligosaccharide (cyclodextrin) inclusion complex formation with chlorogenic acid, the major substrate of apple polyphenol oxidase.

    PubMed

    Irwin, P L; Pfeffer, P E; Doner, L W; Sapers, G M; Brewster, J D; Nagahashi, G; Hicks, K B

    1994-03-18

    The inclusion complexes of cyclomaltohexaose (alpha-CD), cyclomaltoheptaose (beta-CD), cyclomaltooctaose (gamma-CD), and polymerized beta-CD (beta-CDn) with chlorogenic acid (CA), the major substrate of apple fruit polyphenol oxidase (PPO), were studied with regard to pH, ionic strength, and temperature in model buffer systems and apple juice. The thermodynamics of CD.CA inclusion complex formation, which were studied in solution using UV spectrophotometry, displayed enthalpy-entropy compensation typical of processes driven by solvation phenomena. We also found that the apparent association constants (K) of the CD.CA equilibrium were relatively insensitive to pH for beta-CD, compared to alpha- and gamma-CDs, but were subject to substantial enhancement at low ionic strengths. The beta-CD.CA inclusion complex was also characterized for binding geometry and stoichiometry at 9.4 T and 25 degrees C in 0.05 M Na phosphate buffer by 1H NMR spectroscopy. A 1:1 stoichiometric ratio for the complex was found using the method of continuous variations. 1H Spin-lattice relaxation and chemical-shift data indicate that the phenolic ring of CA docks within the cavity of beta-CD. The Ks for beta-, alpha-, and gamma-CD determined in apple juice, which contains a mixture of PPO substrates, were found to correlate with PPO activity-related data. Apple juice, treated with beta-CDn, did not brown until CA was added back. These latter findings strongly argue that the mechanism for inhibition of juice browning with cyclodextrins was mainly due to the binding of PPO substrates and not some other means such as enzyme inactivation via sequestration of Cu2+ by CDs. PMID:8194069

  3. Coencapsulation of oxygen carriers and glucose oxidase in polyelectrolyte complex capsules for the enhancement of D-gluconic acid and delta-gluconolactone production.

    PubMed

    Bucko, Marek; Gemeiner, Peter; Vikartovská, Alica; Mislovicová, Danica; Lacík, Igor; Tkác, Ján

    2010-04-01

    A novel encapsulated oxidative biocatalyst comprising glucose oxidase (GOD) coencapsulated with oxygen carriers within polyelectrolyte complex capsules was developed for the production of D-gluconic acid and delta-gluconolactone. The capsules containing immobilized GOD were produced by polyelectrolyte complexation with sodium alginate (SA) and cellulose sulfate (CS) as polyanions, poly(methylene-co-guanidine) (PMCG) as the polycation, CaCl(2) as the gelling agent and NaCl as the antigelling agent (GOD-SA-CS/PMCG capsules). Poly(dimethylsiloxane) (PDMS) and an emulsion of n-dodecane (DOD) or perfluorodecaline (PFD) with PDMS were used as the oxygen carriers and MnO(2) was used as a hydrogen peroxide decomposition catalyst. Water-soluble PDMS was found to act as both an oxygen carrier and an emulsifier of water-insoluble DOD and PFD. Stable microcapsules could be produced with concentrations of up to 4% (w/w) of PDMS, 10% (w/w) of DOD and PFD, and 25% (w/w) of MnO(2) in the polyanion solution of SA and CS. Roughly a two-fold increase in the GOD activity from 21.0+/-1.1 to 38.4+/-2.0 U*g(-1) and product space-time yields (STY) from 44.3+/-2.0 to 83.4+/-3.4 g*H*day(-1) could be achieved utilizing coencapsulated oxygen carriers compared to GOD encapsulated in the absence of oxygen carriers. This enhanced production does not significantly depend on the selected oxygen carrier under the conditions used in this study. PMID:20222845

  4. Effect of pulsed electric field treatment on enzyme kinetics and thermostability of endogenous ascorbic acid oxidase in carrots (Daucus carota cv. Nantes).

    PubMed

    Leong, Sze Ying; Oey, Indrawati

    2014-03-01

    The objective of this research was to study the enzyme kinetics and thermostability of endogenous ascorbic acid oxidase (AAO) in carrot purée (Daucus carota cv. Nantes) after being treated with pulsed electric field (PEF) processing. Various PEF treatments using electric field strength between 0.2 and 1.2kV/cm and pulsed electrical energy between 1 and 520kJ/kg were conducted. The enzyme kinetics and the kinetics of AAO thermal inactivation (55-70°C) were described using Michaelis-Menten model and first order reaction model, respectively. Overall, the estimated Vmax and KM values were situated in the same order of magnitude as the untreated carrot purée after being exposed to pulsed electrical energy between 1 and 400kJ/kg, but slightly changed at pulsed electrical energy above 500kJ/kg. However, AAO presented different thermostability depending on the electric field strength applied. After PEF treatment at the electric field strength between 0.2 and 0.5kV/cm, AAO became thermolabile (i.e. increase in inactivation rate (k value) at reference temperature) but the temperature dependence of k value (Ea value) for AAO inactivation in carrot purée decreased, indicating that the changes in k values were less temperature dependent. It is obvious that PEF treatment affects the temperature stability of endogenous AAO. The changes in enzyme kinetics and thermostability of AAO in carrot purée could be related to the resulting carrot purée composition, alteration in intracellular environment and the effective concentration of AAO released after being subjected to PEF treatment.

  5. Evidence for association and epistasis at the DAOA/G30 and D-amino acid oxidase loci in an Irish schizophrenia sample.

    PubMed

    Corvin, A; McGhee, K A; Murphy, K; Donohoe, G; Nangle, J M; Schwaiger, S; Kenny, N; Clarke, S; Meagher, D; Quinn, J; Scully, P; Baldwin, P; Browne, D; Walsh, C; Waddington, J L; Morris, D W; Gill, M

    2007-10-01

    The D-amino acid oxidase (DAO) signaling pathway has been implicated in schizophrenia pathogenesis. This may be mediated through modulation of NMDA function by DAO, which is in turn activated by DAO activator (DAOA, formerly G72). Chumakov et al. (2002); PNAS 99: 13675-13680, identifying the novel schizophrenia susceptibility gene DAOA/G30 and a number of independent studies have since reported evidence of association between the DAOA and DAO genes and schizophrenia. However, at least two studies have failed to replicate the epistatic interaction between these loci described in the original report and there have been differences in the associated alleles/haplotypes reported at each locus. In this study, we performed association and epistasis analyses of the DAOA/G30 and DAO loci in a sample of 373 cases with DSM-IV schizophrenia/schizoaffective disorder and 812 controls from the Republic of Ireland. Corrected for the number of tests performed, we found evidence for association between markers at both genes and schizophrenia: DAOA/G30 (P = 0.005, OR = 1.34 (1.09, 1.65)) and DAO (P = 0.003, OR = 1.43 (1.12, 1.84). The data suggest that evidence for association at DAO (marker rs2111902) is more consistent than previously realized, particularly in Caucasian schizophrenia populations. We identified evidence for epistatic interaction between the associated SNPs at DAOA and DAO genes in contributing to schizophrenia risk (OR = 9.3 (1.4, 60.5). Based on these data, more systematic investigation of genes involved in DAO signaling is required.

  6. Dissecting the role of climacteric ethylene in kiwifruit (Actinidia chinensis) ripening using a 1-aminocyclopropane-1-carboxylic acid oxidase knockdown line.

    PubMed

    Atkinson, Ross G; Gunaseelan, Kularajathevan; Wang, Mindy Y; Luo, Luke; Wang, Tianchi; Norling, Cara L; Johnston, Sarah L; Maddumage, Ratnasiri; Schröder, Roswitha; Schaffer, Robert J

    2011-07-01

    During climacteric fruit ripening, autocatalytic (Type II) ethylene production initiates a transcriptional cascade that controls the production of many important fruit quality traits including flavour production and softening. The last step in ethylene biosynthesis is the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene by the enzyme ACC oxidase (ACO). Ten independent kiwifruit (Actinidia chinensis) lines were generated targeting suppression of fruit ripening-related ACO genes and the fruit from one of these lines (TK2) did not produce detectable levels of climacteric ethylene. Ripening behaviour in a population of kiwifruit at harvest is asynchronous, so a short burst of exogenous ethylene was used to synchronize ripening in TK2 and control fruit. Following such a treatment, TK2 and control fruit softened to an 'eating-ripe' firmness. Control fruit produced climacteric ethylene and softened beyond eating-ripe by 5 d. In contrast, TK2 fruit maintained an eating-ripe firmness for >25 d and total volatile production was dramatically reduced. Application of continuous exogenous ethylene to the ripening-arrested TK2 fruit re-initiated fruit softening and typical ripe fruit volatiles were detected. A 17 500 gene microarray identified 401 genes that changed after ethylene treatment, including a polygalacturonase and a pectate lyase involved in cell wall breakdown, and a quinone oxidoreductase potentially involved in volatile production. Many of the gene changes were consistent with the softening and flavour changes observed after ethylene treatment. However, a surprisingly large number of genes of unknown function were also observed, which could account for the unique flavour and textural properties of ripe kiwifruit.

  7. Comparative study on functionalized SBA-15 and SBA-16 nanostructured materials used for immobilization of D-amino acid oxidase

    NASA Astrophysics Data System (ADS)

    Hy, Le Gia; Phuong, Dang Tuyet; Yen, Hoang; Hoan, Nguyen Thi Vuong; Linh, Bui Thi Hai; Thang, Hoang vinh; Hoa, Tran Thi Kim; Thang, Dinh Cao; Nguyen, Vu Thi Hanh; Thao, Phan Thi Hong; Giap, Chu Van; Tuan, Vu Anh

    2008-12-01

    SBA-15 and SBA-16 nanostucrured materials were synthesized via hydrothermal treatment and were functionalized with 3-aminopropyltriethoxysilane (APTES), and vinyltriethoxysilane (VTES). The obtained samples were characterized by different techniques such as XRD, BET, IR and TEM. After functionalization, it showed that these nanostrucrured materials were still maintained the hexagonal pore structure of the parent SBA-15 and cubic cage structure of the parent SBA-16. The non-functionalized pure silica SBA-15 and SBA-16 as well as functionalized SBA-15 and SBA-16 materials were used to immobilize DAAO, which is industrially important enzyme for the production of glutaryl 7-amino cephalosporanic acid (GL-7-ACA) from cephalosporin C (CPC). The obtained results revealed that functionalized SBA- 15 and SBA-16 materials exhibited higher enzymatic activity and stability than those of non-functionalized ones. This might be due to the enhancing of surface hydrophobicity upon functionalization. The surface functionalization of the nanostructured silicas with organic groups can enhance the interaction between enzyme and the supports and consequently increasing the operational stability of the immobilized enzyme. The loading of enzyme on SBA-15 materials was higher than that on SBA-16 samples (both functionalized and non-functionalized types). This might be explained by the difference in pore size and type (cylindrical for SBA-15 and bottle-neck for SBA-16) as well as structure shape (hexagonal for SBA-15 and cubic cage for SBA-16) of both mesoporous materials. Additionally, nature of functionalized groups significantly affected the enzymatic activity. Effects on surface binding force, nature of functional groups, pore size of supports were investigated and discussed.

  8. Bioconversion of α-linolenic acid to n-3 LCPUFA and expression of PPAR-alpha, acyl Coenzyme A oxidase 1 and carnitine acyl transferase I are incremented after feeding rats with α-linolenic acid-rich oils.

    PubMed

    González-Mañán, Daniel; Tapia, Gladys; Gormaz, Juan Guillermo; D'Espessailles, Amanda; Espinosa, Alejandra; Masson, Lilia; Varela, Patricia; Valenzuela, Alfonso; Valenzuela, Rodrigo

    2012-07-01

    High dietary intake of n-6 fatty acids in relation to n-3 fatty acids may generate health disorders, such as cardiovascular and other chronic diseases. Fish consumption rich in n-3 fatty acids is low in Latin America, it being necessary to seek other alternatives to provide α-linolenic acid (ALA), precursor of n-3 LCPUFA (EPA and DHA). Two innovative oils were assayed, chia (Salvia hispanica) and rosa mosqueta (Rosa rubiginosa). This study evaluated hepatic bioconversion of ALA to EPA and DHA, expression of PPAR-α, acyl-Coenzyme A oxidase 1 (ACOX1) and carnitine acyltransferase I (CAT-I), and accumulation of EPA and DHA in plasma and adipose tissue in Sprague-Dawley rats. Three experimental groups were fed 21 days: sunflower oil (SFO, control); chia oil (CO); rosa mosqueta oil (RMO). Fatty acid composition of total lipids and phospholipids from plasma, hepatic and adipose tissue was assessed by gas-liquid chromatography and TLC. Expression of PPAR-α (RT-PCR) and ACOX1 and CAT-I (Western blot). CO and RMO increased plasma, hepatic and adipose tissue levels of ALA, EPA and DHA and decreased n-6:n-3 ratio compared to SFO (p < 0.05, One-way ANOVA and Newman-Keuls test). CO increased levels of ALA and EPA compared to RMO (p < 0.05). No significant differences were observed for DHA levels. CO also increased the expression of PPAR-α, ACOX1 and CAT-I. Only CAT-I levels were increased by RO. CO and RMO may be a nutritional alternative to provide ALA for its bioconversion to EPA and DHA, and to increase the expression of PPAR-α, ACOX1 and CAT-I, especially CO-oil.

  9. Structural Insights into Sulfite Oxidase Deficiency

    SciTech Connect

    Karakas,E.; Wilson, H.; Graf, T.; Xiang, S.; Jaramillo-Busquets, S.; Rajagopalan, K.; Kisker, C.

    2005-01-01

    Sulfite oxidase deficiency is a lethal genetic disease that results from defects either in the genes encoding proteins involved in molybdenum cofactor biosynthesis or in the sulfite oxidase gene itself. Several point mutations in the sulfite oxidase gene have been identified from patients suffering from this disease worldwide. Although detailed biochemical analyses have been carried out on these mutations, no structural data could be obtained because of problems in crystallizing recombinant human and rat sulfite oxidases and the failure to clone the chicken sulfite oxidase gene. We synthesized the gene for chicken sulfite oxidase de novo, working backward from the amino acid sequence of the native chicken liver enzyme by PCR amplification of a series of 72 overlapping primers. The recombinant protein displayed the characteristic absorption spectrum of sulfite oxidase and exhibited steady state and rapid kinetic parameters comparable with those of the tissue-derived enzyme. We solved the crystal structures of the wild type and the sulfite oxidase deficiency-causing R138Q (R160Q in humans) variant of recombinant chicken sulfite oxidase in the resting and sulfate-bound forms. Significant alterations in the substrate-binding pocket were detected in the structure of the mutant, and a comparison between the wild type and mutant protein revealed that the active site residue Arg-450 adopts different conformations in the presence and absence of bound sulfate. The size of the binding pocket is thereby considerably reduced, and its position relative to the cofactor is shifted, causing an increase in the distance of the sulfur atom of the bound sulfate to the molybdenum.

  10. Increased xanthine oxidase in the skin of preeclamptic women.

    PubMed

    Bainbridge, Shannon A; Deng, Jau-Shyong; Roberts, James M

    2009-05-01

    Xanthine oxioreductase is the holoenzyme responsible for terminal purine catabolism. Under conditions of metabolic stress or heightened proinflammatory cytokine production, this enzyme is preferentially in its oxidized form, xanthine oxidase, with catalytic action that generates uric acid and the free radical superoxide. As preeclampsia is characterized by heightened inflammation, oxidative stress, and hyperuricemia, it has been proposed that xanthine oxidase plays a pivotal role in this hypertensive disorder of pregnancy. We sought to determine whether xanthine oxidase protein content was higher in maternal tissue of preeclamptic mothers, compared to healthy pregnant controls, using immunohistochemical analysis of skin biopsies. We further compared xanthine oxidase immunoreactivity in skin biopsies from preeclamptic women and patients with several inflammatory conditions. In preeclamptic women, intense xanthine oxidase immunoreactivity was present within the epidermis. By contrast, only very faint xanthine oxidase staining was observed in skin biopsies from healthy pregnant controls. Further, a role for inflammation in the increase of xanthine oxidase was suggested by similar findings of heightened xanthine oxidase immunoreactivity in the skin biopsies from nonpregnant individuals diagnosed with conditions of systemic inflammation. The finding of increased xanthine oxidase in maternal tissue, most likely as the result of heightened maternal inflammation, suggests maternal xanthine oxidase as a source of free radical and uric acid generation in preeclampsia.

  11. Xanthine oxidase inhibitory activity of alkyl gallates.

    PubMed

    Masuoka, Noriyoshi; Nihei, Ken-ichi; Kubo, Isao

    2006-08-01

    A series (C1-C12) of alkyl gallates was examined for their effects on the activity of xanthine oxidase. Octyl (C8), decyl (C10), and dodecyl (C12) gallates competitively inhibited uric acid formation generated by xanthine oxidase, and the inhibition increased upon increasing the alkyl chain length. Interestingly, neither menthyl nor bornyl gallates inhibited uric acid formation. These data indicate that the hydrophobic alkyl portion is associated with the xanthine-binding site in the Mo-binding domain. It is likely that the linear alkyl portion interacts with the hydrophobic domain close to the binding site, and the hydrophobic interaction is crucial to inhibit the xanthine oxidase reaction. On the other hand, all of gallic acid and its esters equally suppress superoxide anion generation catalyzed by xanthine oxidase at low concentration. The suppression is not due to scavenging activity of these gallates but due to reduction of xanthine oxidase by these gallates. The reduced enzyme catalyzes the reaction to generate hydrogen peroxide and uric acid.

  12. Application of hexafluoroacetone as protecting and activating reagent in amino acid and peptide chemistry.

    PubMed

    Burger, K; Rudolph, M; Fehn, S; Worku, A; Golubev, A

    1995-06-01

    Using hexafluoroacetone as protecting and activating reagent, multifunctional amino acids like aspartic acid can be functionalized regioselectively. This strategy offers i.a. a two-step synthesis for aspartame and preparatively simple access to multifunctional natural and unnatural amino acids, like 4-oxo-L-amino acids, 5-diazo-4-oxo-L-amino acids, 4-substituted L-proline derivatives and various heterocyclic L-amino acids. On application of this strategy to amino diacetic acid N-substituted glycines become readily available.

  13. Fractionation of Dipeptidase Activities of Streptococcus lactis and Dipeptidase Specificity of Some Lactic Acid Bacteria

    PubMed Central

    Sørhaug, Terje; Solberg, Peter

    1973-01-01

    Proteins in sonic extracts of Streptococcus lactis were separated by starch-gel electrophoresis at high voltage. Each slab was sliced longitudinally, and half was stained for peptidases in a mixture containing a peptide, L-amino acid oxidase (snake venom), peroxidase, and o-dianisdine; the other half was stained in amido black for protein. In addition to sonic treatment, trypsin also released enzyme from acetone-treated cells. Glycyl-L-phenylalanine, L-phenylalanyl-glycine, L-alanyl-L-phenylalanine, and L-phenylalanyl-L-alanine served as substrates in characterizing the enzymes. Five different fractions of various specificities appeared in the gels. Broad-range substrate specificities were found for sonic extracts of S. lactis, S. cremoris, S. durans, and Lactobacillus acidophilus. Images PMID:4633426

  14. Activation of polyphenol oxidase of chloroplasts.

    PubMed

    Tolbert, N E

    1973-02-01

    Polyphenol oxidase of leaves is located mainly in chloroplasts isolated by differential or sucrose density gradient centrifugation. This activity is part of the lamellar structure that is not lost on repeated washing of the plastids. The oxidase activity was stable during prolonged storage of the particles at 4 C or -18 C. The Km (dihydroxyphenylalanine) for spinach leaf polyphenol oxidase was 7 mm by a spectrophotometric assay and 2 mm by the manometric assay. Polyphenol oxidase activity in the leaf peroxisomal fraction, after isopycnic centrifugation on a linear sucrose gradient, did not coincide with the peroxisomal enzymes but was attributed to proplastids at nearly the same specific density.Plants were grouped by the latency properties for polyphenol oxidase in their isolated chloroplasts. In a group including spinach, Swiss chard, and beet leaves the plastids immediately after preparation from fresh leaves required a small amount of light for maximal rates of oxidation of dihydroxyphenylalanine. Polyphenol oxidase activity in the dark or light increased many fold during aging of these chloroplasts for 1 to 5 days. Soluble polyphenol oxidase of the cytoplasm was not so stimulated. Chloroplasts prepared from stored leaves were also much more active than from fresh leaves. Maximum rates of dihydroxyphenylalanine oxidation were 2 to 6 mmoles x mg(-1) chlorophyll x hr(-1). Equal stimulation of latent polyphenol oxidase in fresh or aged chloroplasts in this group was obtained by either light, an aged trypsin digest, 3-(4-chlorophenyl)-1, 1-dimethylurea, or antimycin A. A variety of other treatments did not activate or had little effect on the oxidase, including various peptides, salts, detergents, and other proteolytic enzymes.Activation of latent polyphenol oxidase in spinach chloroplasts by trypsin amounted to as much as 30-fold. The trypsin activation occurred even after the trypsin had been treated with 10% trichloroacetic acid, 1.0 n HCl or boiled for 30

  15. Xanthine oxidase inhibitors from Brandisia hancei.

    PubMed

    Kong, L D; Wolfender, J L; Cheng, C H; Hostettmann, K; Tan, R X

    1999-12-01

    Xanthine oxidase is a key enzyme associated with the incidence of hyperuricemia-related disorders. Repeated chromatography of the enzyme inhibitory part of the water extract of the twigs and leaves of Brandisia hancei (Scrophulariaceae) gave a flavone luteolin, an iridoid glycoside mussaenoside, two beta-sitosterol glycosides daucosterol and beta-sitosterol gentiobioside, and five phenylethanoids arenarioside, brandioside, acteoside, 2'-O-acetylacteoside and isoacteoside. Luteolin and isoacteoside inhibited the xanthine oxidase (XO, EC 1.2.3.2) with the IC50 values at 7.83 and 45.48 microM, respectively. Isoacteoside was found to be the first phenylethanoid that decreased substantially the formation of uric acid by inhibiting competitively xanthine oxidase (Ki value: 10.08 microM). Furthermore, the study suggested that the caffeoylation of the 6'-hydroxyl group of the phenylethanoids was essential for the enzyme inhibitory action.

  16. Evaluation of health aspects of kojic acid in food.

    PubMed

    Burdock, G A; Soni, M G; Carabin, I G

    2001-02-01

    Kojic acid is a fungal metabolite commonly produced by many species of Aspergillus, Acetobacter, and Penicillium. The Aspergillus flavus group has traditionally been used in the production of a number of foods, including miso (soybean paste), shoyu (soy sauce), and sake. Kojic acid is widely used as a food additive for preventing enzymatic browning, and in cosmetic preparations as a skin-lightening or bleaching agent. Because kojic acid is often produced during the fermentation of historically used dietary staples, it has a long history of consumption. Various types of compounds, such as glucose, sucrose, acetate, ethanol, arabinose, and xylose, have been used as carbon sources for kojic acid production. Different Aspergillus species are known to produce variable amounts of kojic acid. The mechanism of action of kojic acid is well defined and it has been shown to act as a competitive and reversible inhibitor of animal and plant polyphenol oxidases, xanthine oxidase, and D- and some L-amino acid oxidases. The structure of kojic acid indicates a relatively simple route of metabolism much like dietary hexoses. Acute or subchronic toxicity resulting from an oral dose has not been reported, but convulsions may occur if kojic acid is injected. Results of mutagenicity studies are mixed, but in the in vivo mammalian dominant lethal assay, kojic acid was proven negative. Continuous administration of high doses of kojic acid in mice resulted in induction of thyroid adenomas in both sexes. Kojic acid reversibly affects thyroid function primarily by inhibiting iodine uptake, leading to decreases in T3 and T4 and increase in TSH. Increased TSH from pituitary gland in turn stimulates thyroid hyperplasia. Several lines of evidence indicate that the proliferative effects of kojic acid on thyroid are not related to a genotoxic pathway. The risk of functional inhibition of iodine uptake and its metabolism (organification) and thyroid tumor induction by kojic acid in humans appears

  17. Characterization of ascorbate oxidase from Acremonium sp. HI-25.

    PubMed

    Hirose, J; Sakurai, T; Imamura, K; Watanabe, H; Iwamoto, H; Hiromi, K; Itoh, H; Shin, T; Murao, S

    1994-05-01

    The ascorbate oxidase obtained from a microorganism, Acremonium sp. HI-25 (molecular weight, 80 kDa; monomeric protein), was studied with respect to atomic absorption, EPR, absorption spectra, circular dichroism (CD) spectra, and steady-state kinetics. The enzyme was found to be a multicopper protein, containing four copper atoms of three kinds, types 1, 2, and 3 copper, in the ratio of 1:1:2. The EPR parameters of the type 1 and 2 copper atoms in the ascorbate oxidase are very similar to those in the case of the ascorbate oxidase obtained from cucumber, which is a dimeric protein. The apparent Km and kcat values for ascorbic acid of the ascorbate oxidase from Acremonium sp. HI-25 are almost the same as those of the monomeric unit of the ascorbate oxidase from cucumber. PMID:7961590

  18. Regulation of Ascorbate Oxidase Expression in Pumpkin by Auxin and Copper 1

    PubMed Central

    Esaka, Muneharu; Fujisawa, Kouichi; Goto, Miwa; Kisu, Yasutomo

    1992-01-01

    Ascorbate oxidase expression in pumpkin (Cucurbita spp.) tissues was studied. Specific ascorbate oxidase activities in pumpkin leaf and stem tissues were about 2 and 1.5 times that in the fruit tissues, respectively. In seeds, little ascorbate oxidase activity was detected. Northern blot analyses showed an abundant ascorbate oxidase mRNA in leaf and stem tissues. Fruit tissues had lower levels of ascorbate oxidase mRNA than leaf and stem tissues. Ascorbate oxidase mRNA was not detected in seeds. Specific ascorbate oxidase activity gradually increased during early seedling growth of pumpkin seeds. The increase was accompanied by an increase in ascorbate oxidase mRNA. When ascorbate oxidase activity in developing pumpkin fruits was investigated, the activities in immature fruits that are rapidly growing at 0, 2, 4, and 7 d after anthesis were much higher than those in mature fruits at 14 and 30 d after anthesis. The specific activity and mRNA of ascorbate oxidase markedly increased after inoculation of pumpkin fruit tissues into Murashige and Skoog's culture medium in the presence of an auxin such as 2,4-dichlorophenoxyacetic acid (2,4-D) but not in the absence of 2,4-D. In the presence of 10 mg/L of 2,4-D, ascorbate oxidase mRNA was the most abundant. Thus, ascorbate oxidase is induced by 2,4-D. These results indicate that ascorbate oxidase is involved in cell growth. In pumpkin callus, ascorbate oxidase activity could be markedly increased by adding copper. Furthermore, immunological blotting showed that the amount of ascorbate oxidase protein was also increased by adding copper. However, northern blot analyses showed that ascorbate oxidase mRNA was not increased by adding copper. We suggest that copper may control ascorbate oxidase expression at translation or at a site after translation. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:16652952

  19. High resolution crystal structure of rat long chain hydroxy acid oxidase in complex with the inhibitor 4-carboxy-5-[(4-chlorophenyl)sulfanyl]-1, 2, 3-thiadiazole. Implications for inhibitor specificity and drug design

    SciTech Connect

    Chen, Zhi-wei; Vignaud, Caroline; Jaafar, Adil; Lévy, Bernard; Guéritte, Françoise; Guénard, Daniel; Lederer, Florence; Mathews, F. Scott

    2012-05-24

    Long chain hydroxy acid oxidase (LCHAO) is responsible for the formation of methylguanidine, a toxic compound with elevated serum levels in patients with chronic renal failure. Its isozyme glycolate oxidase (GOX), has a role in the formation of oxalate, which can lead to pathological deposits of calcium oxalate, in particular in the disease primary hyperoxaluria. Inhibitors of these two enzymes may have therapeutic value. These enzymes are the only human members of the family of FMN-dependent L-2-hydroxy acid-oxidizing enzymes, with yeast flavocytochrome b{sub 2} (Fcb2) among its well studied members. We screened a chemical library for inhibitors, using in parallel rat LCHAO, human GOX and the Fcb2 flavodehydrogenase domain (FDH). Among the hits was an inhibitor, CCPST, with an IC{sub 50} in the micromolar range for all three enzymes. We report here the crystal structure of a complex between this compound and LCHAO at 1.3 {angstrom} resolution. In comparison with a lower resolution structure of this enzyme, binding of the inhibitor induces a conformational change in part of the TIM barrel loop 4, as well as protonation of the active site histidine. The CCPST interactions are compared with those it forms with human GOX and those formed by two other inhibitors with human GOX and spinach GOX. These compounds differ from CCPST in having the sulfur replaced with a nitrogen in the five-membered ring as well as different hydrophobic substituents. The possible reason for the {approx}100-fold difference in affinity between these two series of inhibitors is discussed. The present results indicate that specificity is an issue in the quest for therapeutic inhibitors of either LCHAO or GOX, but they may give leads for this quest.

  20. The methanol extract of Euonymus laxiflorus, Rubia lanceolata and Gardenia jasminoides inhibits xanthine oxidase and reduce serum uric acid level in rats.

    PubMed

    Liu, Li-Min; Cheng, Shu-Fen; Shieh, Po-Chuen; Lee, Jang-Chang; Chen, Jih-Jung; Ho, Chi-Tang; Kuo, Sheng-Chu; Kuo, Daih-Huang; Huang, Li-Jiau; Way, Tzong-Der

    2014-08-01

    Chinese herbal medicinal plants, Euonymus laxiflorus (EL), Rubia lanceolata (RL) and Gardenia jasminoides (GJ), have been used wildly to treat arthritis and gout in Taiwan for decades. To understand the beneficial effects of these three plants, their xanthine oxidase (XO) inhibitory activity in vitro and hypouricaemic activity in vivo were investigated. Our results suggested that methanol extracts were better than water extracts for inhibition of XO activity and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, except the water extract of GJ, which exhibited the strongest radical scavenging effect. In animal study, the serum urate level was significantly decreased after oral administration of higher dose (0.39g/kg) methanol extract of the mixture of three plants (ERG). In addition, methanol extract of ERG reduced the pain reaction time in the second phase of formalin induced pain. The results provide useful information on the pharmacological activities of these plants for the potential in treating hyperuricemia.

  1. Role of reactive oxygen species produced by NADPH oxidase in gibberellin biosynthesis during barley seed germination.

    PubMed

    Kai, Kyohei; Kasa, Shinsuke; Sakamoto, Masatsugu; Aoki, Nozomi; Watabe, Gaku; Yuasa, Takashi; Iwaya-Inoue, Mari; Ishibashi, Yushi

    2016-05-01

    NADPH oxidase catalyzes the production of the superoxide anion (O2(-)), a reactive oxygen species (ROS), and regulates the germination of barley (Hordeum vulgare L.). Diphenyleneiodonium (DPI) chloride, an NADPH oxidase inhibitor, delayed barley germination, and exogenous H2O2 (an ROS) partially rescued it. Six enzymes, ent-copalyl diphosphate synthase (CPS), ent-kaurene synthase (KS), ent-kaurene oxidase (KO), ent-kaurenoic acid oxidase (KAO), GA20-oxidase (GA20ox) and GA3-oxidase (GA3ox), catalyze the transformation of trans-geranylgeranyl diphosphate to active gibberellin, which promotes germination. Exogenous H2O2 promoted the expressions of HvKAO1 and HvGA3ox1 in barley embryos. These results suggest that ROS produced by NADPH oxidase are involved in gibberellin biosynthesis through the regulation of HvKAO1 and HvGA3ox1.

  2. Role of reactive oxygen species produced by NADPH oxidase in gibberellin biosynthesis during barley seed germination.

    PubMed

    Kai, Kyohei; Kasa, Shinsuke; Sakamoto, Masatsugu; Aoki, Nozomi; Watabe, Gaku; Yuasa, Takashi; Iwaya-Inoue, Mari; Ishibashi, Yushi

    2016-05-01

    NADPH oxidase catalyzes the production of the superoxide anion (O2(-)), a reactive oxygen species (ROS), and regulates the germination of barley (Hordeum vulgare L.). Diphenyleneiodonium (DPI) chloride, an NADPH oxidase inhibitor, delayed barley germination, and exogenous H2O2 (an ROS) partially rescued it. Six enzymes, ent-copalyl diphosphate synthase (CPS), ent-kaurene synthase (KS), ent-kaurene oxidase (KO), ent-kaurenoic acid oxidase (KAO), GA20-oxidase (GA20ox) and GA3-oxidase (GA3ox), catalyze the transformation of trans-geranylgeranyl diphosphate to active gibberellin, which promotes germination. Exogenous H2O2 promoted the expressions of HvKAO1 and HvGA3ox1 in barley embryos. These results suggest that ROS produced by NADPH oxidase are involved in gibberellin biosynthesis through the regulation of HvKAO1 and HvGA3ox1. PMID:27110861

  3. A biochemical approach to study the role of the terminal oxidases in aerobic respiration in Shewanella oneidensis MR-1.

    PubMed

    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.

  4. Identification of DNA-binding proteins that interact with the 5'-flanking region of the human D-amino acid oxidase gene by pull-down assay coupled with two-dimensional gel electrophoresis and mass spectrometry.

    PubMed

    Tran, Diem Hong; Shishido, Yuji; Chung, Seong Pil; Trinh, Huong Thi Thanh; Yorita, Kazuko; Sakai, Takashi; Fukui, Kiyoshi

    2015-12-10

    D-Amino acid oxidase (DAO) is a flavoenzyme that metabolizes D-amino acids and is expected to be a promising therapeutic target of schizophrenia and glioblastoma. The study of DNA-binding proteins has yielded much information in the regulation of transcription and other biological processes. However, proteins interacting with DAO gene have not been elucidated. Our assessment of human DAO promoter activity using luciferase reporter system indicated the 5'-flanking region of this gene (-4289 bp from transcription initiation site) has a regulatory sequence for gene expression, which is regulated by multi-protein complexes interacting with this region. By using pull-down assay coupled with two-dimensional gel electrophoresis and mass spectrometry, we identified six proteins binding to the 5'-flanking region of the human DAO gene (zinc finger C2HC domain-containing protein 1A; histidine-tRNA ligase, cytoplasmic; molybdenum cofactor biosynthesis protein; 60S ribosomal protein L37; calponin-1; calmodulin binding protein and heterogeneous nuclear ribonucleoprotein A2/B1). These preliminary results will contribute to the advance in the understanding of the potential factors associated with the regulatory mechanism of DAO expression.

  5. Oriented and selective enzyme immobilization on functionalized silica carrier using the cationic binding module Z basic2: design of a heterogeneous D-amino acid oxidase catalyst on porous glass.

    PubMed

    Bolivar, Juan M; Nidetzky, Bernd

    2012-06-01

    D-amino acid oxidase from Trigonopsis variabilis (TvDAO) is applied in industry for the synthesis of pharmaceutical intermediates. Because free TvDAO is extremely sensitive to exposure to gas-liquid interfaces, biocatalytic processing is usually performed with enzyme immobilizates that offer enhanced stability under bubble aeration. We herein present an "Immobilization by Design" approach that exploits engineered charge complementarity between enzyme and carrier to optimize key features of the immobilization of TvDAO. A fusion protein between TvDAO and the positively charged module Z(basic2) was generated, and a corresponding oppositely charged carrier was obtained by derivatization of mesoporous glass with 3-(trihydroxysilyl)-1-propane-sulfonic acid. Using 250 mM NaCl for charge screening at pH 7.0, the Z(basic2) fusion of TvDAO was immobilized directly from E. coli cell extract with almost absolute selectivity and full retention of catalytic effectiveness of the isolated enzyme in solution. Attachment of the homodimeric enzyme to the carrier was quasi-permanent in low-salt buffer but fully reversible upon elution with 5 M NaCl. Immobilized TvDAO was not sensitive to bubble aeration and received substantial (≥ tenfold) stabilization of the activity at 45°C as compared to free enzyme, suggesting immobilization via multisubunit oriented interaction of enzyme with the insoluble carrier. The Z(basic2) enzyme immobilizate was demonstrated to serve as re-usable heterogeneous catalyst for D-amino acid oxidation. Z(basic2) -mediated binding on a sulfonic acid group-containing glass carrier constitutes a generally useful strategy of enzyme immobilization that supports transition from case-specific empirical development to rational design.

  6. Selective monoamine oxidase B inhibition by an Aphanizomenon flos-aquae extract and by its constitutive active principles phycocyanin and mycosporine-like amino acids.

    PubMed

    Scoglio, Stefano; Benedetti, Yanina; Benvenuti, Francesca; Battistelli, Serafina; Canestrari, Franco; Benedetti, Serena

    2014-06-15

    Aphanizomenon flos-aquae (AFA) is a fresh water unicellular blue-green alga that has been traditionally used for over 25 years for its health-enhancing properties. Recent studies have shown the ability of a proprietary AFA extract (Klamin(®)) to improve mood, counteract anxiety, and enhance attention and learning. Aim of this study was to test the monoamine oxidase (MAO) inhibition activity of the same AFA extract and of its constituents phycocyanin (AFA-PC) and mycosporine-like aminoacids (AFA-MAAs). All compounds showed a dose-dependent selective inhibition of MAO-B activity as compared to MAO-A. The IC50 values of the AFA extract (concentration 10 mg/ml), AFA-PC and AFA-MAAs were 6.4 μl/ml, 1.33 μM and 1.98 μM, respectively, evidencing a mixed-type of inhibition for the AFA extract (Ki 0.99 μl/ml), a non-competitive inhibition for AFA-PC (Ki 1.06 μM) and a competitive inhibition for AFA-MAAs (Ki 0.585 μM). These results are important to explain the neuromodulating properties of the AFA extract Klamin(®), which is rich in phenylethylamine, a general neuromodulator, that would nevertheless rapidly destroyed by MAO-B enzymes without the inhibitory activity of the synergic active principles AFA-PC and AFA-MAAs. The present investigation thus proposes the extract as potentially relevant in clinical areas such as mood disorders and neurodegenerative diseases.

  7. NADPH Oxidase NOX5-S and Nuclear Factor κB1 Mediate Acid-Induced Microsomal Prostaglandin E Synthase-1 Expression in Barrett’s Esophageal Adenocarcinoma Cells

    PubMed Central

    Zhou, Xiaoxu; Li, Dan; Resnick, Murray B.; Wands, Jack

    2013-01-01

    The mechanisms of progression from Barrett’s esophagus (BE) to esophageal adenocarcinoma (EA) are not known. Cycloxygenase-2 (COX-2)-derived prostaglandin E2 (PGE2) has been shown to be important in esophageal tumorigenesis. We have shown that COX-2 mediates acid-induced PGE2 production. The prostaglandin E synthase (PGES) responsible for acid-induced PGE2 production in BE, however, is not known. We found that microsomal PGES1 (mPGES1), mPGES2, and cytosolic PGES (cPGES) were present in FLO EA cells. Pulsed acid treatment significantly increased mPGES1 mRNA and protein levels but had little or no effect on mPGES2 or cPGES mRNA. Knockdown of mPGES1 by mPGES1 small interfering RNA (siRNA) blocked acid-induced increase in PGE2 production and thymidine incorporation. Knockdown of NADPH oxidase, NOX5-S, a variant lacking calcium-binding domains, by NOX5 siRNA significantly inhibited acid-induced increase in mPGES1 expression, thymidine incorporation, and PGE2 production. Overexpression of NOX5-S significantly increased the luciferase activity in FLO cells transfected with a nuclear factor κB (NF-κB) in vivo activation reporter plasmid pNF-κB-Luc. Knockdown of NF-κB1 p50 by p50 siRNA significantly decreased acid-induced increase in mPGES1 expression, thymidine incorporation, and PGE2 production. Two novel NF-κB binding elements, GGAGTCTCCC and CGGGACACCC, were identified in the mPGES1 gene promoter. We conclude that mPGES1 mediates acid-induced increase in PGE2 production and cell proliferation. Acid-induced mPGES1 expression depends on activation of NOX5-S and NF-κB1 p50. Microsomal PGES1 may be a potential target to prevent or treat EA. PMID:23439561

  8. Azide inhibition of urate oxidase

    PubMed Central

    Gabison, Laure; Colloc’h, Nathalie; Prangé, Thierry

    2014-01-01

    The inhibition of urate oxidase (UOX) by azide was investigated by X-ray diffraction techniques and compared with cyanide inhibition. Two well characterized sites for reagents are present in the enzyme: the dioxygen site and the substrate-binding site. To examine the selectivity of these sites towards azide inhibition, several crystallization conditions were developed. UOX was co-crystallized with azide (N3) in the presence or absence of either uric acid (UA, the natural substrate) or 8-azaxanthine (8AZA, a competitive inhibitor). In a second set of experiments, previously grown orthorhombic crystals of the UOX–UA or UOX–8AZA complexes were soaked in sodium azide solutions. In a third set of experiments, orthorhombic crystals of UOX with the exchangeable ligand 8-nitroxanthine (8NXN) were soaked in a solution containing uric acid and azide simultaneously (competitive soaking). In all assays, the soaking periods were either short (a few hours) or long (one or two months). These different experimental conditions showed that one or other of the sites, or the two sites together, could be inhibited. This also demonstrated that azide not only competes with dioxygen as cyanide does but also competes with the substrate for its enzymatic site. A model in agreement with experimental data would be an azide in equilibrium between two sites, kinetically in favour of the dioxygen site and thermodynamically in favour of the substrate-binding site. PMID:25005084

  9. Enzymatic preparation of. cap alpha. - and. beta. -deuterated or tritiated amino acids with l-methionine. gamma. -lyase

    SciTech Connect

    Esaki, N.; Sawada, S.; Tanaka, H.; Soda, K.

    1982-01-15

    L-Methionine ..gamma..-lyase catalyzes the exchange of ..cap alpha..- and ..beta..-hydrogens of L-methionine and S-methyl-L-cysteine with deuterium or tritium of solvents. The rate of ..cap alpha..-hydrogen exchange with deuterium was about 40 times faster than that of the elimination reactions. The deuterium and tritium were exchanged also with the ..cap alpha..- and ..beta..-hydrogens of the straight-chain amino acids which do not undergo the elimination: L-alanine, L-..cap alpha..-aminobutyrate, L-norvaline, and L-norleucine. No exchange occurs for the D-isomers, acidic L-amino acids, basic L-amino acids, and branched-chain L-amino acids, although ..cap alpha..-hydrogen of glycine, L-trypotophan, and L-phenylalanine is exchanged slowly. These enzymatic hydrogen-exchange reactions facilitate specific labeling of the L-amino acids with deuterium and tritium.

  10. Crystallization of Mitochondrial Cytochrome Oxidase

    NASA Astrophysics Data System (ADS)

    Ozawa, Takayuki; Tanaka, Masashi; Wakabayashi, Takashi

    1982-12-01

    Cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) was purified from beef heart mitochondria. By washing the oxidase with detergent on a hydrophobic interaction column, phospholipids were depleted to the level of 1 mol of cardiolipin per mol of heme a. Hydrophobic impurities and partially denatured oxidase were separated from the intact oxidase on an affinity column with cytochrome c as the specific ligand. The final preparation of the oxidase contained seven distinct polypeptides. The molecular weight of the oxidase was estimated to be 130,000 from its specific heme a and copper content and from the subunit composition. Crystals of the oxidase were obtained by slow removal of the detergent from the buffer in which the oxidase was dissolved. The needle-shaped crystals were 100 μ m in average length and 5 μ m in width, and they strongly polarized visible light. Electron diffraction patterns were obtained with an unstained glutaraldehyde-fixed single crystal by electron microscopy using 1,000-kV electrons. From electron micrographs and the diffraction patterns of the crystal, it was concluded that the crystal is monoclinic in the space group P21, with unit cell dimensions a = 92 angstrom, b = 84 angstrom, and c = 103 angstrom, and α =β 90 degrees, γ = 126 degrees.

  11. Spectroscopic and genetic evidence for two heme-Cu-containing oxidases in Rhodobacter sphaeroides.

    PubMed Central

    Shapleigh, J P; Hill, J J; Alben, J O; Gennis, R B

    1992-01-01

    It has recently become evident that many bacterial respiratory oxidases are members of a superfamily that is related to the eukaryotic cytochrome c oxidase. These oxidases catalyze the reduction of oxygen to water at a heme-copper binuclear center. Fourier transform infrared (FTIR) spectroscopy has been used to examine the heme-copper-containing respiratory oxidases of Rhodobacter sphaeroides Ga. This technique monitors the stretching frequency of CO bound at the oxygen binding site and can be used to characterize the oxidases in situ with membrane preparations. Oxidases that have a heme-copper binuclear center are recognizable by FTIR spectroscopy because the bound CO moves from the heme iron to the nearby copper upon photolysis at low temperature, where it exhibits a diagnostic spectrum. The FTIR spectra indicate that the binuclear center of the R. sphaeroides aa3-type cytochrome c oxidase is remarkably similar to that of the bovine mitochondrial oxidase. Upon deletion of the ctaD gene, encoding subunit I of the aa3-type oxidase, substantial cytochrome c oxidase remains in the membranes of aerobically grown R. sphaeroides. This correlates with a second wild-type R. sphaeroides is grown photosynthetically, the chromatophore membranes lack the aa3-type oxidase but have this second heme-copper oxidase. Subunit I of the heme-copper oxidase superfamily contains the binuclear center. Amino acid sequence alignments show that this subunit is structurally very highly conserved among both eukaryotic and prokaryotic species. The polymerase chain reaction was used to show that the chromosome of R. sphaeroides contains at least one other gene that is a homolog of ctaD, the gene encoding subunit I of the aa3-type cytochrome c oxidase.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:1313003

  12. The Escherichia coli CydX protein is a member of the CydAB cytochrome bd oxidase complex and is required for cytochrome bd oxidase activity.

    PubMed

    VanOrsdel, Caitlin E; Bhatt, Shantanu; Allen, Rondine J; Brenner, Evan P; Hobson, Jessica J; Jamil, Aqsa; Haynes, Brittany M; Genson, Allyson M; Hemm, Matthew R

    2013-08-01

    Cytochrome bd oxidase operons from more than 50 species of bacteria contain a short gene encoding a small protein that ranges from ∼30 to 50 amino acids and is predicted to localize to the cell membrane. Although cytochrome bd oxidases have been studied for more than 70 years, little is known about the role of this small protein, denoted CydX, in oxidase activity. Here we report that Escherichia coli mutants lacking CydX exhibit phenotypes associated with reduced oxidase activity. In addition, cell membrane extracts from ΔcydX mutant strains have reduced oxidase activity in vitro. Consistent with data showing that CydX is required for cytochrome bd oxidase activity, copurification experiments indicate that CydX interacts with the CydAB cytochrome bd oxidase complex. Together, these data support the hypothesis that CydX is a subunit of the CydAB cytochrome bd oxidase complex that is required for complex activity. The results of mutation analysis of CydX suggest that few individual amino acids in the small protein are essential for function, at least in the context of protein overexpression. In addition, the results of analysis of the paralogous small transmembrane protein AppX show that the two proteins could have some overlapping functionality in the cell and that both have the potential to interact with the CydAB complex.

  13. NikD, AN UNUSUAL AMINO ACID OXIDASE ESSENTIAL FOR NIKKOMYCIN BIOSYNTHESIS: STRUCTURES OF CLOSED AND OPEN FORMS AT 1.15 AND 1.90 Å RESOLUTION

    PubMed Central

    Carrell, Christopher J.; Bruckner, Robert C.; Venci, David; Zhao, Gouhua; Jorns, Marilyn Schuman; Mathews, F. Scott

    2009-01-01

    Summary NikD is an unusual amino acid oxidizing enzyme that contains covalently bound FAD, catalyzes a 4-electron oxidation of piperideine-2-carboxylic acid to picolinate and plays a critical role in the biosynthesis of nikkomycin antibiotics. Crystal structures of closed and open forms of nikD, a two-domain enzyme, have been determined to resolutions of 1.15 and 1.9 Å, respectively. The two forms differ by an 11° rotation of the catalytic domain with respect to the FAD-binding domain. The active site is inaccessible to solvent in the closed form; an endogenous ligand, believed to be picolinate, is bound close to and parallel with the flavin ring, an orientation compatible with redox catalysis. The active site is solvent accessible in the open form but the picolinate ligand is approximately perpendicular to the flavin ring and a tryptophan is stacked above the flavin ring. NikD also contains a mobile cation binding loop. PMID:17697998

  14. Effects of anthocyanins from purple sweet potato (Ipomoea batatas L. cultivar Eshu No. 8) on the serum uric acid level and xanthine oxidase activity in hyperuricemic mice.

    PubMed

    Zhang, Zi-Cheng; Su, Guan-Hua; Luo, Chun-Li; Pang, Ya-Lu; Wang, Lin; Li, Xing; Wen, Jia-Hao; Zhang, Jiu-Liang

    2015-09-01

    This study was aimed at evaluating the hypouricemic effect of the anthocyanin-rich purple sweet potato extract (APSPE). In vitro, APSPE has been proved to significantly inhibit XO activity in a dose-dependent manner. In vivo, APSPE could not only inhibit the XO activity in mouse liver, but also reduce the serum uric acid level in hyperuricemic mice and affect the expression of mRNA levels of related renal transporters, such as mURAT1, mGLUT9, mOAT1 and mOCTN2. Moreover, APSPE could effectively regulate BUN and Cr levels to normal and decrease the inflammatory cellular influx in the tubule of the hyperuricemic mice. This study indicates the potential clinical utility of APSPE as a safe and effective anti-hyperuricemia bioactive agent or functional food. PMID:26201407

  15. Effects of anthocyanins from purple sweet potato (Ipomoea batatas L. cultivar Eshu No. 8) on the serum uric acid level and xanthine oxidase activity in hyperuricemic mice.

    PubMed

    Zhang, Zi-Cheng; Su, Guan-Hua; Luo, Chun-Li; Pang, Ya-Lu; Wang, Lin; Li, Xing; Wen, Jia-Hao; Zhang, Jiu-Liang

    2015-09-01

    This study was aimed at evaluating the hypouricemic effect of the anthocyanin-rich purple sweet potato extract (APSPE). In vitro, APSPE has been proved to significantly inhibit XO activity in a dose-dependent manner. In vivo, APSPE could not only inhibit the XO activity in mouse liver, but also reduce the serum uric acid level in hyperuricemic mice and affect the expression of mRNA levels of related renal transporters, such as mURAT1, mGLUT9, mOAT1 and mOCTN2. Moreover, APSPE could effectively regulate BUN and Cr levels to normal and decrease the inflammatory cellular influx in the tubule of the hyperuricemic mice. This study indicates the potential clinical utility of APSPE as a safe and effective anti-hyperuricemia bioactive agent or functional food.

  16. Sodium iron EDTA and ascorbic acid, but not polyphenol oxidase treatment, counteract the strong inhibitory effect of polyphenols from brown sorghum on the absorption of fortification iron in young women.

    PubMed

    Cercamondi, Colin I; Egli, Ines M; Zeder, Christophe; Hurrell, Richard F

    2014-02-01

    In addition to phytate, polyphenols (PP) might contribute to low Fe bioavailability from sorghum-based foods. To investigate the inhibitory effects of sorghum PP on Fe absorption and the potential enhancing effects of ascorbic acid (AA), NaFeEDTA and the PP oxidase enzyme laccase, we carried out three Fe absorption studies in fifty young women consuming dephytinised Fe-fortified test meals based on white and brown sorghum varieties with different PP concentrations. Fe absorption was measured as the incorporation of stable Fe isotopes into erythrocytes. In study 1, Fe absorption from meals with 17 mg PP (8·5%) was higher than that from meals with 73 mg PP (3·2%) and 167 mg PP (2·7%; P< 0·001). Fe absorption from meals containing 73 and 167 mg PP did not differ (P= 0·9). In study 2, Fe absorption from NaFeEDTA-fortified meals (167 mg PP) was higher than that from the same meals fortified with FeSO₄ (4·6 v. 2·7%; P< 0·001), but still it was lower than that from FeSO₄-fortified meals with 17 mg PP (10·7%; P< 0·001). In study 3, laccase treatment decreased the levels of PP from 167 to 42 mg, but it did not improve absorption compared with that from meals with 167 mg PP (4·8 v. 4·6%; P= 0·4), whereas adding AA increased absorption to 13·6% (P< 0·001). These findings suggest that PP from brown sorghum contribute to low Fe bioavailability from sorghum foods and that AA and, to a lesser extent, NaFeEDTA, but not laccase, have the potential to overcome the inhibitory effect of PP and improve Fe absorption from sorghum foods.

  17. Abscisic acid and aldehyde oxidase activity in maize ear leaf and grain relative to post-flowering photosynthetic capacity and grain-filling rate under different water/nitrogen treatments.

    PubMed

    Qin, Shujun; Zhang, Zongzheng; Ning, Tangyuan; Ren, Shizhong; Su, Licheng; Li, Zengjia

    2013-09-01

    This study investigated changes in leaf abscisic acid (ABA) concentrations and grain ABA concentrations in two maize cultivars and analyzed the following relationships under different water/nitrogen treatments: leaf ABA concentrations and photosynthetic parameters; leaf ABA concentrations and grain ABA concentrations; leaf/grain ABA concentrations and grain-filling parameters; and aldehyde oxidase (AO, EC 1.2.3.1) activities and ABA concentrations. The ear leaf average AO activities and ABA concentrations were lower in the controlled release urea treatments compared with the conventional urea treatments. The average AO activities in the grains were higher in the controlled release urea treatments, and the ABA concentrations were significantly increased at 11-30 DAF. The Pn and ABA concentrations in ear leaves were negatively correlated. And the Gmean were positively correlated with the grain ABA concentrations at 11-30 DAF and negatively correlated with the leaf ABA concentrations at 20 and 40-50 DAF. The grain ABA concentrations and leaf ABA concentrations were positively correlated. Thus, the Gmean were closely related to the AO activities and to the ear leaf and grain ABA concentrations. As compared to other treatments, the subsoiling and controlled release urea treatment promoted the uptake of water and nitrogen by maize, increased the photosynthetic capacity of the ear leaves, increased the grain-filling rate, and improved the movement of photosynthetic assimilates toward the developing grains. In the cultivar Z958, higher ABA concentrations in grains at 11-30 DAF and lower ABA concentrations in ear leaves during the late grain-filling stage, resulted in higher grain-filling rate and increased accumulation of photosynthetic products (relative to the cultivar D3). PMID:23770596

  18. D-Serine metabolism in C6 glioma cells: Involvement of alanine-serine-cysteine transporter (ASCT2) and serine racemase (SRR) but not D-amino acid oxidase (DAO)

    PubMed Central

    Sikka, Pilleriin; Walker, Rosie; Cockayne, Rebecca; Wood, Matthew JA; Harrison, Paul J; Burnet, Philip WJ

    2010-01-01

    D-serine is an endogenous N-methyl-D-aspartate (NMDA) receptor coagonist. It is synthesized from L-serine by serine racemase (SRR), but many aspects of its metabolism remain unclear, especially in the forebrain, which lacks active D-amino acid oxidase (DAO), the major D-serine degradative enzyme. Candidate mechanisms include SRR operating in α,β-eliminase mode (converting D-serine to pyruvate) and regulation by serine transport, in which the alanine-serine-cysteine transporter ASCT2 is implicated. Here we report studies in C6 glioma cells, which “simulate” the forebrain, in that the cells express SRR and ASCT2 but lack DAO activity. We measured D-serine, ASCT2, SRR, and DAO expression and DAO activity in two situations: after incubation of cells for 48 hr with serine isomers and after increased or decreased SRR expression by transfection and RNA interference, respectively. Incubation with serine enantiomers decreased [3H]D-serine uptake and ASCT2 mRNA and increased SRR immunoreactivity but did not alter DAO immunoreactivity, and DAO activity remained undetectable. SRR overexpression increased D-serine and pyruvate and decreased [3H]D-serine uptake and ASCT2 mRNA but did not affect DAO. SRR knockdown did not alter any of the parameters. Our data suggest that D-serine transport mediated by ASCT2 contributes prominently to D-serine homeostasis when DAO activity is absent. The factors regulating D-serine are important for understanding normal NMDA receptor function and because D-serine, along with DAO and SRR, is implicated in the pathogenesis and treatment of schizophrenia. © 2010 Wiley-Liss, Inc. PMID:20091774

  19. Abscisic acid and aldehyde oxidase activity in maize ear leaf and grain relative to post-flowering photosynthetic capacity and grain-filling rate under different water/nitrogen treatments.

    PubMed

    Qin, Shujun; Zhang, Zongzheng; Ning, Tangyuan; Ren, Shizhong; Su, Licheng; Li, Zengjia

    2013-09-01

    This study investigated changes in leaf abscisic acid (ABA) concentrations and grain ABA concentrations in two maize cultivars and analyzed the following relationships under different water/nitrogen treatments: leaf ABA concentrations and photosynthetic parameters; leaf ABA concentrations and grain ABA concentrations; leaf/grain ABA concentrations and grain-filling parameters; and aldehyde oxidase (AO, EC 1.2.3.1) activities and ABA concentrations. The ear leaf average AO activities and ABA concentrations were lower in the controlled release urea treatments compared with the conventional urea treatments. The average AO activities in the grains were higher in the controlled release urea treatments, and the ABA concentrations were significantly increased at 11-30 DAF. The Pn and ABA concentrations in ear leaves were negatively correlated. And the Gmean were positively correlated with the grain ABA concentrations at 11-30 DAF and negatively correlated with the leaf ABA concentrations at 20 and 40-50 DAF. The grain ABA concentrations and leaf ABA concentrations were positively correlated. Thus, the Gmean were closely related to the AO activities and to the ear leaf and grain ABA concentrations. As compared to other treatments, the subsoiling and controlled release urea treatment promoted the uptake of water and nitrogen by maize, increased the photosynthetic capacity of the ear leaves, increased the grain-filling rate, and improved the movement of photosynthetic assimilates toward the developing grains. In the cultivar Z958, higher ABA concentrations in grains at 11-30 DAF and lower ABA concentrations in ear leaves during the late grain-filling stage, resulted in higher grain-filling rate and increased accumulation of photosynthetic products (relative to the cultivar D3).

  20. Identification of Genes Involved in Indole-3-Acetic Acid Biosynthesis by Gluconacetobacter diazotrophicus PAL5 Strain Using Transposon Mutagenesis

    PubMed Central

    Rodrigues, Elisete P.; Soares, Cleiton de Paula; Galvão, Patrícia G.; Imada, Eddie L.; Simões-Araújo, Jean L.; Rouws, Luc F. M.; de Oliveira, André L. M.; Vidal, Márcia S.; Baldani, José I.

    2016-01-01

    Gluconacetobacter diazotrophicus is a beneficial nitrogen-fixing endophyte found in association with sugarcane plants and other important crops. Beneficial effects of G. diazotrophicus on sugarcane growth and productivity have been attributed to biological nitrogen fixation process and production of phytohormones especially indole-3-acetic acid (IAA); however, information about the biosynthesis and function of IAA in G. diazotrophicus is still scarce. Therefore, the aim of this work was to identify genes and pathways involved in IAA biosynthesis in this bacterium. In our study, the screening of two independent Tn5 mutant libraries of PAL5T strain using the Salkowski colorimetric assay revealed two mutants (Gdiaa34 and Gdiaa01), which exhibited 95% less indolic compounds than the parental strain when grown in LGIP medium supplemented with L-tryptophan. HPLC chromatograms of the wild-type strain revealed the presence of IAA and of the biosynthetic intermediates indole-3-pyruvic acid (IPyA) and indole-3-lactate (ILA). In contrast, the HPLC profiles of both mutants showed no IAA but only a large peak of non-metabolized tryptophan and low levels of IPyA and ILA were detected. Molecular characterization revealed that Gdiaa01 and Gdiaa34 mutants had unique Tn5 insertions at different sites within the GDI2456 open read frame, which is predicted to encode a L-amino acid oxidase (LAAO). GDI2456 (lao gene) forms a cluster with GDI2455 and GDI2454 ORFs, which are predicted to encode a cytochrome C and an RidA protein, respectively. RT-qPCR showed that transcript levels of lao. cccA, and ridA genes were reduced in the Gdiaa01 as compared to PAL5T. In addition, rice plants inoculated with Gdiaa01 showed significantly smaller root development (length, surface area, number of forks and tips) than those plants inoculated with PAL5T. In conclusion, our study demonstrated that G. diazotrophicus PAL5T produces IAA via the IPyA pathway in cultures supplemented with tryptophan and

  1. Origin, Microbiology, Nutrition, and Pharmacology of D-Amino Acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Exposure of food proteins to certain processing conditions induces two major chemical changes: racemization of all L-amino acids (LAA) to D-amino acids (DAA) and concurrent formation of crosslinked amino acids such as lysinoalanine (LAL). The diet contains both processing-induced and naturally-form...

  2. Involvement of NADH Oxidase in Biofilm Formation in Streptococcus sanguinis

    PubMed Central

    Ge, Xiuchun; Shi, Xiaoli; Shi, Limei; Liu, Jinlin; Stone, Victoria; Kong, Fanxiang; Kitten, Todd; Xu, Ping

    2016-01-01

    Biofilms play important roles in microbial communities and are related to infectious diseases. Here, we report direct evidence that a bacterial nox gene encoding NADH oxidase is involved in biofilm formation. A dramatic reduction in biofilm formation was observed in a Streptococcus sanguinis nox mutant under anaerobic conditions without any decrease in growth. The membrane fluidity of the mutant bacterial cells was found to be decreased and the fatty acid composition altered, with increased palmitic acid and decreased stearic acid and vaccenic acid. Extracellular DNA of the mutant was reduced in abundance and bacterial competence was suppressed. Gene expression analysis in the mutant identified two genes with altered expression, gtfP and Idh, which were found to be related to biofilm formation through examination of their deletion mutants. NADH oxidase-related metabolic pathways were analyzed, further clarifying the function of this enzyme in biofilm formation. PMID:26950587

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

    PubMed Central

    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. PMID:25763711

  4. Inhibition of peroxisomal fatty acyl-CoA oxidase by antimycin A.

    PubMed Central

    Vamecq, J; Schepers, L; Parmentier, G; Mannaerts, G P

    1987-01-01

    Peroxisomal fatty acyl-CoA oxidase was inhibited by micromolar concentrations of antimycin A, an inhibitor of mitochondrial respiration. The inhibition was observed with all three substrates tested, i.e. palmitoyl-CoA, trihydroxycoprostanoyl-CoA and hexadecanedioyl-CoA. The peroxisomal D-amino acid oxidase was also inhibited by antimycin, but the peroxisomal L-alpha-hydroxyacid oxidase and uric acid oxidase and the mitochondrial monoamine oxidase were not. The degree of inhibition of acyl-CoA oxidase by antimycin was strongly dependent on the amount of cellular protein present in the assay mixture: at a fixed antimycin concentration, the inhibition was gradually lost with increasing protein concentrations. At a fixed cellular protein concentration in the assay mixtures, the mitochondrial oxidation of glutamate or palmitoylcarnitine was inhibited at antimycin concentrations that were much lower than those required for the inhibition of fatty acyl-CoA oxidase. Our results, nevertheless, demonstrate that antimycin A must be used with caution, when it is added to homogenates or subcellular fractions in order to distinguish between mitochondrial and peroxisomal fatty acid oxidation. PMID:3435468

  5. Hordeum vulgare Seedlings Amine Oxidase

    PubMed Central

    Cogoni, Antonina; Piras, Carla; Farci, Raffaele; Melis, Antonello; Floris, Giovanni

    1990-01-01

    Although no amine oxidase could be detected in crude extracts, the enzyme has been purified to apparent homogeneity from Hordeum vulgare seedlings using ammonium sulfate precipitation and chromatography on DEAE cellulose, Hydroxylapatite, and Sephadex G200 columns. Gel filtration experiments indicate a molecular weight of about 150,000. The pH optimum of the enzyme was found to be 7.5 in potassium phosphate buffer. The spectrum of ultraviolet and visible regions were similar to Cuamine oxidase from Leguminosae. PMID:16667542

  6. [Molecular docking analysis of xanthine oxidase inhibition by constituents of cichory].

    PubMed

    Wang, Xue-jie; Lin, Zhi-jian; Zhang, Bing; Zhu, Chun-sheng; Niu, Hong-juan; Zhou, Yue; Nie, An-zheng; Wang, Yu

    2015-10-01

    Human xanthine oxidase is considered to be a target for therapy of hyperuricemia. Cichorium intybus is a Chinese plant medicine which widely used in Xinjiang against various diseases. In order to screen the inhibitors of xanthine oxidase from C. intybus and to explore main pharmacological actions of cichory a compound collection of C. intybus was built via consulting related references about chemical research on cichory. The three-dimensional crystal structure of xanthine oxidase (PDB code: 1N5X) from Protein Data Bank was downloaded.. Autodock 4.2 was employed to screen the inhibitors of xanthine oxidase from cichory 70 compounds were found to possess quite low binding free energy comparing with TEI (febuxostat). C. intybus contains constituents possessing potential inhibitive activity against xanthine oxidase. It can explain the main pharmacological actions of cichory which can significantly lower the level of serum uric acid. PMID:26975108

  7. [Molecular docking analysis of xanthine oxidase inhibition by constituents of cichory].

    PubMed

    Wang, Xue-jie; Lin, Zhi-jian; Zhang, Bing; Zhu, Chun-sheng; Niu, Hong-juan; Zhou, Yue; Nie, An-zheng; Wang, Yu

    2015-10-01

    Human xanthine oxidase is considered to be a target for therapy of hyperuricemia. Cichorium intybus is a Chinese plant medicine which widely used in Xinjiang against various diseases. In order to screen the inhibitors of xanthine oxidase from C. intybus and to explore main pharmacological actions of cichory a compound collection of C. intybus was built via consulting related references about chemical research on cichory. The three-dimensional crystal structure of xanthine oxidase (PDB code: 1N5X) from Protein Data Bank was downloaded.. Autodock 4.2 was employed to screen the inhibitors of xanthine oxidase from cichory 70 compounds were found to possess quite low binding free energy comparing with TEI (febuxostat). C. intybus contains constituents possessing potential inhibitive activity against xanthine oxidase. It can explain the main pharmacological actions of cichory which can significantly lower the level of serum uric acid.

  8. Expression of alternative oxidase in tomato

    SciTech Connect

    Kakefuda, M.; McIntosh, L. )

    1990-05-01

    Tomato fruit ripening is characterized by an increase in ethylene biosynthesis, a burst in respiration (i.e. the climacteric), fruit softening and pigmentation. As whole tomatoes ripened from mature green to red, there was an increase in the alternative oxidase capacity. Aging pink tomato slices for 24 and 48 hrs also showed an increase of alternative oxidase and cytochrome oxidase capacities. Monoclonal antibodies prepared to the Sauromatum guttatum alternative oxidase were used to follow the appearance of alternative oxidase in tomato fruits. There is a corresponding increase in a 36kDa protein with an increase in alternative oxidase capacity. Effects of ethylene and norbornadiene on alternative oxidase capacity were also studied. We are using an alternative oxidase cDNA clone from potato to study the expression of mRNA in ripening and wounded tomatoes to determine if the gene is transcriptionally regulated.

  9. d-Amino Acid Catabolism Is Common Among Soil-Dwelling Bacteria

    PubMed Central

    Radkov, Atanas D; McNeill, Katlyn; Uda, Koji; Moe, Luke A

    2016-01-01

    Soil and rhizosphere environments were examined in order to determine the identity and relative abundance of bacteria that catabolize d- and l-amino acids as the sole source of carbon and nitrogen. All substrates were readily catabolized by bacteria from both environments, with most d-amino acids giving similar CFU counts to their l-amino acid counterparts. CFU count ratios between l- and d-amino acids typically ranged between 2 and 1. Isolates were phylogenetically typed in order to determine the identity of d-amino acid catabolizers. Actinobacteria, specifically the Arthrobacter genus, were abundant along with members of the α- and β-Proteobacteria classes. PMID:27169790

  10. NADPH oxidases in Eukaryotes: red algae provide new hints!

    PubMed

    Hervé, Cécile; Tonon, Thierry; Collén, Jonas; Corre, Erwan; Boyen, Catherine

    2006-03-01

    The red macro-alga Chondrus crispus is known to produce superoxide radicals in response to cell-free extracts of its green algal pathogenic endophyte Acrochaete operculata. So far, no enzymes involved in this metabolism have been isolated from red algae. We report here the isolation of a gene encoding a homologue of the respiratory burst oxidase gp91(phox) in C. crispus, named Ccrboh. This single copy gene encodes a polypeptide of 825 amino acids. Search performed in available genome and EST algal databases identified sequences showing common features of NADPH oxidases in other algae such as the red unicellular Cyanidioschyzon merolae, the economically valuable red macro-alga Porphyra yezoensis and the two diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. Domain organization and phylogenetic relationships with plant, animal, fungal and algal NADPH oxidase homologues were analyzed. Transcription analysis of the C. crispus gene revealed that it was over-transcribed during infection of C. crispus gametophyte by the endophyte A. operculata, and after incubation in presence of atrazine, methyl jasmonate and hydroxyperoxides derived from C20 polyunsaturated fatty acids (PUFAs). These results also illustrate the interest of exploring the red algal lineage for gaining insight into the deep evolution of NADPH oxidases in Eukaryotes.

  11. Urate oxidase knockdown decreases oxidative stress in a murine hepatic cell line

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Humans, birds, and some primates do not express the uric acid degrading enzyme urate oxidase (UOX) and, as a result, have plasma uric acid concentrations higher than UOX expressing animals. Although high uric acid concentrations are suggested to increase the antioxidant defense system and provide a...

  12. Hepatocyte NADPH Oxidase 4 Regulates Stress Signaling, Fibrosis, and Insulin Sensitivity During Development of Steatohepatitis in Mice

    PubMed Central

    Bettaieb, Ahmed; Jiang, Joy X.; Sasaki, Yu; Chao, Tzu-I; Kiss, Zsofia; Chen, Xiangling; Tian, Jijing; Katsuyama, Masato; Yabe-Nishimura, Chihiro; Xi, Yannan; Szyndralewiez, Cedric; Schröder, Kathrin; Shah, Ajay; Brandes, Ralph P.; Haj, Fawaz G.; Török, Natalie J.

    2015-01-01

    Background & Aims Reactive oxidative species (ROS) are believed to be involved in the progression of non-alcoholic steatohepatitis (NASH). However, little is known about the sources of ROS in hepatocytes or their role in disease progression. We studied the effects of NADPH oxidase 4 (NOX4) in liver tissues from patients with NASH and mice with steatohepatitis. Methods Liver biopsy samples were obtained from 5 patients with NASH, as well as 4 patients with simple steatosis and 5 patients without steatosis (controls) from the University of California, Davis Cancer Center Biorepository. Mice with hepatocyte-specific deletion of NOX4 (NOX4hepKO) and NOX4floxp+/+ C57BL/6 mice (controls) were given fast food diets (supplemented with high-fructose corn syrup) or choline-deficient L-amino acid-defined to induce steatohepatitis, or control diets, for 20 weeks. A separate group of mice were given the NOX4 inhibitor (GKT137831). Liver tissues were collected and immunoblot analyses were performed determine levels of NOX4, markers of inflammation and fibrosis, double-stranded RNA-activated protein kinase (PKR), and phospho-eIF-2alpha kinase (PERK)-mediated stress signaling pathways. We performed hyperinsulinemic-euglycemic clamp studies and immunoprecipitation analyses to determine the oxidation and phosphatase activity of PP1C. Results Levels of NOX4 were increased in patients with NASH, compared with controls. Hepatocyte-specific deletion of NOX4 reduced oxidative stress, lipid peroxidation, and liver fibrosis in mice with diet-induced steatohepatitis. A small molecule inhibitor of NOX4 reduced liver inflammation and fibrosis and increased insulin sensitivity in mice with diet-induced steatohepatitis. In primary hepatocytes, NOX4 reduced the activity of the phosphatase PP1C, prolonging activation of PKR and PERK-mediated stress signaling. Mice with hepatocyte-specific deletion of NOX4 and mice given GKT137831had increased insulin sensitivity. Conclusion NOX4 regulates

  13. A Role for Reactive Oxygen Species Produced by NADPH Oxidases in the Embryo and Aleurone Cells in Barley Seed Germination

    PubMed Central

    Ishibashi, Yushi; Kasa, Shinsuke; Sakamoto, Masatsugu; Aoki, Nozomi; Kai, Kyohei; Yuasa, Takashi; Hanada, Atsushi; Yamaguchi, Shinjiro; Iwaya-Inoue, Mari

    2015-01-01

    Reactive oxygen species (ROS) promote the germination of several seeds, and antioxidants suppress it. However, questions remain regarding the role and production mechanism of ROS in seed germination. Here, we focused on NADPH oxidases, which produce ROS. After imbibition, NADPH oxidase mRNAs were expressed in the embryo and in aleurone cells of barley seed; these expression sites were consistent with the sites of ROS production in the seed after imbibition. To clarify the role of NADPH oxidases in barley seed germination, we examined gibberellic acid (GA) / abscisic acid (ABA) metabolism and signaling in barley seeds treated with diphenylene iodonium chloride (DPI), an NADPH oxidase inhibitor. DPI significantly suppressed germination, and suppressed GA biosynthesis and ABA catabolism in embryos. GA, but not ABA, induced NADPH oxidase activity in aleurone cells. Additionally, DPI suppressed the early induction of α-amylase by GA in aleurone cells. These results suggest that ROS produced by NADPH oxidases promote GA biosynthesis in embryos, that GA induces and activates NADPH oxidases in aleurone cells, and that ROS produced by NADPH oxidases induce α-amylase in aleurone cells. We conclude that the ROS generated by NADPH oxidases regulate barley seed germination through GA / ABA metabolism and signaling in embryo and aleurone cells. PMID:26579718

  14. A Role for Reactive Oxygen Species Produced by NADPH Oxidases in the Embryo and Aleurone Cells in Barley Seed Germination.

    PubMed

    Ishibashi, Yushi; Kasa, Shinsuke; Sakamoto, Masatsugu; Aoki, Nozomi; Kai, Kyohei; Yuasa, Takashi; Hanada, Atsushi; Yamaguchi, Shinjiro; Iwaya-Inoue, Mari

    2015-01-01

    Reactive oxygen species (ROS) promote the germination of several seeds, and antioxidants suppress it. However, questions remain regarding the role and production mechanism of ROS in seed germination. Here, we focused on NADPH oxidases, which produce ROS. After imbibition, NADPH oxidase mRNAs were expressed in the embryo and in aleurone cells of barley seed; these expression sites were consistent with the sites of ROS production in the seed after imbibition. To clarify the role of NADPH oxidases in barley seed germination, we examined gibberellic acid (GA) / abscisic acid (ABA) metabolism and signaling in barley seeds treated with diphenylene iodonium chloride (DPI), an NADPH oxidase inhibitor. DPI significantly suppressed germination, and suppressed GA biosynthesis and ABA catabolism in embryos. GA, but not ABA, induced NADPH oxidase activity in aleurone cells. Additionally, DPI suppressed the early induction of α-amylase by GA in aleurone cells. These results suggest that ROS produced by NADPH oxidases promote GA biosynthesis in embryos, that GA induces and activates NADPH oxidases in aleurone cells, and that ROS produced by NADPH oxidases induce α-amylase in aleurone cells. We conclude that the ROS generated by NADPH oxidases regulate barley seed germination through GA / ABA metabolism and signaling in embryo and aleurone cells.

  15. A Role for Reactive Oxygen Species Produced by NADPH Oxidases in the Embryo and Aleurone Cells in Barley Seed Germination.

    PubMed

    Ishibashi, Yushi; Kasa, Shinsuke; Sakamoto, Masatsugu; Aoki, Nozomi; Kai, Kyohei; Yuasa, Takashi; Hanada, Atsushi; Yamaguchi, Shinjiro; Iwaya-Inoue, Mari

    2015-01-01

    Reactive oxygen species (ROS) promote the germination of several seeds, and antioxidants suppress it. However, questions remain regarding the role and production mechanism of ROS in seed germination. Here, we focused on NADPH oxidases, which produce ROS. After imbibition, NADPH oxidase mRNAs were expressed in the embryo and in aleurone cells of barley seed; these expression sites were consistent with the sites of ROS production in the seed after imbibition. To clarify the role of NADPH oxidases in barley seed germination, we examined gibberellic acid (GA) / abscisic acid (ABA) metabolism and signaling in barley seeds treated with diphenylene iodonium chloride (DPI), an NADPH oxidase inhibitor. DPI significantly suppressed germination, and suppressed GA biosynthesis and ABA catabolism in embryos. GA, but not ABA, induced NADPH oxidase activity in aleurone cells. Additionally, DPI suppressed the early induction of α-amylase by GA in aleurone cells. These results suggest that ROS produced by NADPH oxidases promote GA biosynthesis in embryos, that GA induces and activates NADPH oxidases in aleurone cells, and that ROS produced by NADPH oxidases induce α-amylase in aleurone cells. We conclude that the ROS generated by NADPH oxidases regulate barley seed germination through GA / ABA metabolism and signaling in embryo and aleurone cells. PMID:26579718

  16. Degradation of pentachlorophenol by potato polyphenol oxidase.

    PubMed

    Hou, Mei-Fang; Tang, Xiao-Yan; Zhang, Wei-De; Liao, Lin; Wan, Hong-Fu

    2011-11-01

    In this study, polyphenol oxidase (PPO) was extracted from commercial potatoes. Degradation of pentachlorophenol by potato PPO was investigated. The experimental results show that potato PPO is more active in weak acid than in basic condition and that the optimum pH for the reaction is 5.0. The degradation of pentachlorophenol by potato PPO reaches a maximum at 298 K. After reaction for 1 h, the removal of both pentachlorophenol and total organic carbon is >70% with 6.0 units/mL potato PPO at pH 5.0 and 298 K. Pentachlorophenol can be degraded through dechlorination and ring-opening by potato PPO. The work demonstrates that pentachlorophenol can be effectively eliminated by crude potato PPO. PMID:21967325

  17. Xanthine oxidase inhibitory lanostanoids from Ganoderma tsugae.

    PubMed

    Lin, Kai-Wei; Chen, Yen-Ting; Yang, Shyh-Chyun; Wei, Bai-Luh; Hung, Chi-Feng; Lin, Chun-Nan

    2013-09-01

    Two new lanostanoids, 3α-acetoxy-22-oxo-5α-lanosta-8,24-dien-21-oic acid, named tsugaric acid D (1) and 16α-hydroxy-3-oxo-5α-lanosta-6,8,24(24(1))-trien-21-oic acid, named tsugaric acid E (2) were isolated from the fruit bodies of Ganoderma tsugae. The structures 1 and 2 were determined by spectroscopic methods. Compound 1 and known compounds 3 and 6 exhibited significant inhibitory effects on xanthine oxidase (XO) activity with an IC50 values of 90.2±24.2, 116.1±3.0, and 181.9±5.8 μM, respectively. Known compound 5 was able to protect human keratinocytes against damage induced by UVB light, which showed 5 could protect keratinocytes from photodamage. The 1 and 5 μM 1 combined with 5 μM cisplatin, respectively, enhanced the cytotoxicity induced by cisplatin. It suggested that 1 and 5 μM 1 combined with low dose of cisplatin may enhance the therapeutic efficacy of cisplatin and reduce side effect and cisplatin resistant.

  18. Structures and Mechanism of the Monoamine Oxidase Family

    PubMed Central

    Gaweska, Helena; Fitzpatrick, Paul F.

    2011-01-01

    Members of the monoamine oxidase family of flavoproteins catalyze the oxidation of primary and secondary amines, polyamines, amino acids, and methylated lysine side chains in proteins. The enzymes have similar overall structures, with conserved FAD-binding domains and varied substrate-binding sites. Multiple mechanisms have been proposed for the catalytic reactions of these enzymes. The present review compares the structures of different members of the family and the various mechanistic proposals. PMID:22022344

  19. Updates on industrial production of amino acids using Corynebacterium glutamicum.

    PubMed

    Wendisch, Volker F; Jorge, João M P; Pérez-García, Fernando; Sgobba, Elvira

    2016-06-01

    L-Amino acids find various applications in biotechnology. L-Glutamic acid and its salts are used as flavor enhancers. Other L-amino acids are used as food or feed additives, in parenteral nutrition or as building blocks for the chemical and pharmaceutical industries. L-amino acids are synthesized from precursors of central carbon metabolism. Based on the knowledge of the biochemical pathways microbial fermentation processes of food, feed and pharma amino acids have been developed. Production strains of Corynebacterium glutamicum, which has been used safely for more than 50 years in food biotechnology, and Escherichia coli are constantly improved using metabolic engineering approaches. Research towards new processes is ongoing. Fermentative production of L-amino acids in the million-ton-scale has shaped modern biotechnology and its markets continue to grow steadily. This review focusses on recent achievements in strain development for amino acid production including the use of CRISPRi/dCas9, genome-reduced strains, biosensors and synthetic pathways to enable utilization of alternative carbon sources. PMID:27116971

  20. Effect of d-amino acids on IgE binding to peanut allergens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    D-amino acids are formed when L-amino acids are exposed to heat. The objective was to determine the existence of D-amino acids in roasted peanut and their effect on IgE binding. Raw and roasted peanut protein extracts were hydrolyzed in 6 N HCL under vacuum. The hydrolysates were then analyzed for D...

  1. Tyrosine codon corresponds to topa quinone at the active site of copper amine oxidases.

    PubMed

    Mu, D; Janes, S M; Smith, A J; Brown, D E; Dooley, D M; Klinman, J P

    1992-04-25

    The recently discovered organic cofactor of bovine serum amine oxidase, topa quinone, is an uncommon amino acid residue in the polypeptide backbone (Janes, S. M., Mu, D., Wemmer, D., Smith, A. J., Kaur, S., Maltby, D., Burlingame, A. L., and Klinman, J. P. (1990) Science 248, 981-987). The amine oxidase gene from the yeast Hansenula polymorpha has been cloned and sequenced (Bruinenberg, P. G., Evers, M., Waterham, H. R., Kuipers, J., Arnberg, A. C., and Geert, A. B. (1989) Biochim. Biophys. Acta 1008, 157-167). In order to understand the incorporation of topa quinone in eukaryotes, we have isolated yeast amine oxidase from H. polymorpha. Following protocols established with bovine serum amine oxidase, yeast amine oxidase was derivatized with [14C]phenylhydrazine, followed by thermolytic digestion and isolation of a dominant radiolabeled peptide by high pressure liquid chromatography. Comparison of resonance Raman spectra for this peptide to spectra of a model compound demonstrates that topa quinone is the cofactor. By alignment of a DNA-derived yeast amine oxidase sequence with the topa quinone-containing peptide sequence, it is found that the tyrosine codon, UAC, corresponds to topa quinone in the mature protein. In a similar manner, alignment of a tryptic peptide from bovine serum amine oxidase implicates tyrosine as the precursor to topa quinone in mammals.

  2. Incorporation of copper into lysyl oxidase.

    PubMed

    Kosonen, T; Uriu-Hare, J Y; Clegg, M S; Keen, C L; Rucker, R B

    1997-10-01

    Lysyl oxidase is a copper-dependent enzyme involved in extracellular processing of collagens and elastin. Although it is known that copper is essential for the functional activity of the enzyme, there is little information on the incorporation of copper. In the present study we examined the insertion of copper into lysyl oxidase using 67Cu in cell-free transcription/translation assays and in normal skin fibroblast culture systems. When a full-length lysyl oxidase cDNA was used as a template for transcription/translation reactions in vitro, unprocessed prolysyl oxidase appeared to bind copper. To examine further the post-translational incorporation of copper into lysyl oxidase, confluent skin fibroblasts were incubated with inhibitors of protein synthesis (cycloheximide, 10 microg/ml), glycosylation (tunicamycin, 10 microg/ml), protein secretion (brefeldin A, 10 microg/ml) and prolysyl oxidase processing (procollagen C-peptidase inhibitor, 2.5 microg/ml) together with 300 microCi of carrier-free 67Cu. It was observed that protein synthesis was a prerequisite for copper incorporation, but inhibition of glycosylation by tunicamycin did not affect the secretion of 67Cu as lysyl oxidase. Brefeldin A inhibited the secretion of 67Ci-labelled lysyl oxidase by 46%, but the intracellular incorporation of copper into lysyl oxidase was not affected. In addition, the inhibition of the extracellular proteolytic processing of prolysyl oxidase to lysyl oxidase had minimal effects on the secretion of protein-bound 67Cu. Our results indicate that, similar to caeruloplasmin processing [Sato and Gitlin (1991) J. Biol. Chem. 266, 5128-5134], copper is inserted into prolysyl oxidase independently of glycosylation. PMID:9355764

  3. HypC, the Anthrone Oxidase Involved in Aflatoxin Biosynthesis▿ †

    PubMed Central

    Ehrlich, Kenneth C.; Li, Ping; Scharfenstein, Leslie; Chang, Perng-Kuang

    2010-01-01

    On the basis of gene disruption and enzyme activity, hypC, an open reading frame in the region between the pksA (aflC) and nor-1 (aflD) genes in the aflatoxin biosynthesis gene cluster, encodes a 17-kDa oxidase that converts norsolorinic acid anthrone to norsolorinic acid. PMID:20348292

  4. Elicitation of dihydrobenzophenanthridine oxidase in Sanguinaria canadensis cell cultures.

    PubMed

    Ignatov, A; Clark, W G; Cline, S D; Psenak, M; Krueger, J; Coscia, C J

    1996-12-01

    Dihydrobenzophenanthridine (DHBP) oxidase catalyses the last step in the biogenesis of the benzo[c]phenanthridine alkaloid sanguinarine. Addition of autoclaved fungal preparations or putative plant defence signalling intermediates (jasmonic acid (JA), methyl jasmonate (MeJ), acetylsalicylic acid (ASA)) to Sanguinaria canadensis cell suspension cultures elicited an increase in the activity of DHBP oxidase. MeJ and ASA were better inducers of oxidase activity than were the fungal elicitor and JA. Enzyme-specific activity could be induced in a dose- and time-dependent manner up to 4- to 14-fold, respectively, when cells were treated with MeJ or with ASA. A change in total enzyme activity in cultured cells was observed only at the highest concentration of MeJ and not at any level of ASA tested. The results suggest that MeJ and ASA may play a role in the S. canadensis defence against pathogens by eliciting the enzymes involved in the synthesis of the phytoalexin benzophenanthridine alkaloids.

  5. Monoamine Oxidase Inhibitors: Clinical Review

    PubMed Central

    Remick, Ronald A.; Froese, Colleen

    1990-01-01

    Monoamine oxidase inhibitors (MAOIs) are effective antidepressant agents. They are increasingly and effectively used in a number of other psychiatric and non-psychiatric medical syndromes. Their potential for serious toxicity (i.e., hypertensive reaction) is far less than original reports suggest, and newer reversible substrate-specific MAOIs may offer even less toxicity. The author reviews the pharmacology, mechanism of action, clinical indications, and dosing strategies of MAOIs. The common MAOI side-effects (hypotension, weight gain, sexual dysfunction, insomnia, daytime sedation, myoclonus, and hypertensive episodes) are described and management techniques suggested. Recent clinical developments involving MAOIs are outlined. PMID:21233984

  6. Glucose oxidase activity of actinomycetes.

    PubMed

    St Vlahov, S

    1978-01-01

    The ability of 311 actiomycete, belonging to 12 species to produce glucose oxidase was studied. It was found that 174 of them formed exoenzymes on solid medium and 133 in liquid medium. The composition of the nutrient medium has an essential effect on the amount of enzyme formed. Strains with considerably higher activity form a greater amount of exoenzymes on soya meal medium and on synthetic medium with KNO2. The highest activity of the culture liquid of some strains was observed between the 6th and 7th day of cultivation. During this phase of growth the highest productivity of the biomas was established. PMID:76424

  7. Characterization of a cytochrome a1 that functions as a ubiquinol oxidase in Acetobacter aceti.

    PubMed

    Fukaya, M; Tayama, K; Tamaki, T; Ebisuya, H; Okumura, H; Kawamura, Y; Horinouchi, S; Beppu, T

    1993-07-01

    The terminal oxidase for ethanol oxidation in Acetobacter aceti was purified as a complex consisting of four subunits (subunits I, II, III, and IV) with molecular masses of 72, 34, 21, and 13 kDa, respectively. Spectrophotometric analysis and catalytic properties determined with the purified enzyme showed that it belonged to a family of cytochrome a1 (ba)-type ubiquinol oxidases. A polymerase chain reaction with two oligonucleotides designed for amino acid sequences that are conserved in subunit I of the aa3-type cytochrome c oxidases from various origins and of an Escherichia coli o (bo)-type ubiquinol oxidase was used for cloning the cytochrome a1 gene. A 0.5-kb fragment thus amplified was used as the probe to clone a 4.5-kb KpnI fragment that contained a putative open reading frame for the whole subunit I gene. The molecular weight and amino acid composition of the product of this open reading frame (cyaA) were the same as those of the purified protein from A. aceti. The amino acid sequence of CyaA was homologous to that of subunit I of the E. coli o-type ubiquinol oxidase. Nucleotide sequence analysis of the region neighboring the cyaA gene revealed that the genes (cyaB, cyaC, and cyaD) encoding the other three subunits (subunits II, III, and IV) were clustered upstream and downstream of the cyaA gene in the order cyaB, cyaA, cyaC, and cyaD and with the same transcription polarity, forming an operon. As expected from the enzymatic properties, CyaB, CyaC, and CyaD showed great similarity in amino acid sequence to the corresponding sununits of the E. coli o-type ubiquinol oxidase and as(3)-type cytochrome c oxidases.

  8. 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.

  9. Enantiomer-specific selection of amino acids.

    PubMed

    Ren, Xueying; Tellez, Luis A; de Araujo, Ivan E

    2013-12-01

    Dietary intake of L-amino acids impacts on several physiological functions, including the control of gastrointestinal motility, pancreatic secretion, and appetite. However, the biological mechanisms regulating behavioral predilections for certain amino acid types remain poorly understood. We tested the hypothesis that, in mice, the potency with which a given glucogenic amino acid increases glucose utilization reflects its rewarding properties. We have found that: (1) during long-, but not short-, term preference tests, L-alanine and L-serine were preferred over their D-enantiomer counterparts, while no such effect was observed for L-threonine vs. D-threonine; (2) these behavioral patterns were closely associated with the ability of L-amino acids to promote increases in respiratory exchange ratios such that those, and only those, L-amino acids able to promote increases in respiratory exchange ratios were preferred over their D-isomers; (3) these behavioral preferences were independent of gustatory influences, since taste-deficient Trpm5 knockout mice displayed ingestive responses very similar to those of their wild-type counterparts. We conclude that the ability to promote increases in respiratory exchange ratios enhances the reward value of nutritionally relevant amino acids and suggest a mechanistic link between substrate utilization and amino acid preferences.

  10. Immunological comparison of sulfite oxidase

    SciTech Connect

    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 inhibited 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.

  11. Mitochondrial Cytochrome c Oxidase Deficiency

    PubMed Central

    Rak, Malgorzata; Bénit, Paule; Chrétien, Dominique; Bouchereau, Juliette; Schiff, Manuel; El-Khoury, Riyad; Tzagoloff, Alexander; Rustin, Pierre

    2016-01-01

    As with other mitochondrial respiratory chain components, marked clinical and genetic heterogeneity is observed in patients with a cytochrome c oxidase deficiency. This constitutes a considerable diagnostic challenge and raises a number of puzzling questions. So far, pathological mutations have been reported in more than 30 genes, in both mitochondrial and nuclear DNA, affecting either structural subunits of the enzyme or proteins involved in its biogenesis. In this review, we discuss the possible causes of the discrepancy between the spectacular advances made in the identification of the molecular bases of cytochrome oxidase deficiency and the lack of any efficient treatment in diseases resulting from such deficiencies. This brings back many unsolved questions related to the frequent delay of clinical manifestation, variable course and severity, and tissue-involvement often associated with these diseases. In this context, we stress the importance to study different models of these diseases, but also discuss the limitations encountered in most available disease models. In the future, with the possible exception of replacement therapy using genes, cells or organs, a better understanding of underlying mechanism(s) of these mitochondrial diseases is presumably required to develop efficient therapy. PMID:26846578

  12. Chemopreventive mechanisms of α-keto acid metabolites of naturally occurring organoselenium compounds.

    PubMed

    Pinto, John T; Lee, Jeong-In; Sinha, Raghu; MacEwan, Melanie E; Cooper, Arthur J L

    2011-06-01

    Previous studies on the chemopreventive mechanisms of dietary selenium have focused on its incorporation into antioxidative selenoproteins, such as glutathione peroxidase and thioredoxin reductase. Several studies, however, have revealed that dietary selenium in the form of L-selenomethionine and the 21st amino acid, selenocysteine, also have intrinsic anti-cancer properties. Biochemical mechanisms previously investigated to contribute to their anticancer effects involve β- and γ-lyase reactions. Some pyridoxal 5'-phosphate (PLP)-containing enzymes can catalyze a β-lyase reaction with Se-methyl-L-selenocysteine (MSC) generating pyruvate and ammonia. Other PLP-enzymes can catalyze a γ-lyase reaction with L-selenomethionine (SM) generating α-ketobutyrate and ammonia. In both cases, a purported third product is methylselenol (CH(3)SeH). Although not directly quantifiable, as a result of its extreme hydrophobicity and high vapor pressure, CH(3)SeH has been indirectly observed to act through the alteration of protein-sulfhydryl moieties on redox-responsive signal and transcription factors, thereby maintaining a non-proliferative intracellular environment. We have considered the possibility that α-keto acid analogues of MSC (i.e., methylselenopyruvate; MSP) and SM (i.e., α-keto-γ-methylselenobutyrate; KMSB), generated via a transamination and/or L-amino acid oxidase reaction may also be chemoprotective. Indeed, these compounds were shown to increase the level of histone-H3 acetylation in human prostate and colon cancer cells. MSP and KMSB structurally resemble butyrate, an inhibitor of several histone deacetylases. Thus, the seleno α-keto acid metabolites of MSC and SM, along with CH(3)SeH derived from β- and γ-lyase reactions, may be potential direct-acting metabolites of organoselenium that lead to de-repression of silenced tumor suppressor proteins and/or regulation of genes and signaling molecules.

  13. Spinach thylakoid polyphenol oxidase isolation, activation, and properties of the native chloroplast enzyme

    SciTech Connect

    Golbeck, J.H.; Cammarata, K.V.

    1981-05-01

    Polyphenol oxidase activity (E.C. 1.14,18.1) has been found in two enzyme species isolated from thylakoid membranes of spinach chloroplasts. The proteins were released from the membrane by sonication and purified >900-fold by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography. The enzymes appear to be the tetramer and monomer of a subunit with a molecular weight of 42,500 as determined by lithium dodecyl sulfate gel electrophoresis. Sonication releases polyphenol oxidase from the membrane largely in the latent state. In the absence of added fatty acids, the isolated enzyme spontaneously, but slowly, activates with time. Purified polyphenol oxidase utilizes o-diphenols as substrates and shows no detectable levels of monophenol or p-diphenol oxidase activities. Suitable substrates include chlorogenic acid, catechol, caffeic acid, pyrogallol, and dopamine; however, the enzyme is substrate-inhibited by the last four at concentrations near their K/sub m/. A large seasonal variation in polyphenol oxidase activity may result from a decrease in enzyme content rather than inhibition of the enzyme present.

  14. The C-terminal region controls correct folding of genus Trametes pyranose 2-oxidases.

    PubMed

    Maresová, Helena; Palyzová, Andrea; Kyslík, Pavel

    2007-06-30

    The pyranose 2-oxidases from Trametes ochracea and Trametes pubescens share markedly similar amino acid sequences with identity of 93.4%. When expressed from the recombinant plasmids based on the same vector in the Escherichia coli host strain BL21(DE3) at higher growth temperatures, they differ strikingly in the formation of the inclusion bodies. Upon overexpression in the cultures performed at 28 degrees C, the specific activity of pyranose 2-oxidase from T. pubescens was eight times higher than that from T. ochracea: 93% of pyranose 2-oxidase from T. ochracea and only 15% of that from T. pubescens was present in the form of inclusion bodies. To ascertain the cause of this difference, both cloned genes were shuffled. Site-directed recombination of p2o cDNAs revealed that DNA constructs ending with 3' end of p2o cDNA from T. pubescens code for proteins that are folded into an active form to the greater extent, regardless of the gene expression level. "In silicio" analysis of physico-chemical properties of the protein sequences of pyranose 2-oxidases revealed that the sequence of amino acid residues 368-430, constituting the small, head domain of pyranose 2-oxidase from T. pubescens, affects positively the enzyme folding at higher cultivation temperatures. The domain differs in six amino acid residues from that of T. ochracea.

  15. Phenol oxidase activity in secondary transformed peat-moorsh soils

    NASA Astrophysics Data System (ADS)

    Styła, K.; Szajdak, L.

    2009-04-01

    The chemical composition of peat depends on the geobotanical conditions of its formation and on the depth of sampling. The evolution of hydrogenic peat soils is closely related to the genesis of peat and to the changes in water conditions. Due to a number of factors including oscillation of ground water level, different redox potential, changes of aerobic conditions, different plant communities, and root exudes, and products of the degradation of plant remains, peat-moorsh soils may undergo a process of secondary transformation conditions (Sokolowska et al. 2005; Szajdak et al. 2007). Phenol oxidase is one of the few enzymes able to degrade recalcitrant phenolic materials as lignin (Freeman et al. 2004). Phenol oxidase enzymes catalyze polyphenol oxidation in the presence of oxygen (O2) by removing phenolic hydrogen or hydrogenes to from radicals or quinines. These products undergo nucleophilic addition reactions in the presence or absence of free - NH2 group with the eventual production of humic acid-like polymers. The presence of phenol oxidase in soil environments is important in the formation of humic substances a desirable process because the carbon is stored in a stable form (Matocha et al. 2004). The investigations were carried out on the transect of peatland 4.5 km long, located in the Agroecological Landscape Park host D. Chlapowski in Turew (40 km South-West of Poznań, West Polish Lowland). The sites of investigation were located along Wyskoć ditch. The following material was taken from four chosen sites marked as Zbechy, Bridge, Shelterbelt and Hirudo in two layers: cartel (0-50cm) and cattle (50-100cm). The object of this study was to characterize the biochemical properties by the determination of the phenol oxidize activity in two layers of the four different peat-moors soils used as meadow. The phenol oxidase activity was determined spectrophotometrically by measuring quinone formation at λmax=525 nm with catechol as substrate by method of Perucci

  16. Evolution of the primate cytochrome c oxidase subunit II gene.

    PubMed

    Adkins, R M; Honeycutt, R L

    1994-03-01

    We examined the nucleotide and amino acid sequence variation of the cytochrome c oxidase subunit II (COII) gene from 25 primates (4 hominoids, 8 Old World monkeys, 2 New World monkeys, 2 tarsiers, 7 lemuriforms, 2 lorisiforms). Marginal support was found for three phylogenetic conclusions: (1) sister-group relationship between tarsiers and a monkey/ape clade, (2) placement of the aye-aye (Daubentonia) sister to all other strepsirhine primates, and (3) rejection of a sister-group relationship of dwarf lemurs (i.e., Cheirogaleus) with lorisiform primates. Stronger support was found for a sister-group relationship between the ring-tail lemur (Lemur catta) and the gentle lemurs (Hapalemur). In congruence with previous studies on COII, we found that the monkeys and apes have undergone a nearly two-fold increase in the rate of amino acid replacement relative to other primates. Although functionally important amino acids are generally conserved among all primates, the acceleration in amino acid replacements in higher primates is associated with increased variation in the amino terminal end of the protein. Additionally, the replacement of two carboxyl-bearing residues (glutamate and aspartate) at positions 114 and 115 may provide a partial explanation for the poor enzyme kinetics in cross-reactions between the cytochromes c and cytochrome c oxidases of higher primates and other mammals. PMID:8006990

  17. Diamine Oxidase in Cotyledons of Pisum sativum Develops as a Result of the Supply of Oxygen through the Embryonic Axis during Germination 1

    PubMed Central

    Hirasawa, Eiji

    1988-01-01

    The activity of diamine oxidase (EC 1.4.3.6.) in pea, Pisum sativum cv Alaska, cotyledons was studied. The rapid hydration caused by soaking seeds in water, the excision of the embryonic axis, and the suppression of the elongation of the embryonic axis by indoleacetic acid generate anaerobic conditions in these cotyledons that suppress diamine oxidase activity. These results show that oxygen is essential for the induction of diamine oxidase activity in pea cotyledons. During germination cotyledonary diamine oxidase develops as a result of the supply of oxygen through the embryonic axis of the intact pea seedling. PMID:16666323

  18. A role for active oxygen species as second messengers in the induction of alternative oxidase gene expression in Petunia hybrida cells.

    PubMed

    Wagner, A M

    1995-07-17

    Incubation of Petunia hybrida cells with H2O2 leads to an increase in alternative oxidase activity measured after 24 h. This increased activity is accompanied by an increase in alternative oxidase protein. A model is presented for the regulation of alternative oxidase protein synthesis in which active oxygen species and especially H2O2 play a crucial role as second messengers in the signal transducing pathway from the mitochondria to the nucleus. It is proposed that also the induction of the alternative oxidase by salicylic acid is mediated via H2O2.

  19. Crystal Structures of Intermediates in the Nitroalkane Oxidase Reaction

    SciTech Connect

    Heroux, A.; Bozinovski, D; Valley, M; Fitzpatrick, P; Orville, A

    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. The 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.

  20. Human lysyl oxidase-like 2.

    PubMed

    Moon, Hee-Jung; Finney, Joel; Ronnebaum, Trey; Mure, Minae

    2014-12-01

    Lysyl oxidase like-2 (LOXL2) belongs to the lysyl oxidase (LOX) family, which comprises Cu(2+)- and lysine tyrosylquinone (LTQ)-dependent amine oxidases. LOXL2 is proposed to function similarly to LOX in the extracellular matrix (ECM) by promoting crosslinking of collagen and elastin. LOXL2 has also been proposed to regulate extracellular and intracellular cell signaling pathways. Dysregulation of LOXL2 has been linked to many diseases, including cancer, pro-oncogenic angiogenesis, fibrosis and heart diseases. In this review, we will give an overview of the current understandings and hypotheses regarding the molecular functions of LOXL2.

  1. An alternative oxidase monoclonal antibody recognises a highly conserved sequence among alternative oxidase subunits.

    PubMed

    Finnegan, P M; Wooding, A R; Day, D A

    1999-03-19

    The alternative oxidase is found in the inner mitochondrial membranes of plants and some fungi and protists. A monoclonal antibody raised against the alternative oxidase from the aroid lily Sauromatum guttatum has been used extensively to detect the enzyme in these organisms. Using an immunoblotting strategy, the antibody binding site has been localised to the sequence RADEAHHRDVNH within the soybean alternative oxidase 2 protein. Examination of sequence variants showed that A2 and residues C-terminal to H7 are required for recognition by the monoclonal antibody raised against the alternative oxidase. The recognition sequence is highly conserved among all alternative oxidase proteins and is absolutely conserved in 12 of 14 higher plant sequences, suggesting that this antibody will continue to be extremely useful in studying the expression and synthesis of the alternative oxidase.

  2. Prokaryotic origins for the mitochondrial alternative oxidase and plastid terminal oxidase nuclear genes.

    PubMed

    Finnegan, Patrick M; Umbach, Ann L; Wilce, Jackie A

    2003-12-18

    The mitochondrial alternative oxidase is a diiron carboxylate quinol oxidase (Dox) found in plants and some fungi and protists, but not animals. The plastid terminal oxidase is distantly related to alternative oxidase and is most likely also a Dox protein. Database searches revealed that the alpha-proteobacterium Novosphingobium aromaticivorans and the cyanobacteria Nostoc sp. PCC7120, Synechococcus sp. WH8102 and Prochlorococcus marinus subsp. pastoris CCMP1378 each possess a Dox homolog. Each prokaryotic protein conforms to the current structural models of the Dox active site and phylogenetic analyses suggest that the eukaryotic Dox genes arose from an ancestral prokaryotic gene.

  3. Regulation of NADPH oxidases in skeletal muscle.

    PubMed

    Ferreira, Leonardo F; Laitano, Orlando

    2016-09-01

    The only known function of NAD(P)H oxidases is to produce reactive oxygen species (ROS). Skeletal muscles express three isoforms of NAD(P)H oxidases (Nox1, Nox2, and Nox4) that have been identified as critical modulators of redox homeostasis. Nox2 acts as the main source of skeletal muscle ROS during contractions, participates in insulin signaling and glucose transport, and mediates the myocyte response to osmotic stress. Nox2 and Nox4 contribute to skeletal muscle abnormalities elicited by angiotensin II, muscular dystrophy, heart failure, and high fat diet. Our review addresses the expression and regulation of NAD(P)H oxidases with emphasis on aspects that are relevant to skeletal muscle. We also summarize: i) the most widely used NAD(P)H oxidases activity assays and inhibitors, and ii) studies that have defined Nox enzymes as protagonists of skeletal muscle redox homeostasis in a variety of health and disease conditions. PMID:27184955

  4. Abiotic racemization kinetics of amino acids in marine sediments.

    PubMed

    Steen, Andrew D; Jørgensen, Bo Barker; Lomstein, Bente Aa

    2013-01-01

    The ratios of d- versus l-amino acids can be used to infer the sources and composition of sedimentary organic matter. Such inferences, however, rely on knowing the rates at which amino acids in sedimentary organic matter racemize abiotically between the d- and the l-forms. Based on a heating experiment, we report kinetic parameters for racemization of aspartic acid, glutamic acid, serine, and alanine in bulk sediment from Aarhus Bay, Denmark, taken from the surface, 30 cm, and 340 cm depth below seafloor. Extrapolation to a typical cold deep sea sediment temperature of 3°C suggests racemization rate constants of 0.50×10(-5)-11×10(-5) yr(-1). These results can be used in conjunction with measurements of sediment age to predict the ratio of d:l amino acids due solely to abiotic racemization of the source material, deviations from which can indicate the abundance and turnover of active microbial populations.

  5. Involvement of alternative oxidase in the regulation of growth, development, and resistance to oxidative stress of Sclerotinia sclerotiorum.

    PubMed

    Xu, Ting; Yao, Fei; Liang, Wu-Sheng; Li, Yong-Hong; Li, Dian-Rong; Wang, Hao; Wang, Zheng-Yi

    2012-08-01

    Sclerotinia sclerotiorum is a cosmopolitan, filamentous, fungal pathogen that can cause serious disease in many kinds of crops. Alternative oxidase is the terminal oxidase of the alternative mitochondrial respiratory pathway in fungi and higher plants. We report the presence of this alternative pathway respiration and demonstrate its expression in two isolates of S. sclerotiorum under unstressed, normal culture conditions. Application of salicylhydroxamic acid, a specific inhibitor of alternative oxidase, severely inhibited the mycelial growth of S. sclerotiorum both on potato dextrose agar plates and in liquid culture media. Inhibition of alternative oxidase could influence the growth pattern of S. sclerotiorum, as salicylhydroxamic acid treatment induced obvious aerial mycelia growing on potato dextrose agar plates. Under the treatment with salicylhydroxamic acid, S. sclerotiorum formed sclerotia much more slowly than the control. Treatment with hydrogen peroxide in millimolar concentrations greatly decreased the growth rate of mycelia and delayed the formation of sclerotia in both tested S. sclerotiorum isolates. As well, this treatment obviously increased their alternative pathway respiration and the levels of both mRNA and protein of the alternative oxidase. These results indicate that alternative oxidase is involved in the regulation of growth, development, and resistance to oxidative stress of S. sclerotiorum. PMID:22923107

  6. Heme/copper terminal oxidases

    SciTech Connect

    Ferguson-Miller, S.; Babcock, G.T.

    1996-11-01

    Spatially well-organized electron-transfer reactions in a series of membrane-bound redox proteins form the basis for energy conservation in both photosynthesis and respiration. The membrane-bound nature of the electron-transfer processes is critical, as the free energy made available in exergonic redox chemistry is used to generate transmembrane proton concentration and electrostatic potential gradients. These gradients are subsequently used to drive ATP formation, which provides the immediate energy source for constructive cellular processes. The terminal heme/copper oxidases in respiratory electron-transfer chains illustrate a number of the thermodynamic and structural principles that have driven the development of respiration. This class of enzyme reduces dioxygen to water, thus clearing the respiratory system of low-energy electrons so that sustained electron transfer and free-energy transduction can occur. By using dioxygen as the oxidizing substrate, free-energy production per electron through the chain is substantial, owing to the high reduction potential of O{sub 2} (0.815 V at pH 7). 122 refs.

  7. Effects of Biota orientalis extract and its flavonoid constituents, quercetin and rutin on serum uric acid levels in oxonate-induced mice and xanthine dehydrogenase and xanthine oxidase activities in mouse liver.

    PubMed

    Zhu, Ji Xiao; Wang, Ying; Kong, Ling Dong; Yang, Cheng; Zhang, Xin

    2004-07-01

    The hypouricemic actions of Biota orientalis (BO) extract and its flavonoid constituents quercetin and rutin, were in vivo examined using oxonate-induced hyperuricemic mice. Quercetin and rutin, when administered three times orally to the oxonate-induced hyperuricemic mice, were able to elicit dose-dependent hypouricemic effects. The effects of quercetin and rutin were more potent than that of Biota orientalis extract at the same dose of 100 mg/kg. At doses of 50 mg/kg of quercetin or above, or at doses of 100 mg/kg of rutin or above, the serum urate levels of the oxonate-pretreated mice were not different from normal mice. In addition, Biota orientalis extract, quercetin and rutin, when tested in vivo on mouse liver homogenates, elicited significant inhibitory actions on the xanthine dehydrogenase/xanthine oxidase (XDH/XO) activities. The effects of quercetin and rutin resulted less potent than that of allopurinol. However, intraperitoneal administration at the same scheme did not produce any observable hypouricemic effect. These hypouricemic effects are partly due to the inhibition of XDH/XO activities in mouse liver. The pharmacological profile of the flavonoids is partly different from that of allopurinol. Such hypouricemic action and inhibition of the enzyme activity of quercetin and rutin may be responsible for a part of the beneficial effects of Biota orientalis extract on hyperuricemia and gout. The effects of quercetin and rutin on serum urate levels in hyperuricemic mice induced by oxonate and the inhibition of enzyme activities in mouse liver are discussed in relation to their absorption and metabolism, and their potential application to treat gout and hyperuricemia.

  8. Childhood encephalomyopathy with cytochrome c oxidase deficiency, ataxia, muscle wasting, and mental impairment.

    PubMed

    Angelini, C; Bresolin, N; Pegolo, G; Bet, L; Rinaldo, P; Trevisan, C; Vergani, L

    1986-08-01

    The son of third cousins was normal until age 2 when he had difficulty walking. At age 8 there was limb weakness, ataxia, loss of tendon reflexes, dislalia, and he was mildly retarded. During fasting, urinary organic acid excretion was abnormally high. Cytochrome c oxidase activity in muscle was 7% of the normal mean. The enzyme in platelets was 16% of controls with a decreased cytochrome aa3 peak. These data suggest an autosomal recessive transmission of this variant of cytochrome c oxidase deficiency.

  9. Effects of ascorbic acid on carcinogenicity and acute toxicity of nickel subsulfide, and on tumor transplants growth in gulonolactone oxidase knock-out mice and wild-type C57BL mice

    SciTech Connect

    Kasprzak, Kazimierz S.; Diwan, Bhalchandra A.; Kaczmarek, Monika Z.; Logsdon, Daniel L.; Fivash, Mathew J.; Salnikow, Konstantin

    2011-11-15

    The aim of this study was to test a hypothesis that ascorbate depletion could enhance carcinogenicity and acute toxicity of nickel. Homozygous L-gulono- < gamma > -lactone oxidase gene knock-out mice (Gulo-/- mice) unable to produce ascorbate and wild-type C57BL mice (WT mice) were injected intramuscularly with carcinogenic nickel subsulfide (Ni{sub 3}S{sub 2}), and observed for the development of injection site tumors for 57 weeks. Small pieces of one of the induced tumors were transplanted subcutaneously into separate groups of Gulo-/- and WT mice and the growth of these tumors was measured for up to 3 months. The two strains of mice differed significantly with regard to (1) Ni{sub 3}S{sub 2} carcinogenesis: Gulo-/- mice were 40% more susceptible than WT mice; and (2) transplanted tumors development: Gulo-/- mice were more receptive to tumor growth than WT mice, but only in terms of a much shorter tumor latency; later in the exponential phase of growth, the growth rates were the same. And, with adequate ascorbate supplementation, the two strains were equally susceptible to acute toxicity of Ni{sub 3}S{sub 2}. Statistically significant effects of dietary ascorbate dosing levels were the following: (1) reduction in ascorbate supplementation increased acute toxicity of Ni{sub 3}S{sub 2} in Gulo-/- mice; (2) ascorbate supplementation extended the latency of transplanted tumors in WT mice. In conclusion, the lack of endogenous ascorbate synthesis makes Gulo-/- mice more susceptible to Ni{sub 3}S{sub 2} carcinogenesis. Dietary ascorbate tends to attenuate acute toxicity of Ni{sub 3}S{sub 2} and to extend the latency of transplanted tumors. The latter effects may be of practical importance to humans and thus deserve further studies. -- Highlights: Black-Right-Pointing-Pointer Ascorbate depletion enhances carcinogenicity and acute toxicity of nickel. Black-Right-Pointing-Pointer Gulo-/- mice unable to synthesize ascorbate were used in this study. Black

  10. Plant sulfite oxidase as novel producer of H2O2: combination of enzyme catalysis with a subsequent non-enzymatic reaction step.

    PubMed

    Hänsch, Robert; Lang, Christina; Riebeseel, Erik; Lindigkeit, Rainer; Gessler, Arthur; Rennenberg, Heinz; Mendel, Ralf R

    2006-03-10

    Sulfite oxidase (EC 1.8.3.1) from the plant Arabidopsis thaliana is the smallest eukaryotic molybdenum enzyme consisting of a molybdenum cofactor-binding domain but lacking the heme domain that is known from vertebrate sulfite oxidase. While vertebrate sulfite oxidase is a mitochondrial enzyme with cytochrome c as the physiological electron acceptor, plant sulfite oxidase is localized in peroxisomes and does not react with cytochrome c. Here we describe results that identified oxygen as the terminal electron acceptor for plant sulfite oxidase and hydrogen peroxide as the product of this reaction in addition to sulfate. The latter finding might explain the peroxisomal localization of plant sulfite oxidase. 18O labeling experiments and the use of catalase provided evidence that plant sulfite oxidase combines its catalytic reaction with a subsequent non-enzymatic step where its reaction product hydrogen peroxide oxidizes another molecule of sulfite. In vitro, for each catalytic cycle plant SO will bring about the oxidation of two molecules of sulfite by one molecule of oxygen. In the plant, sulfite oxidase could be responsible for removing sulfite as a toxic metabolite, which might represent a means to protect the cell against excess of sulfite derived from SO2 gas in the atmosphere (acid rain) or during the decomposition of sulfur-containing amino acids. Finally we present a model for the metabolic interaction between sulfite and catalase in the peroxisome.

  11. Rat pristanoyl-CoA oxidase. cDNA cloning and recognition of its C-terminal (SQL) by the peroxisomal-targeting signal 1 receptor.

    PubMed

    Vanhooren, J C; Fransen, M; de Béthune, B; Baumgart, E; Baes, M; Torrekens, S; Van Leuven, F; Mannaerts, G P; Van Veldhoven, P P

    1996-07-15

    The composite pristanoyl-CoA oxidase cDNA sequence, derived from two overlapping clones from a rat liver cDNA library and a 5'-RACE (rapid amplification of cDNA ends) PCR fragment, consisted of 2600 bases and contained an open reading frame of 2100 bases, encoding a protein of 700 amino acids with a calculated molecular mass of 78445 Da. This value is somewhat larger than the reported molecular mass of 70 kDa as determined earlier by SDS-gel electrophoresis. The amino acid identity with rat palmitoyl-CoA oxidase was rather low (28%) and barely higher than that with the yeast acyl-CoA oxidases (20%), suggesting that the palmitoyl-CoA oxidase/pristanoyl-CoA oxidase duplication occurred early in evolution. The carboxy-terminal tripeptide of pristanoyl-CoA oxidase was SQL. In vitro studies with the bacterially expressed human peroxisomal-targeting signal-1 import receptor indicated that SQL functions as a peroxisome-targeting signal. Northern analysis of tissues from control and clofibrate treated rats demonstrated that the pristanoyl-CoA oxidase gene is transcribed in liver and extrahepatic tissues and that transcription is not enhanced by treatment of rats with peroxisome proliferators. No mRNA could be detected by northern analysis of human tissues, suggesting that the human pristanoyl-CoA oxidase gene, if present, is only poorly or not transcribed.

  12. Identification of crypto- and neochlorogenic lactones as potent xanthine oxidase inhibitors in roasted coffee beans.

    PubMed

    Honda, Sari; Miura, Yukari; Masuda, Akiko; Masuda, Toshiya

    2014-01-01

    Xanthine oxidase (XO) inhibitory activity has been found in boiling water extracts from roasted coffee beans. Therefore, assay-guided purification of the extracts was performed using size-exclusion column chromatography, and subsequently with reversed phase HPLC to afford lactone derivatives of chlorogenic acids. Among the tested lactones, crypto- and neochlorogenic lactones showed potent XO inhibitory activities compared with three major chlorogenic acids found in coffee beans. These XO inhibitory lactones may ameliorate gout and hyperuricemia in humans who drink coffee.

  13. Role of IAA-Oxidase in Abscission Control in Cotton 123

    PubMed Central

    Schwertner, Harvey A.; Morgan, Page W.

    1966-01-01

    The potential role of indoleactic acid (IAA)-oxidase as an in vivo abscission regulating system in the cotton (Gossypium hirsutum L.) cotyledonary explant was investigated. Phenols (usually monophenols), which are cofactors of cotton IAA-oxidase in vitro, accelerated abscission. Phenols (usually orthodihydroxyphenols), which inhibit cotton IAA-oxidase in vitro, inhibited abscission. Inhibition or stimulation of abscission was accomplished by phenols both with and without IAA. Results were similar when treatments were applied as lanolin pastes to the cut petiole ends or as solutions in which explants were submerged. An abscission accelerating phenol stimulated the decarboxylation of IAA-1-14C by explants and an abscission inhibiting phenol inhibited the decarboxylation of IAA-1-14C. The mechanism of abscission regulation by the phenolic compounds was concluded to involve auxin destruction via IAA-oxidase. In addition to the direct relationship of this study to abscission, the results support the more general hypothesis that IAA-oxidase acts in vivo to regulate auxin levels. PMID:16656432

  14. Amyloid-β peptide binds to cytochrome C oxidase subunit 1.

    PubMed

    Hernandez-Zimbron, Luis Fernando; Luna-Muñoz, Jose; Mena, Raul; Vazquez-Ramirez, Ricardo; Kubli-Garfias, Carlos; Cribbs, David H; Manoutcharian, Karen; Gevorkian, Goar

    2012-01-01

    Extracellular and intraneuronal accumulation of amyloid-beta aggregates has been demonstrated to be involved in the pathogenesis of Alzheimer's disease (AD). However, the precise mechanism of amyloid-beta neurotoxicity is not completely understood. Previous studies suggest that binding of amyloid-beta to a number of macromolecules has deleterious effects on cellular functions. Mitochondria were found to be the target for amyloid-beta, and mitochondrial dysfunction is well documented in AD. In the present study we have shown for the first time that Aβ 1-42 bound to a peptide comprising the amino-terminal region of cytochrome c oxidase subunit 1. Phage clone, selected after screening of a human brain cDNA library expressed on M13 phage and bearing a 61 amino acid fragment of cytochrome c oxidase subunit 1, bound to Aβ 1-42 in ELISA as well as to Aβ aggregates present in AD brain. Aβ 1-42 and cytochrome c oxidase subunit 1 co-immunoprecipitated from mitochondrial fraction of differentiated human neuroblastoma cells. Likewise, molecular dynamics simulation of the cytochrome c oxidase subunit 1 and the Aβ 1-42 peptide complex resulted in a reliable helix-helix interaction, supporting the experimental results. The interaction between Aβ 1-42 and cytochrome c oxidase subunit 1 may explain, in part, the diminished enzymatic activity of respiratory chain complex IV and subsequent neuronal metabolic dysfunction observed in AD.

  15. Fibromodulin Interacts with Collagen Cross-linking Sites and Activates Lysyl Oxidase*

    PubMed Central

    Bihan, Dominique; Bonna, Arkadiusz; Rubin, Kristofer; Farndale, Richard W.

    2016-01-01

    The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites. PMID:26893379

  16. Fibromodulin Interacts with Collagen Cross-linking Sites and Activates Lysyl Oxidase.

    PubMed

    Kalamajski, Sebastian; Bihan, Dominique; Bonna, Arkadiusz; Rubin, Kristofer; Farndale, Richard W

    2016-04-01

    The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites.

  17. Amyloid-β Peptide Binds to Cytochrome C Oxidase Subunit 1

    PubMed Central

    Hernandez-Zimbron, Luis Fernando; Luna-Muñoz, Jose; Mena, Raul; Vazquez-Ramirez, Ricardo; Kubli-Garfias, Carlos; Cribbs, David H.; Manoutcharian, Karen; Gevorkian, Goar

    2012-01-01

    Extracellular and intraneuronal accumulation of amyloid-beta aggregates has been demonstrated to be involved in the pathogenesis of Alzheimer's disease (AD). However, the precise mechanism of amyloid-beta neurotoxicity is not completely understood. Previous studies suggest that binding of amyloid-beta to a number of macromolecules has deleterious effects on cellular functions. Mitochondria were found to be the target for amyloid-beta, and mitochondrial dysfunction is well documented in AD. In the present study we have shown for the first time that Aβ 1–42 bound to a peptide comprising the amino-terminal region of cytochrome c oxidase subunit 1. Phage clone, selected after screening of a human brain cDNA library expressed on M13 phage and bearing a 61 amino acid fragment of cytochrome c oxidase subunit 1, bound to Aβ 1–42 in ELISA as well as to Aβ aggregates present in AD brain. Aβ 1–42 and cytochrome c oxidase subunit 1 co-immunoprecipitated from mitochondrial fraction of differentiated human neuroblastoma cells. Likewise, molecular dynamics simulation of the cytochrome c oxidase subunit 1 and the Aβ 1–42 peptide complex resulted in a reliable helix-helix interaction, supporting the experimental results. The interaction between Aβ 1–42 and cytochrome c oxidase subunit 1 may explain, in part, the diminished enzymatic activity of respiratory chain complex IV and subsequent neuronal metabolic dysfunction observed in AD. PMID:22927926

  18. Hydrogen peroxide generation by monoamine oxidases in rat white adipocytes: role on cAMP production.

    PubMed

    Raimondi, L; Banchelli, G; Sgromo, L; Pirisino, R; Ner, M; Parini, A; Cambon, C

    2000-05-01

    In rat, white adipocytes monoamine oxidases (EC 1.4.3.4.) generate hydrogen peroxide (H(2)O(2)). Recent studies suggested that, in addition to its toxic features, H(2)O(2) may behave as a cell second messenger. In the present study, using fluorimetric and chemiluminescence (CL) assays, we showed that tyramine degradation by monoamine oxidases in intact adipocytes resulted in the concentration-dependent generation of H(2)O(2). In addition, we found that, in the presence of low tyramine concentrations, forskolin-dependent cAMP production was significantly increased as compared to that of the control and this increase was prevented by the monoamine oxidase inhibitor pargyline or by the H(2)O(2) trapping system homovanillic acid-peroxidase. Finally, we demonstrated that tyramine degradation by monoamine oxidases increased the ability of isoproterenol to induce cell lipolysis. Taken together, these data suggest that H(2)O(2) produced during substrate degradation by monoamine oxidases may participate in the regulation of adipocyte metabolism.

  19. Novel deletion in a patient with an isolated peroxisoml acyl-CoA oxidase deficiency

    SciTech Connect

    Poll-The, B.T.; Fournier, B.; Clevers, H.; Wanders, R.J.A.

    1994-09-01

    Disorders with defective peroxisome assembly are associated with multiple peroxisomal enzymatic abnormalities. Besides these diseases patients have been described suspected of having a single enzyme defect in the peroxisomal {beta}-oxidation pathway. Laboratory findings for these patients include elevated plasma very long chain fatty acids (VLCFA) and impaired VLCFA oxidation in fibroblasts. Complementation analysis between these patients and those with a proven single enzyme deficiency, using peroxisomal {beta}-oxidation of VLCFA as the criterion for complementation, has been used to show whether the patients are deficient in acyl-CoA oxidase, peroxisomal trifunctional protein or thiolase activity. Fibroblasts from a patient showing the clinical and biochemical abnormalities of isolated acyl-CoA oxidase deficiency (using cell complementation) were analyzed at the molecular level. Isolation of RNA from patient`s fibroblasts was followed by random reverse transcription of RNA and PCR amplification. PCR products were blotted and hybridized with the human acyl-CoA oxidase cDNA. A fragment 150 bp shorter than normal was found. Upon sequencing, exon 7 was found to be deleted leading to a frameshift in the acyl-CoA oxidase mRNA. Southern blot analysis of the patient`s DNA did not reveal any deletion in contrast to two siblings previously reported as having a deletion of at least 17 kb in the acyl-CoA oxidase gene.

  20. 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.

  1. 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.

  2. Chemical Evidence for Potent Xanthine Oxidase Inhibitory Activity of Ethyl Acetate Extract of Citrus aurantium L. Dried Immature Fruits.

    PubMed

    Liu, Kun; Wang, Wei; Guo, Bing-Hua; Gao, Hua; Liu, Yang; Liu, Xiao-Hong; Yao, Hui-Li; Cheng, Kun

    2016-03-02

    Xanthine oxidase is a key enzyme which can catalyze hypoxanthine and xanthine to uric acid causing hyperuricemia in humans. Xanthine oxidase inhibitory activities of 24 organic extracts of four species belonging to Citrus genus of the family Rutaceae were assayed in vitro. Since the ethyl acetate extract of C. aurantium dried immature fruits showed the highest xanthine oxidase inhibitory activity, chemical evidence for the potent inhibitory activity was clarified on the basis of structure identification of the active constituents. Five flavanones and two polymethoxyflavones were isolated and evaluated for inhibitory activity against xanthine oxidase in vitro. Of the compounds, hesperetin showed more potent inhibitory activity with an IC50 value of 16.48 μM. For the first time, this study provides a rational basis for the use of C. aurantium dried immature fruits against hyperuricemia.

  3. NADPH oxidases: new actors in thyroid cancer?

    PubMed

    Ameziane-El-Hassani, Rabii; Schlumberger, Martin; Dupuy, Corinne

    2016-08-01

    Hydrogen peroxide (H2O2) is a crucial substrate for thyroid peroxidase, a key enzyme involved in thyroid hormone synthesis. However, as a potent oxidant, H2O2 might also be responsible for the high level of oxidative DNA damage observed in thyroid tissues, such as DNA base lesions and strand breakages, which promote chromosomal instability and contribute to the development of tumours. Although the role of H2O2 in thyroid hormone synthesis is well established, its precise mechanisms of action in pathological processes are still under investigation. The NADPH oxidase/dual oxidase family are the only oxidoreductases whose primary function is to produce reactive oxygen species. As such, the function and expression of these enzymes are tightly regulated. Thyrocytes express dual oxidase 2, which produces most of the H2O2 for thyroid hormone synthesis. Thyrocytes also express dual oxidase 1 and NADPH oxidase 4, but the roles of these enzymes are still unknown. Here, we review the structure, expression, localization and function of these enzymes. We focus on their potential role in thyroid cancer, which is characterized by increased expression of these enzymes. PMID:27174022

  4. Mitochondrial targeting of human protoporphyrinogen oxidase.

    PubMed

    Davids, Lester M; Corrigall, Anne V; Meissner, Peter N

    2006-05-01

    Variegate porphyria is an autosomal dominant disorder of heme metabolism resulting from a deficiency in protoporphyrinogen oxidase, an enzyme located on the inner mitochondrial membrane. This study examined the effect of three South African VP-causing mutations (H20P, R59W, R168C) on mitochondrial targeting. Only H20P did not target, and of eight protoporphyrinogen oxidase-GFP chimeric fusion proteins created, N-terminal residues 1-17 were found to be the minimal protoporphyrinogen oxidase sequence required for efficient mitochondrial targeting. Removal of this N-terminal sequence displayed mitochondrial localization, suggesting internal mitochondrial targeting signals. In addition, six constructs were engineered to assess the effect of charge and helicity on mitochondrial targeting of the protein. Of those engineered, only the PPOX20/H20P-GFP construct abolished mitochondrial targeting, presumably through disruption of the protoporphyrinogen oxidase alpha-helix. Based on our results we propose a mechanism for protoporphyrinogen oxidase targeting to the mitochondrion.

  5. Immunoblot analyses of the elicited Sanguinaria canadensis enzyme, dihydrobenzophenanthridine oxidase: evidence for resolution from a polyphenol oxidase isozyme.

    PubMed

    Ignatov, A; Neuman, M C; Barg, R; Krueger, R J; Coscia, C J

    1997-11-15

    In our initial purification of dihydrobenzophenanthridine oxidase from Sanguinaria canadensis plant cell cultures, we reported that our most purified preparations contained a major band at 77 kDa and minor lower Mr bands. Here we present evidence on highly purified dihydrobenzophenanthridine oxidase from elicited S. canadensis cultures to indicate that this enzyme is the 77-kDa protein and that lower Mr bands include an isozyme(s) of the polyphenol oxidase family that copurifies with it. An antibody raised against the 77-kDa protein and an anti-polyphenol oxidase antibody that recognizes a 70-kDa band were used to monitor chromatographic fractions by immunoblot analysis of the oxidases. Oxidase-containing eluates from DEAE-Sephadex, CM, and HiTrap blue were compared to corresponding flow-through fractions. Bands at 77 and 88 kDa were detected with anti-dihydrobenzophenanthridine oxidase antibody in eluates displaying high dihydrobenzophenanthridine oxidase activity. Polyphenol oxidase specific activity and immunoreactivity partitioned both in flow-through and eluate fractions of the CM and HiTrap columns. Estimation of the dihydrobenzophenanthridine oxidase and polyphenol oxidase specific activities for each step showed increasing enrichment of alkaloidal enzyme accompanied by variable dihydrobenzophenanthridine oxidase/polyphenol oxidase activity ratios. Taken together these observations indicate that the dihydrobenzophenanthridine and polyphenol oxidases have Mr values of 77 and 70 kDa, respectively, and the two enzymes are different entities.

  6. ALTERNATIVE OXIDASE: From Gene to Function.

    PubMed

    Vanlerberghe, Greg C.; McIntosh, Lee

    1997-06-01

    Plants, some fungi, and protists contain a cyanide-resistant, alternative mitochondrial respiratory pathway. This pathway branches at the ubiquinone pool and consists of an alternative oxidase encoded by the nuclear gene Aox1. Alternative pathway respiration is only linked to proton translocation at Complex 1 (NADH dehydrogenase). Alternative oxidase expression is influenced by stress stimuli-cold, oxidative stress, pathogen attack-and by factors constricting electron flow through the cytochrome pathway of respiration. Control is exerted at the levels of gene expression and in response to the availability of carbon and reducing potential. Posttranslational control involves reversible covalent modification of the alternative oxidase and activation by specific carbon metabolites. This dynamic system of coarse and fine control may function to balance upstream respiratory carbon metabolism and downstream electron transport when these coupled processes become imbalanced as a result of changes in the supply of, or demand for, carbon, reducing power, and ATP.

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

    SciTech Connect

    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-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 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 figs.

  8. Titanium Dioxide Nanoparticles Increase Superoxide Anion Production by Acting on NADPH Oxidase

    PubMed Central

    Trepout, Sylvain; Wien, Frank; Marco, Sergio

    2015-01-01

    Titanium dioxide (TiO2) anatase nanoparticles (NPs) are metal oxide NPs commercialized for several uses of everyday life. However their toxicity has been poorly investigated. Cellular internalization of NPs has been shown to activate macrophages and neutrophils that contribute to superoxide anion production by the NADPH oxidase complex. Transmission electron micrososcopy images showed that the membrane fractions were close to the NPs while fluorescence indicated an interaction between NPs and cytosolic proteins. Using a cell-free system, we have investigated the influence of TiO2 NPs on the behavior of the NADPH oxidase. In the absence of the classical activator molecules of the enzyme (arachidonic acid) but in the presence of TiO2 NPs, no production of superoxide ions could be detected indicating that TiO2 NPs were unable to activate by themselves the complex. However once the NADPH oxidase was activated (i.e., by arachidonic acid), the rate of superoxide anion production went up to 140% of its value without NPs, this effect being dependent on their concentration. In the presence of TiO2 nanoparticles, the NADPH oxidase produces more superoxide ions, hence induces higher oxidative stress. This hyper-activation and the subsequent increase in ROS production by TiO2 NPs could participate to the oxidative stress development. PMID:26714308

  9. Titanium Dioxide Nanoparticles Increase Superoxide Anion Production by Acting on NADPH Oxidase.

    PubMed

    Masoud, Rawand; Bizouarn, Tania; Trepout, Sylvain; Wien, Frank; Baciou, Laura; Marco, Sergio; Houée Levin, Chantal

    2015-01-01

    Titanium dioxide (TiO2) anatase nanoparticles (NPs) are metal oxide NPs commercialized for several uses of everyday life. However their toxicity has been poorly investigated. Cellular internalization of NPs has been shown to activate macrophages and neutrophils that contribute to superoxide anion production by the NADPH oxidase complex. Transmission electron micrososcopy images showed that the membrane fractions were close to the NPs while fluorescence indicated an interaction between NPs and cytosolic proteins. Using a cell-free system, we have investigated the influence of TiO2 NPs on the behavior of the NADPH oxidase. In the absence of the classical activator molecules of the enzyme (arachidonic acid) but in the presence of TiO2 NPs, no production of superoxide ions could be detected indicating that TiO2 NPs were unable to activate by themselves the complex. However once the NADPH oxidase was activated (i.e., by arachidonic acid), the rate of superoxide anion production went up to 140% of its value without NPs, this effect being dependent on their concentration. In the presence of TiO2 nanoparticles, the NADPH oxidase produces more superoxide ions, hence induces higher oxidative stress. This hyper-activation and the subsequent increase in ROS production by TiO2 NPs could participate to the oxidative stress development. PMID:26714308

  10. Alternative oxidase in animals: unique characteristics and taxonomic distribution.

    PubMed

    McDonald, Allison E; Vanlerberghe, Greg C; Staples, James F

    2009-08-01

    Alternative oxidase (AOX), a ubiquinol oxidase, introduces a branch point into the respiratory electron transport chain, bypassing complexes III and IV and resulting in cyanide-resistant respiration. Previously, AOX was thought to be limited to plants and some fungi and protists but recent work has demonstrated the presence of AOX in most kingdoms of life, including animals. In the present study we identified AOX in 28 animal species representing nine phyla. This expands the known taxonomic distribution of AOX in animals by 10 species and two phyla. Using bioinformatics we found AOX gene sequences in members of the animal phyla Porifera, Placozoa, Cnidaria, Mollusca, Annelida, Nematoda, Echinodermata, Hemichordata and Chordata. Using reverse-transcriptase polymerase chain reaction (RT-PCR) with degenerate primers designed to recognize conserved regions of animal AOX, we demonstrated that AOX genes are transcribed in several animals from different phyla. An analysis of full-length AOX sequences revealed an amino acid motif in the C-terminal region of the protein that is unique to animal AOXs. Animal AOX also lacks an N-terminal cysteine residue that is known to be important for AOX enzyme regulation in plants. We conclude that the presence of AOX is the ancestral state in animals and hypothesize that its absence in some lineages, including vertebrates, is due to gene loss events. PMID:19648408

  11. Purification and biochemical characterization of pyruvate oxidase from Lactobacillus plantarum.

    PubMed

    Sedewitz, B; Schleifer, K H; Götz, F

    1984-10-01

    Pyruvate oxidase (EC 1.2.3.3) was isolated and characterized from Lactobacillus plantarum. The enzyme catalyzes the oxidative decarboxylation of pyruvate in the presence of phosphate and oxygen, yielding acetyl phosphate, carbon dioxide, and hydrogen peroxide. This pyruvate oxidase is a flavoprotein, with the relatively tightly bound cofactors flavin adenine dinucleotide, thiamine pyrophosphate, and a divalent metal ion, with Mn2+ being the most effective. The enzyme is only slightly inhibited by EDTA, implying that the enzyme-bound metal ion is poorly accessible to EDTA. Only under relatively drastic conditions, such as acid ammonium sulfate precipitation, could a colorless and entirely inactive apoenzyme be obtained. A partial reactivation of the enzyme was only possible by the combined addition of flavin adenine dinucleotide, thiamine pyrophosphate, and MnSO4. The enzyme has a molecular weight of ca. 260,000 and consists of four subunits with apparently identical molecular weights of 68,000. For catalytic activity the optimum pH is 5.7, and the optimum temperature is 30 degrees C. The Km values for pyruvate, phosphate, and arsenate are 0.4, 2.3, and 1.2 mM, respectively. The substrate specificity revealed that the enzyme reacts also with certain aldehydes and that phosphate can be replaced by arsenate. In addition to oxygen, several artificial compounds can function as electron acceptors.

  12. Iron regulates xanthine oxidase activity in the lung.

    PubMed

    Ghio, Andrew J; Kennedy, Thomas P; Stonehuerner, Jacqueline; Carter, Jacqueline D; Skinner, Kelly A; Parks, Dale A; Hoidal, John R

    2002-09-01

    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 oxidoreductase activity in cultured V79 cells was increased with exposure to ferric ammonium sulfate and inhibited by deferoxamine. Lung XO and total xanthine oxidoreductase activities were reduced in rats fed an iron-depleted diet and increased in rats supplemented with iron, without change in the ratio of XO to total oxidoreductase. Intratracheal injection of an iron salt or silica-iron, but not aluminum salts or silica-zinc, significantly increased rat lung XO and total xanthine oxidoreductase activities, immunoreactive xanthine oxidoreductase, and the concentration of urate in bronchoalveolar fluid. These results suggest the possibility that the production of uric acid, a major chelator of iron in extracellular fluid, is directly influenced by iron-mediated regulation of the expression and/or activity of its enzymatic source, xanthine oxidase.

  13. Alternative oxidase in animals: unique characteristics and taxonomic distribution.

    PubMed

    McDonald, Allison E; Vanlerberghe, Greg C; Staples, James F

    2009-08-01

    Alternative oxidase (AOX), a ubiquinol oxidase, introduces a branch point into the respiratory electron transport chain, bypassing complexes III and IV and resulting in cyanide-resistant respiration. Previously, AOX was thought to be limited to plants and some fungi and protists but recent work has demonstrated the presence of AOX in most kingdoms of life, including animals. In the present study we identified AOX in 28 animal species representing nine phyla. This expands the known taxonomic distribution of AOX in animals by 10 species and two phyla. Using bioinformatics we found AOX gene sequences in members of the animal phyla Porifera, Placozoa, Cnidaria, Mollusca, Annelida, Nematoda, Echinodermata, Hemichordata and Chordata. Using reverse-transcriptase polymerase chain reaction (RT-PCR) with degenerate primers designed to recognize conserved regions of animal AOX, we demonstrated that AOX genes are transcribed in several animals from different phyla. An analysis of full-length AOX sequences revealed an amino acid motif in the C-terminal region of the protein that is unique to animal AOXs. Animal AOX also lacks an N-terminal cysteine residue that is known to be important for AOX enzyme regulation in plants. We conclude that the presence of AOX is the ancestral state in animals and hypothesize that its absence in some lineages, including vertebrates, is due to gene loss events.

  14. Copper-dependent inhibition of cytochrome c oxidase by Abeta(1-42) requires reduced methionine at residue 35 of the Abeta peptide.

    PubMed

    Crouch, Peter J; Barnham, Kevin J; Duce, James A; Blake, Rachel E; Masters, Colin L; Trounce, Ian A

    2006-10-01

    By altering key amino acid residues of the Alzheimer's disease-associated amyloid-beta peptide, we investigated the mechanism through which amyloid-beta inhibits cytochrome c oxidase (EC 1.9.3.1). Native amyloid-beta inhibited cytochrome oxidase by up to 65%, and the level of inhibition was determined by the period of amyloid-beta ageing before the cytochrome oxidase assay. Substituting tyrosine-10 with alanine did not affect maximal enzyme inhibition, but the altered peptide required a longer period of ageing. By contrast, oxidizing the sulfur of methionine-35 to a sulfoxide, or substituting methionine-35 with valine, completely abrogated the peptide's inhibitory potential towards cytochrome oxidase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the loss of inhibitory potential towards cytochrome oxidase with the methionine-35-altered peptides did not correlate with a substantially different distribution of amyloid-beta oligomeric species. Although the amyloid-beta-mediated inhibition of cytochrome oxidase was completely dependent on the presence of divalent Cu2+, it was not supported by monovalent Cu+, and experiments with catalase and H2O2 indicated that the mechanism of cytochrome oxidase inhibition does not involve amyloid-beta-mediated H2O2 production. We propose that amyloid-beta-mediated inhibition of cytochrome oxidase is dependent on the peptide's capacity to bind, then reduce Cu2+, and that it may involve the formation of a redox active amyloid-beta-methionine radical. PMID:16987248

  15. Forage polyphenol oxidase and ruminant livestock nutrition

    PubMed Central

    Lee, Michael R. F.

    2014-01-01

    Polyphenol oxidase (PPO) is predominately associated with the detrimental effect of browning fruit and vegetables, however, interest within PPO containing forage crops (crops to be fed to animals) has grown since the browning reaction was associated with reduced nitrogen (N) losses in silo and the rumen. The reduction in protein breakdown in silo of red clover (high PPO forage) increased the quality of protein, improving N-use efficiency [feed N into product N (e.g., Milk): NUE] when fed to ruminants. A further benefit of red clover silage feeding is a significant reduction in lipolysis (cleaving of glycerol-based lipid) in silo and an increase in the deposition of beneficial C18 polyunsaturated fatty acid (PUFA) in animal products, which has also been linked to PPO activity. PPOs protection of plant protein and glycerol based-PUFA in silo is related to the deactivation of plant proteases and lipases. This deactivation occurs through PPO catalyzing the conversion of diphenols to quinones which bind with cellular nucleophiles such as protein reforming a protein-bound phenol (PBP). If the protein is an enzyme (e.g., protease or lipase) the complexing denatures the enzyme. However, PPO is inactive in the anaerobic rumen and therefore any subsequent protection of plant protein and glycerol based-PUFA in the rumen must be as a result of events that occurred to the forage pre-ingestion. Reduced activity of plant proteases and lipases would have little effect on NUE and glycerol based-PUFA in the rumen due to the greater concentration of rumen microbial proteases and lipases. The mechanism for PPOs protection of plant protein in the rumen is a consequence of complexing plant protein, rather than protease deactivation per se. These complexed proteins reduce protein digestibility in the rumen and subsequently increase undegraded dietary protein flow to the small intestine. The mechanism for protecting glycerol-based PUFA has yet to be fully elucidated but may be associated

  16. Isoprenoid metabolism is required for stimulation of the respiratory burst oxidase of HL-60 cells.

    PubMed Central

    Bokoch, G M; Prossnitz, V

    1992-01-01

    The formation of oxygen radicals by phagocytic cells occurs through the activation of a multiple-component NADPH oxidase system. An unidentified low molecular weight GTP-binding protein has been proposed to modulate the activity of the NADPH oxidase. The low molecular weight GTP-binding proteins undergo posttranslational processing, including an initial covalent incorporation of an isoprenyl group. To test whether such an isoprenylation reaction might be required for the activity of the oxidase, we utilized compactin and lovastatin as inhibitors of the isoprenylation pathway. Treatment of DMSO-differentiated HL-60 cells with compactin produced a concentration-dependent inhibition of O2- formation in response to FMLP or phorbol myristate acetate. Cell viability was not affected nor was normal differentiation of the HL-60 cells into a neutrophil-like cell. The inhibitory effect of compactin was specifically prevented by addition of exogenous mevalonic acid to the HL-60 cells, indicating that the inhibitory effects of the drug were due to blockade of the pathway leading to isoprenoid synthesis. Addition of cholesterol, ubiquinone, or dolichol, which are also downstream products of the isoprenoid pathway, did not override the inhibitory effects of the drug. Subcellular fractions were prepared from compactin-treated cells, and the location of the compactin-sensitive factor was determined by complementation analysis in a cell-free NADPH oxidase system. The inhibited factor was localized to the HL-60 cytosol. These data suggest that an isoprenoid pathway intermediate is necessary for activation of the phagocyte NADPH oxidase. This is likely to represent the requirement for an isoprenoid moiety in the posttranslational modification of a low molecular weight GTP-binding protein. Our studies provide support for the involvement of such a low molecular weight GTP-binding protein in NADPH oxidase activation. Images PMID:1310693

  17. [Establishment of double targets of high throughput screening model for xanthine oxidase inhibitors and superoxide anion scavengers].

    PubMed

    Xie, Tao; Qin, Zhi-Zhen; Zhou, Rui; Zhao, Ying; Du, Guan-hua

    2015-04-01

    A double targets of high throughput screening model for xanthine oxidase inhibitors and superoxide anion scavengers was established. In the reaction system of xanthine oxidase, WST-1 works as the probe for the ultra oxygen anion generation, and product uric acid works as xanthine oxidase activity indicator. By using SpectraMax M5 continuous spectrum enzyme sign reflectoscope reflector, the changes of these indicators' concentration were observed and the influence factors of this reaction system to establish the high throughput screening model were studied. And the model is confirmed by positive drugs. In the reaction system, the final volume of reaction system is 50 μL and the concentrations of xanthine oxidase is 4 mU x mL(-1), xanthine 250 μmol x L(-1) and WST-1 100 μmol x L(-1), separately. The Z'-factor of model for xanthine oxidase inhibitors is 0.537 4, S/N is 47.519 9; the Z'-factor of model for superoxide anion scavengers is 0.507 4, S/N is 5.388 9. This model for xanthine oxidase inhibitors and superoxide anion scavengers has more common characteristics of the good stability, the fewer reagent types and quantity, the good repeatability, and so on. And it can be widely applied in high-throughput screening research.

  18. 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

  19. Extracellular oxidases of the lignin-degrading fungus Panus tigrinus.

    PubMed

    Cadimaliev, D A; Revin, V V; Atykyan, N A; Samuilov, V D

    2005-06-01

    Two extracellular oxidases (laccases) were isolated from the extracellular fluid of the fungus Panus (Lentinus) tigrinus cultivated in low-nitrogen medium supplemented with birch sawdust. The enzymes were purified by successive chromatography on columns with TEAE-cellulose and DEAE-Toyopearl 650M. Both oxidases catalyze oxidation of pyrocatechol and ABTS. Moreover, oxidase 1 also catalyzes oxidation of guaiacol, o-phenylenediamine, and syringaldazine. The enzymes have identical pH (7.0) and temperature (60-65 degrees C) optimums. Absorption spectra of the oxidases differ from the spectra of typical "blue" laccases and are similar to the spectrum of yellow oxidase. PMID:16038613

  20. Partial characterization of polyphenol oxidase activity in raspberry fruits.

    PubMed

    González, E M; de Ancos, B; Cano, M P

    1999-10-01

    A partial characterization of polyphenol oxidase (PPO) activity in raspberry fruits is described. Two early cultivars harvested in May/June (Heritage and Autumm Bliss) and two late cultivars harvested in October-November (Ceva and Rubi) were analyzed for PPO activity. Stable and highly active PPO extracts were obtained using insoluble poly(vinylpyrrolidone) (PVP) and Triton X-100 in sodium phosphate, pH 7.0 buffer. Polyacrylamide gel electrophoresis of raspberry extracts under nondenaturing conditions resolved in one band (R(f)()(1) = 0.25). Raspberry PPO activity has pH optima of 8.0 and 5.5, both with catechol (0.1 M). Maximum activity was with D-catechin (catecholase activity), followed by p-coumaric acid (cresolase activity). Heritage raspberry also showed PPO activity toward 4-methylcatechol. Ceva and Autumm Bliss raspberries showed the higher PPO activity using catechol as substrate.

  1. Xanthine oxidase inhibitory activity of Lychnophora species from Brazil ("Arnica").

    PubMed

    Filha, Z S Ferraz; Vitolo, I F; Fietto, L G; Lombardi, J A; Saúde-Guimarães, D A

    2006-08-11

    Twenty-two extracts from five Lychnophora species and one Lychnophoriopsis species, traditionally used in Brazil as analgesic, anti-inflammatory, and to treat bruise and rheumatism were examined for the inhibition of xanthine oxidase (XO), the enzyme that catalyses the metabolism of hypoxanthine and xanthine into uric acid. Sixteen extracts were tested. All of them were found to have excellent XO inhibitory activity, with inhibitions greater than 38% at 100 microg/mL in the assay mixture. The most active plants examined were Lychnophora trichocarpha, Lychnophora ericoides, Lychnophora staavioides and Lychnophoriopsis candelabrum, with inhibitions of 77%, 78%, 66% and 63% at 100 microg/mL, respectively, and IC(50) values of 6.16, 8.28, 33.97 and 37.70 microg/mL, respectively.

  2. The Next Generation MOD: A Microchip Amino Acid Analyzer for Detecting Extraterrestrial Life

    NASA Technical Reports Server (NTRS)

    Mathies, R. A.; Hutt, L. D.; Bada, J. L.; Glavin, D.; Grunthaner, F. J.; Grunthaner, P. J.

    2000-01-01

    The MOD (Mars Organic Detector) instrument which has selected for the definition phase of the BEDS package on the 2005 Mars Explorer Program spacecraft is designed to simply detect the presence of amino acids in Martian surface samples at a sensitivity of a few parts per billion (ppb). An additional important aspect of amino acid analyses of Martian samples is identifying and quantifying which compounds are present, and also distinguishing those produced abiotically from those synthesized by either extinct or extant life. Amino acid homochirality provides an unambiguous way of distinguishing between abiotic vs. biotic origins. Proteins made up of mixed D- and L-amino acids would not likely have been efficient catalysts in early organisms because they could not fold into bioactive configurations such as the a-helix. However, enzymes made up of all D-amino acids function just as well as those made up of only L-amino acids, but the two enzymes use the opposite stereoisomeric substrates. There are no biochemical reasons why L-amino acids would be favored over Damino acids. On Earth, the use of only L-amino acids in proteins by life is probably simply a matter of chance. We assume that if proteins and enzymes were a component of extinct or extant life on Mars, then amino acid homochirality would have been a requirement. However, the possibility that Martian life was (or is) based on D-amino acids would be equal to that based on L-amino acids. The detection of a nonracemic mixture of amino acids in a Martian sample would be strong evidence for the presence of an extinct or extant biota on Mars. The finding of an excess of D-amino acids would provide irrefutable evidence of unique Martian life that could not have been derived from seeding the planet with terrestrial life (or the seeding of the Earth with Martian life). In contrast, the presence of racemic amino acids, along with non-protein amino acids such as alpha-aminoisobutyric acid and isovaline, would be indicative

  3. 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 b558; and (iii) to determine the S100A8 consensus site involved in cytochrome b558/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 b558 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 activation

  4. Isozymes of Ipomoea batatas catechol oxidase differ in catalase-like activity.

    PubMed

    Gerdemann, C; Eicken, C; Magrini, A; Meyer, H E; Rompel, A; Spener, F; Krebs, B

    2001-07-01

    The amino acid sequences of two isozymes of catechol oxidase from sweet potatoes (Ipomoea batatas) were determined by Edman degradation of BrCN cleavage fragments of the native protein and by sequencing of amplified cDNA fragments. Sequence alignment and phylogenetic analysis of plant catechol oxidases revealed about 80% equidistance between the two I. batatas catechol oxidases and approximately 40--60% to catechol oxidases of other plants. When H(2)O(2) was applied as substrate the 39 kDa isozyme, but not the 40 kDa isozyme, showed catalase-like activity. The structure of the 40 kDa isozyme was modeled on the basis of the published crystal structure of the 39 kDa isozyme [T. Klabunde et al., Nat. Struct. Biol. 5 (1998) 1084]. The active site model closely resembled that of the 39 kDa isozyme determined by crystallography, except for a mutation of Thr243 (40 kDa isozyme) to Ile241 (39 kDa isozyme) close to the dimetal center. This residue difference affects the orientation of the Glu238/236 residue, which is thought to be responsible for the catalase-like activity of the 39 kDa isozyme for which a catalytic mechanism is proposed. PMID:11451442

  5. Evaluation of Rhesus Monkey and Guinea Pig Hepatic Cytosol Fractions as Models for Human Aldehyde Oxidase

    PubMed Central

    Choughule, Kanika V.; Barr, John T.

    2013-01-01

    Aldehyde oxidase (AOX) is a cytosolic enzyme expressed across a wide range of species, including guinea pig and rhesus monkey. These species are believed to be the best preclinical models for studying human AOX-mediated metabolism. We compared AOX activity in rhesus monkeys, guinea pigs, and humans using phthalazine and N-[2-(dimethylamino)ethyl]acridone-4-carboxamide (DACA) as substrates and raloxifene as an inhibitor. Michaelis-Menten kinetics was observed for phthalazine oxidation in rhesus monkey, guinea pig, and human liver cytosol, whereas substrate inhibition was seen with DACA oxidase activity in all three livers. Raloxifene inhibited phthalazine and DACA oxidase activity uncompetitively in guinea pig, whereas mixed-mode inhibition was seen in rhesus monkey. Our analysis of the primary sequence alignment of rhesus monkey, guinea pig, and human aldehyde oxidase isoform 1 (AOX1) along with homology modeling has led to the identification of several amino acid residue differences within the active site and substrate entrance channel of AOX1. We speculate that some of these residues might be responsible for the differences observed in activity. Overall, our data indicate that rhesus monkeys and guinea pigs would overestimate intrinsic clearance in humans and would be unsuitable to use as animal models. Our study also showed that AOX metabolism in species is substrate-dependent and no single animal model can be reliably used to predict every drug response in humans. PMID:23918666

  6. Isozymes of Ipomoea batatas catechol oxidase differ in catalase-like activity.

    PubMed

    Gerdemann, C; Eicken, C; Magrini, A; Meyer, H E; Rompel, A; Spener, F; Krebs, B

    2001-07-01

    The amino acid sequences of two isozymes of catechol oxidase from sweet potatoes (Ipomoea batatas) were determined by Edman degradation of BrCN cleavage fragments of the native protein and by sequencing of amplified cDNA fragments. Sequence alignment and phylogenetic analysis of plant catechol oxidases revealed about 80% equidistance between the two I. batatas catechol oxidases and approximately 40--60% to catechol oxidases of other plants. When H(2)O(2) was applied as substrate the 39 kDa isozyme, but not the 40 kDa isozyme, showed catalase-like activity. The structure of the 40 kDa isozyme was modeled on the basis of the published crystal structure of the 39 kDa isozyme [T. Klabunde et al., Nat. Struct. Biol. 5 (1998) 1084]. The active site model closely resembled that of the 39 kDa isozyme determined by crystallography, except for a mutation of Thr243 (40 kDa isozyme) to Ile241 (39 kDa isozyme) close to the dimetal center. This residue difference affects the orientation of the Glu238/236 residue, which is thought to be responsible for the catalase-like activity of the 39 kDa isozyme for which a catalytic mechanism is proposed.

  7. 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

  8. 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

  9. Lysyl oxidase isoforms in gastric cancer.

    PubMed

    Añazco, Carolina; Delgado-López, Fernando; Araya, Paulina; González, Ileana; Morales, Erik; Pérez-Castro, Ramón; Romero, Jacqueline; Rojas, Armando

    2016-09-01

    Gastric cancer (GC) is the fifth most frequent cancer in the world and shows the highest incidence in Latin America and Asia. An increasing amount of evidence demonstrates that lysyl oxidase isoforms, a group of extracellular matrix crosslinking enzymes, should be considered as potential biomarkers and therapeutic targets in GC. In this review, we focus on the expression levels of lysyl oxidase isoforms, its functions and the clinical implications in GC. Finding novel proteins related to the processing of these extracellular matrix enzymes might be helpful in the design of new therapies, which, in combination with classic pharmacology, could be used to delay the progress of this aggressive cancer and offer a wider temporal window for clinical intervention. PMID:27564724

  10. Imaging Monoamine Oxidase in the Human Brain

    SciTech Connect

    Fowler, J. S.; Volkow, N. D.; Wang, G-J.; Logan, Jean

    1999-11-10

    Positron emission tomography (PET) studies mapping monoamine oxidase in the human brain have been used to measure the turnover rate for MAO B; to determine the minimum effective dose of a new MAO inhibitor drug lazabemide and to document MAO inhibition by cigarette smoke. These studies illustrate the power of PET and radiotracer chemistry to measure normal biochemical processes and to provide information on the effect of drug exposure on specific molecular targets.

  11. 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.

  12. Characterization of polyphenol oxidase activity in Ataulfo mango.

    PubMed

    Cheema, Summervir; Sommerhalter, Monika

    2015-03-15

    Crude extracts of Ataulfo exhibited polyphenol oxidase (PPO) activity with pyrogallol, 3-methylcatechol, catechol, gallic acid, and protocatechuic acid. The substrate dependent pH optima ranged from pH 5.4 to 6.4 with Michaelis-Menten constants between 0.84 ± 0.09 and 4.6 ± 0.7 mM measured in MES or phosphate buffers. The use of acetate buffers resulted in larger Michaelis-Menten constants, up to 14.62 ± 2.03 mM. Sodium ascorbate, glutathione, and kojic acid are promising inhibitors to prevent enzymatic browning in Ataulfo. PPO activity increased with ripeness and was always higher in the skin compared to the pulp. Sodium dodecyl sulphate (SDS) enhanced PPO activity, with pulp showing a stronger increase than skin. SDS-PAGE gels stained for catecholase activity showed multiple bands, with the most prominent bands at apparent molecular weights of 53, 112, and 144 kDa.

  13. 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.

  14. Characterization of polyphenol oxidase activity in Ataulfo mango.

    PubMed

    Cheema, Summervir; Sommerhalter, Monika

    2015-03-15

    Crude extracts of Ataulfo exhibited polyphenol oxidase (PPO) activity with pyrogallol, 3-methylcatechol, catechol, gallic acid, and protocatechuic acid. The substrate dependent pH optima ranged from pH 5.4 to 6.4 with Michaelis-Menten constants between 0.84 ± 0.09 and 4.6 ± 0.7 mM measured in MES or phosphate buffers. The use of acetate buffers resulted in larger Michaelis-Menten constants, up to 14.62 ± 2.03 mM. Sodium ascorbate, glutathione, and kojic acid are promising inhibitors to prevent enzymatic browning in Ataulfo. PPO activity increased with ripeness and was always higher in the skin compared to the pulp. Sodium dodecyl sulphate (SDS) enhanced PPO activity, with pulp showing a stronger increase than skin. SDS-PAGE gels stained for catecholase activity showed multiple bands, with the most prominent bands at apparent molecular weights of 53, 112, and 144 kDa. PMID:25308684

  15. Isolation and characterization of a potato cDNA corresponding to a 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene differentially activated by stress.

    PubMed

    Zanetti, María Eugenia; Terrile, María Cecilia; Arce, Débora; Godoy, Andrea Verónica; Segundo, Blanca San; Casalongué, Claudia

    2002-12-01

    1-Aminocyclopropane-1-carboxylate (ACC) oxidase enzyme catalyses the final step in ethylene biosynthesis, converting 1-aminocyclopropane-1-carboxylic acid to ethylene. A cDNA clone encoding an ACC oxidase, ST-ACO3, was isolated from potato (Solanum tuberosum L.) by differential screening of a Fusarium eumartii infected-tuber cDNA library. The deduced amino acid sequence exhibited similarity to other ACC oxidase proteins from several plants species. Northern blot analysis revealed that the ST-ACO3 mRNA level increased in potato tubers upon inoculation with F. eumartii, as well as after treatment with salicylic acid and indole-3-acetic acid, suggesting a cross-talk between different signalling pathways involved in the defence response of potato tubers against F. eumartii attack.

  16. Stability Constants of Mixed Ligand Complexes of Nickel(II) with Adenine and Some Amino Acids

    PubMed Central

    Türkel, Naciye

    2015-01-01

    Nickel is one of the essential trace elements found in biological systems. It is mostly found in nickel-based enzymes as an essential cofactor. It forms coordination complexes with amino acids within enzymes. Nickel is also present in nucleic acids, though its function in DNA or RNA is still not clearly understood. In this study, complex formation tendencies of Ni(II) with adenine and certain L-amino acids such as aspartic acid, glutamic acid, asparagine, leucine, phenylalanine, and tryptophan were investigated in an aqueous medium. Potentiometric equilibrium measurements showed that both binary and ternary complexes of Ni(II) form with adenine and the above-mentioned L-amino acids. Ternary complexes of Ni(II)-adenine-L-amino acids are formed by stepwise mechanisms. Relative stabilities of the ternary complexes are compared with those of the corresponding binary complexes in terms of Δlog10⁡K, log10⁡X, and % RS values. It was shown that the most stable ternary complex is Ni(II):Ade:L-Asn while the weakest one is Ni(II):Ade:L-Phe in aqueous solution used in this research. In addition, results of this research clearly show that various binary and ternary type Ni(II) complexes are formed in different concentrations as a function of pH in aqueous solution. PMID:26843852

  17. Purification and characterisation of polyphenol oxidase (PPO) from eggplant (Solanum melongena).

    PubMed

    Mishra, Bibhuti B; Gautam, Satyendra; Sharma, Arun

    2012-10-15

    Eggplant (Solanum melongena) is a very rich source of polyphenol oxidase (PPO), which negatively affects its quality upon cutting and postharvest processing due to enzymatic browning. PPO inhibitors, from natural or synthetic sources, are used to tackle this problem. One isoform of PPO was 259-fold purified using standard chromatographic procedures. The PPO was found to be a 112 kDa homodimer. The enzyme showed very low K(m) (0.34 mM) and high catalytic efficiency (3.3×10(6)) with 4-methyl catechol. The substrate specificity was in the order: 4-methyl catechol>tert-butylcatechol>dihydrocaffeic acid>pyrocatechol. Cysteine hydrochloride, potassium metabilsulphite, ascorbic acid, erythorbic acid, resorcylic acid and kojic acid showed competitive inhibition, whereas, citric acid and sodium azide showed mixed inhibition of PPO activity. Cysteine hydrochloride was found to be an excellent inhibitor with the low inhibitor constant of 1.8 μM.

  18. Purification and characterisation of polyphenol oxidase (PPO) from eggplant (Solanum melongena).

    PubMed

    Mishra, Bibhuti B; Gautam, Satyendra; Sharma, Arun

    2012-10-15

    Eggplant (Solanum melongena) is a very rich source of polyphenol oxidase (PPO), which negatively affects its quality upon cutting and postharvest processing due to enzymatic browning. PPO inhibitors, from natural or synthetic sources, are used to tackle this problem. One isoform of PPO was 259-fold purified using standard chromatographic procedures. The PPO was found to be a 112 kDa homodimer. The enzyme showed very low K(m) (0.34 mM) and high catalytic efficiency (3.3×10(6)) with 4-methyl catechol. The substrate specificity was in the order: 4-methyl catechol>tert-butylcatechol>dihydrocaffeic acid>pyrocatechol. Cysteine hydrochloride, potassium metabilsulphite, ascorbic acid, erythorbic acid, resorcylic acid and kojic acid showed competitive inhibition, whereas, citric acid and sodium azide showed mixed inhibition of PPO activity. Cysteine hydrochloride was found to be an excellent inhibitor with the low inhibitor constant of 1.8 μM. PMID:23442630

  19. Comparison of glucose oxidases from Penicillium adametzii, Penicillium Funiculosum and Aspergillus Niger in the design of amperometric glucose biosensors.

    PubMed

    Ramanavicius, Arunas; Voronovic, Jaroslav; Semashko, Tatiana; Mikhailova, Raisa; Kausaite-Minkstimiene, Asta; Ramanaviciene, Almira

    2014-01-01

    The properties of amperometric glucose biosensors based on three different glucose oxidases and various redox mediators were evaluated. Glucose oxidases (GOx) from Penicillium adametzii, Penicillium funiculosum and Aspergillus niger and artificial redox mediators, such as ferrocene, ferrocenecarboxaldehyde, α-methylferrocene methanol and ferrocenecarboxylic acid, were used for modifying the graphite rod electrode and amperometrical reagent-less glucose detection. The obtained results were compared using N-methylphenazonium methyl sulphate in the solution. Taking into account the experimental kinetic parameters and the stability of the tested enzymatic electrodes, GOx from Penicillium funiculosum proved to be more suitable for glucose biosensor design in comparison with other evaluated enzymes. PMID:25492463

  20. Comparison of kinetic properties of amine oxidases from sainfoin and lentil and immunochemical characterization of copper/quinoprotein amine oxidases.

    PubMed

    Zajoncová, L; Frébort, I; Luhová, L; Sebela, M; Galuszka, P; Pec, P

    1999-01-01

    Kinetic properties of novel amine oxidase isolated from sainfoin (Onobrychis viciifolia) were compared to those of typical plant amine oxidase (EC 1.4.3.6) from lentil (Lens culinaris). The amine oxidase from sainfoin was active toward substrates, such as 1,5-diaminopentane (cadaverine) with K(m) of 0.09 mM and 1,4-diaminobutane (putrescine) with K(m) of 0.24 mM. The maximum rate of oxidation for cadaverine at saturating concentration was 2.7 fold higher than that of putrescine. The amine oxidase from lentil had the maximum rate for putrescine comparable to the rate of sainfoin amine oxidase with the same substrate. Both amine oxidases, like other plant Cu-amine oxidases, were inhibited by substrate analogs (1,5-diamino-3-pentanone, 1,4-diamino-2-butanone and aminoguanidine), Cu2+ chelating agents (diethyltriamine, 1,10-phenanthroline, 8-hydroxyquinoline, 2,2'-bipyridyl, imidazole, sodium cyanide and sodium azide), some alkaloids (L-lobeline and cinchonine), some lathyrogens (beta-aminopropionitrile and aminoacetonitrile) and other inhibitors (benzamide oxime, acetone oxime, hydroxylamine and pargyline). Tested by Ouchterlony's double diffusion in agarose gel, polyclonal antibodies against the amine oxidase from sainfoin, pea and grass pea cross-reacted with amine oxidases from several other Fabaceae and from barley (Hordeum vulgare) of Poaceae, while amine oxidase from the filamentous fungus Aspergillus niger did not cross-react at all. However, using Western blotting after SDS-PAGE with rabbit polyclonal antibodies against the amine oxidase from Aspergillus niger, some degree of similarity of plant amine oxidases from sainfoin, pea, field pea, grass pea, fenugreek, common melilot, white sweetclover and Vicia panonica with the A. niger amine oxidase was confirmed. PMID:10092944

  1. Pathological changes in platelet histamine oxidases in atopic eczema

    PubMed Central

    Ionescu, Gruia

    1993-01-01

    Increased plasma histamine levels were associated with significantly lowered diamine and type B monoamine oxidase activities in platelet-rich plasma of atopic eczema (AE) patients. The diamine oxidase has almost normal cofactor levels (pyridoxal phosphate and Cu2+) but the cofactor levels for type B monoamine oxidase (flavin adenine dinucleotide and Fe2+) are lowered. The biogenic amines putrescine, cadaverine, spermidine, spermine, tyramine and serotonin in the sera, as well as dopamine and epinephrine in EDTA-plasma were found to be normal. It is unlikely, therefore, that these amines are responsible for the decreased activities of monoamine and diamine oxidase in these patients. The most likely causative factors for the inhibition of the diamine oxidase are nicotine, alcohol, food additives and other environmental chemicals, or perhaps a genetic defect of the diamine oxidase. PMID:18475554

  2. Proton-pumping mechanism of cytochrome c oxidase: a kinetic master-equation approach.

    PubMed

    Kim, Young C; Hummer, Gerhard

    2012-04-01

    Cytochrome c oxidase is an efficient energy transducer that reduces oxygen to water and converts the released chemical energy into an electrochemical membrane potential. As a true proton pump, cytochrome c oxidase translocates protons across the membrane against this potential. Based on a wealth of experiments and calculations, an increasingly detailed picture of the reaction intermediates in the redox cycle has emerged. However, the fundamental mechanism of proton pumping coupled to redox chemistry remains largely unresolved. Here we examine and extend a kinetic master-equation approach to gain insight into redox-coupled proton pumping in cytochrome c oxidase. Basic principles of the cytochrome c oxidase proton pump emerge from an analysis of the simplest kinetic models that retain essential elements of the experimentally determined structure, energetics, and kinetics, and that satisfy fundamental physical principles. The master-equation models allow us to address the question of how pumping can be achieved in a system in which all reaction steps are reversible. Whereas proton pumping does not require the direct modulation of microscopic reaction barriers, such kinetic gating greatly increases the pumping efficiency. Further efficiency gains can be achieved by partially decoupling the proton uptake pathway from the active-site region. Such a mechanism is consistent with the proposed Glu valve, in which the side chain of a key glutamic acid shuttles between the D channel and the active-site region. We also show that the models predict only small proton leaks even in the absence of turnover. The design principles identified here for cytochrome c oxidase provide a blueprint for novel biology-inspired fuel cells, and the master-equation formulation should prove useful also for other molecular machines. . PMID:21946020

  3. In vitro antimalarial and xanthine oxidase inhibition of 2-Aminoanthraquinone.

    PubMed

    Rauf, Abdur; Khan, Rehan; Khan, Haroon; Jehan, Noor; Akram, Mohammad; Ahmad, Zarka; Muhammad, Naveed; Farooq, Umar; Khan, Ajmal

    2016-03-01

    In the present research study 2-Aminoanthraquinone were scrutinized for their antimalarial and Xanthine oxidase inhibitor potential. It demonstrated marked concentration dependent antimalarial activity with maximum effect of 89.06% and with IC50 of 34.17 µM. Regarding Xanthine oxidase inhibitor activity, it evoked significant effect with 57.45% activity with IC50 value of 81.57.19 μM. In conclusion, 2-Aminoanthraquinone showed potent antimalarial and xanthine oxidase inhibitory activity. PMID:27087090

  4. 4-Coumaroyl coenzyme A 3-hydroxylase activity from cell cultures of Lithospermum erythrorhizon and its relationship to polyphenol oxidase.

    PubMed

    Wang, Z X; Li, S M; Löscher, R; Heide, L

    1997-11-15

    A 4-coumaroyl-CoA 3-hydroxylase activity was purified 4600-fold from cell cultures of Lithospermum erythrorhizon. The enzyme showed a molecular mass of 42,400 +/- 1700 Da in gel chromatography and required ascorbate, NADH, or NADPH as cofactors. 4-Coumaroyl-CoA, 4-coumarate, p-cresol, and several other phenolic substances, but not tyrosine, were accepted as substrates for the hydroxylation. Besides hydroxylase activity, the enzyme showed diphenol oxidase activity. Both activities were inhibited by diethyldithiocarbamate or beta-mercaptoethanol, although at different concentrations. The enzyme showed striking similarity to a 4-coumaroyl-glucose 3-hydroxylase from sweet potato (Ipomoe batatas) roots, which has reportedly been purified to homogeneity and identified as a specific enzyme of chlorogenic acid biosynthesis. Close examination and comparison to a commercially available polyphenol oxidase, however, suggest that the enzyme activities purified from both Lithospermum and sweet potato are polyphenol oxidases rather than specific enzymes of secondary metabolism. PMID:9367532

  5. Crystal Structure of a Two-domain Multicopper Oxidase

    PubMed Central

    Lawton, Thomas J.; Sayavedra-Soto, Luis A.; Arp, Daniel J.; Rosenzweig, Amy C.

    2009-01-01

    The two-domain multicopper oxidases are proposed to be key intermediates in the evolution of three-domain multicopper oxidases. A number of two-domain multicopper oxidases have been identified from genome sequences and are classified as type A, type B, or type C on the basis of the predicted location of the type 1 copper center. The crystal structure of blue copper oxidase, a type C two-domain multicopper oxidase from Nitrosomonas europaea, has been determined to 1.9 Å resolution. Blue copper oxidase is a trimer, of which each subunit comprises two cupredoxin domains. Each subunit houses a type 1 copper site in domain 1 and a type 2/type 3 trinuclear copper cluster at the subunit-subunit interface. The coordination geometry at the trinuclear copper site is consistent with reduction of the copper ions. Although the overall architecture of blue copper oxidase is similar to nitrite reductases, detailed structural alignments show that the fold and domain orientation more closely resemble the three-domain multicopper oxidases. These observations have important implications for the evolution of nitrite reductases and multicopper oxidases. PMID:19224923

  6. A highly enantioselective amino acid-catalyzed route to functionalized alpha-amino acids.

    PubMed

    Córdova, Armando; Notz, Wolfgang; Zhong, Guofu; Betancort, Juan M; Barbas, Carlos F

    2002-03-01

    The development of syntheses providing enantiomerically pure alpha-amino acids has intrigued generations of chemists and been the subject of intense research. This report describes a general approach to functionalized alpha-amino acids based on catalytic asymmetric synthesis. Proline catalyzed Mannich-type reactions of N-PMP-protected alpha-imino ethyl glyoxylate with a variety of unmodified ketones to provide functionalized alpha-amino acids in high yields with excellent regio-, diastereo-, and enantioselectivities. Study of seven examples yielded six with product ee values of > or = 99%. In reactions involving ketone donors where diastereoisomeric products could be formed, two adjacent stereogenic centers were created simultaneously upon carbon-carbon bond formation with complete syn-stereocontrol. Significantly, this methodology utilizes readily available and rather inexpensive starting materials, does not require any preactivation of substrates or metal ion assistance, and can be carried out on a gram scale under operationally simple reaction conditions. The keto-functionality present in the products provides a particularly attractive site for versatile modifications. This study compliments and extends our bioorganic approach to asymmetric synthesis to a versatile synthon class. Given that we have shown that a variety of optically active amino acids can be synthesized with proline catalysis, where an L-amino acid begets other L-amino acids, our results may stimulate thoughts concerning prebiotic syntheses of optically active amino acids based on this route.

  7. Glucose Biosensor Based on Immobilization of Glucose Oxidase in Platinum Nanoparticles/Graphene/Chitosan Nanocomposite Film

    SciTech Connect

    Wu, Hong; Wang, Jun; Kang, Xinhuang; Wang, Chong M.; Wang, Donghai; Liu, Jun; Aksay, Ilhan A.; Lin, Yuehe

    2009-09-01

    The bionanocomposite film consisting of glucose oxidase/Pt/functional graphene sheets/chitosan (GOD/Pt/FGS/chitosan) for glucose sensing was described. With the electrocatalytic synergy of FGS and Pt nanoparticles to hydrogen peroxide, a sensitive biosensor with detection limit of 0.6 µM glucose was achieved. The biosensor also had good reproducibility, long term stability and negligible interfering signals from ascorbic acid and uric acid comparing to the response to glucose. The large surface area and good conductivity of graphene suggests that graphene is a potential candidate for sensor material. The hybrid nanocomposite glucose sensor provides new opportunity for clinical diagnosis and point-of-care applications.

  8. The cyclope gene of Drosophila encodes a cytochrome c oxidase subunit VIc homolog.

    PubMed

    Szuplewski, S; Terracol, R

    2001-08-01

    Cytochrome c oxidase is the terminal enzyme of the mitochondrial electron transfer chain. In eukaryotes, the enzyme is composed of 3 mitochondrial DNA-encoded subunits and 7-10 (in mammals) nuclear DNA-encoded subunits. This enzyme has been extensively studied in mammals and yeast but, in Drosophila, very little is known and no mutant has been described so far. Here we report the genetic and molecular characterization of mutations in cyclope (cype) and the cloning of the gene encoding a cytochrome c oxidase subunit VIc homolog. cype is an essential gene whose mutations are lethal and show pleiotropic phenotypes. The 77-amino acid peptide encoded by cype is 46% identical and 59% similar to the human subunit (75 amino acids). The transcripts are expressed maternally and throughout development in localized regions. They are found predominantly in the central nervous system of the embryo; in the central region of imaginal discs; in the germarium, follicular, and nurse cells of the ovary; and in testis. A search in the Genome Annotation Database of Drosophila revealed the absence of subunit VIIb and the presence of 9 putative nuclear cytochrome c oxidase subunits with high identity scores when compared to the 10 human subunits. PMID:11514451

  9. The cyclope gene of Drosophila encodes a cytochrome c oxidase subunit VIc homolog.

    PubMed Central

    Szuplewski, S; Terracol, R

    2001-01-01

    Cytochrome c oxidase is the terminal enzyme of the mitochondrial electron transfer chain. In eukaryotes, the enzyme is composed of 3 mitochondrial DNA-encoded subunits and 7-10 (in mammals) nuclear DNA-encoded subunits. This enzyme has been extensively studied in mammals and yeast but, in Drosophila, very little is known and no mutant has been described so far. Here we report the genetic and molecular characterization of mutations in cyclope (cype) and the cloning of the gene encoding a cytochrome c oxidase subunit VIc homolog. cype is an essential gene whose mutations are lethal and show pleiotropic phenotypes. The 77-amino acid peptide encoded by cype is 46% identical and 59% similar to the human subunit (75 amino acids). The transcripts are expressed maternally and throughout development in localized regions. They are found predominantly in the central nervous system of the embryo; in the central region of imaginal discs; in the germarium, follicular, and nurse cells of the ovary; and in testis. A search in the Genome Annotation Database of Drosophila revealed the absence of subunit VIIb and the presence of 9 putative nuclear cytochrome c oxidase subunits with high identity scores when compared to the 10 human subunits. PMID:11514451

  10. Functional incorporation of beef-heart cytochrome c oxidase into membranes of Streptococcus cremoris.

    PubMed

    Driessen, A J; de Vrij, W; Konings, W N

    1986-02-01

    Beef heart mitochondrial cytochrome c oxidase has been incorporated into membrane vesicles derived from the homofermentative lactic acid bacterium Streptococcus cremoris. Proteoliposomes containing cytochrome c oxidase were fused with the bacterial membrane vesicles by means of a freeze/thaw sonication technique. Evidence that membrane fusion has taken place is presented by the demonstration that nonexchangeable fluorescent phospholipid probes, originally present only in the bacterial membrane or only in the liposomal membrane, are diluted in the membrane after fusion and, by sucrose gradient centrifugation, indicating a buoyant density of the membranes after fusion in between those of the starting membrane preparations. The fused membranes are endowed with a relatively low ion permeability which makes it possible to generate a high proton motive force (100 mV, inside negative and alkaline) by cytochrome-c-oxidase-mediated oxidation of the electron donor system ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine/cytochrome c. In the fused membranes this proton motive force can drive the uptake of several amino acids via secondary transport systems. The incorporation procedure described for primary proton pumps in biological membranes opens attractive possibilities for studies of proton-motive-force-dependent processes in isolated membrane vesicles from bacterial or eukaryotic origin which lack a suitable proton-motive-force-generating system.

  11. Nox NADPH Oxidases and the Endoplasmic Reticulum

    PubMed Central

    Araujo, Thaís L.S.; Abrahão, Thalita B.

    2014-01-01

    Abstract Significance: Understanding isoform- and context-specific subcellular Nox reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase compartmentalization allows relevant functional inferences. This review addresses the interplay between Nox NADPH oxidases and the endoplasmic reticulum (ER), an increasingly evident player in redox pathophysiology given its role in redox protein folding and stress responses. Recent Advances: Catalytic/regulatory transmembrane subunits are synthesized in the ER and their processing includes folding, N-glycosylation, heme insertion, p22phox heterodimerization, as shown for phagocyte Nox2. Dual oxidase (Duox) maturation also involves the regulation by ER-resident Duoxa2. The ER is the activation site for some isoforms, typically Nox4, but potentially other isoforms. Such location influences redox/Nox-mediated calcium signaling regulation via ER targets, such as sarcoendoplasmic reticulum calcium ATPase (SERCA). Growing evidence suggests that Noxes are integral signaling elements of the unfolded protein response during ER stress, with Nox4 playing a dual prosurvival/proapoptotic role in this setting, whereas Nox2 enhances proapoptotic signaling. ER chaperones such as protein disulfide isomerase (PDI) closely interact with Noxes. PDI supports growth factor-dependent Nox1 activation and mRNA expression, as well as migration in smooth muscle cells, and PDI overexpression induces acute spontaneous Nox activation. Critical Issues: Mechanisms of PDI effects include possible support of complex formation and RhoGTPase activation. In phagocytes, PDI supports phagocytosis, Nox activation, and redox-dependent interactions with p47phox. Together, the results implicate PDI as possible Nox organizer. Future Directions: We propose that convergence between Noxes and ER may have evolutive roots given ER-related functional contexts, which paved Nox evolution, namely calcium signaling and pathogen killing. Overall, the interplay between

  12. Cell growth and enzyme synthesis of a mutant of Arthrobacter sp. (DSM 3747) used for the production of L-amino acids from D,L-5-monosubstituted hydantoins.

    PubMed

    Syldatk, C; Mackowiak, V; Höke, H; Gross, C; Dombach, G; Wagner, F

    1990-06-01

    A microorganism with the ability to form L-tryptophan from D,L-5-(3-indolyl-methyl)hydantoin (D,L-5-IMH) was isolated and identified as Arthrobacter sp. (DSM 3747). After isolation of a mutant with high tryptophan production activity but low tryptophan degradation, cultural conditions were optimized to achieve high amounts of biomass with good specific activities concerning the enzymatic hydantoin-cleaving reactions. The ability of the microorganism to perform these bioconversions was found to be inducible by D,L-5-IMH as well as to be dependent on the presence of Mn2+. The highest specific D,L-5-IMH-cleaving activity of the cells was observed in the exponential phase of growth. The addition of yeast extract to the mineral salts medium was found to be essential for obtaining biomass concentrations of about 25 g l-1 cell dry mass by bioreactor cultivations. In order to obtain a constantly high growth rate, feeding of the C-source was pO2-controlled. The inducer D,L-5-IMH had to be continuously fed to prevent a decline of the L-tryptophan-forming enzyme activities, because it was subjected to degradation with the enzymes induced and higher concentrations of D,L-5-IMH aggravated the growth significantly. The synthesis of the enzymes was also inducible, when inducer and Mn2+ were not added until the late growth phase. Using this process, the consumption of D,L-5-IMH was reduced remarkably. So, under these conditions biomass concentrations of 25 g l-1 cell dry weight with a specific enzymatic activity of 0.20 mmol g-1 h-1 (tryptophan per dry mass per time) could be obtained within 13 h. Using 1 g l-1 of the chemically modified inducer D,L-5-(3-indolylmethyl)-3-N-methylhydantoin, which was not degradable by the microorganisms, a biomass concentration of 28 g l-1 cell dry weight with a specific activity of 0.34 mmol g-1 h-1 (tryptophan per dry mass per time) could be obtained within 28 h.

  13. Xanthine oxidase inhibitory activity of Vietnamese medicinal plants.

    PubMed

    Nguyen, Mai Thanh Thi; Awale, Suresh; Tezuka, Yasuhiro; Tran, Quan Le; Watanabe, Hiroshi; Kadota, Shigetoshi

    2004-09-01

    Among 288 extracts, prepared from 96 medicinal plants used in Vietnamese traditional medicine to treat gout and related symptoms, 188 demonstrated xanthine oxidase (XO) inhibitory activity at 100 microg/ml, with 46 having greater than 50% inhibition. At 50 microg/ml, 168 of the extracts were active, with 21 possessing more than 50% inhibition. At 25 microg/ml, 146 extracts exhibited inhibitory activity, with 8 showing over 50% inhibition, while 126 extracts presented activity at 10 microg/ml, with 2 having greater than 50% inhibition. The MeOH extracts of Artemisia vulgaris, Caesalpinia sappan (collected at the Seven-Mountain area), Blumea balsamifera (collected in Lam Dong province), Chrysanthemum sinense and MeOH-H(2)O extract of Tetracera scandens (Khanh Hoa province) exhibited strong XO inhibitory activity with IC(50) values less than 20 microg/ml. The most active extract was the MeOH extract of the flower of C. sinense with an IC(50) value of 5.1 microg/ml. Activity-guided fractionation of the MeOH extract led to the isolation of caffeic acid (1), luteolin (2), eriodictyol (3), and 1,5-di-O-caffeoylquinic acid (4). All these compounds showed significant XO inhibitory activity in a concentration-dependent manner, and the activity of 2 was more potent (IC(50) 1.3 microM) than the clinically used drug, allopurinol (IC(50) 2.5 microM). PMID:15340229

  14. Inhibitory effects of cardols and related compounds on superoxide anion generation by xanthine oxidase.

    PubMed

    Masuoka, Noriyoshi; Nihei, Ken-ichi; Maeta, Ayami; Yamagiwa, Yoshiro; Kubo, Isao

    2015-01-01

    5-Pentadecatrienylresorcinol, isolated from cashew nuts and commonly known as cardol (C₁₅:₃), prevented the generation of superoxide radicals catalysed by xanthine oxidase without the inhibition of uric acid formation. The inhibition kinetics did not follow the Michelis-Menten equation, but instead followed the Hill equation. Cardol (C₁₀:₀) also inhibited superoxide anion generation, but resorcinol and cardol (C₅:₀) did not inhibit superoxide anion generation. The related compounds 3,5-dihydroxyphenyl alkanoates and alkyl 2,4-dihydroxybenzoates, had more than a C9 chain, cooperatively inhibited but alkyl 3,5-dihydroxybenzoates, regardless of their alkyl chain length, did not inhibit the superoxide anion generation. These results suggested that specific inhibitors for superoxide anion generation catalysed by xanthine oxidase consisted of an electron-rich resorcinol group and an alkyl chain having longer than C9 chain. PMID:25053055

  15. Involvement of NADH Oxidase in Competition and Endocarditis Virulence in Streptococcus sanguinis

    PubMed Central

    Ge, Xiuchun; Yu, Yang; Zhang, Min; Chen, Lei; Chen, Weihua; Elrami, Fadi; Kong, Fanxiang; Kitten, Todd

    2016-01-01

    Here, we report for the first time that the Streptococcus sanguinis nox gene encoding NADH oxidase is involved in both competition with Streptococcus mutans and virulence for infective endocarditis. An S. sanguinis nox mutant was found to fail to inhibit the growth of Streptococcus mutans under microaerobic conditions. In the presence of oxygen, the recombinant Nox protein of S. sanguinis could reduce oxygen to water and oxidize NADH to NAD+. The oxidation of NADH to NAD+ was diminished in the nox mutant. The nox mutant exhibited decreased levels of extracellular H2O2; however, the intracellular level of H2O2 in the mutant was increased. Furthermore, the virulence of the nox mutant was attenuated in a rabbit endocarditis model. The nox mutant also was shown to be more sensitive to blood killing, oxidative and acid stresses, and reduced growth in serum. Thus, NADH oxidase contributes to multiple phenotypes related to competitiveness in the oral cavity and systemic virulence. PMID:26930704

  16. Activity of carbohydrate oxidases as influenced by wheat flour dough components.

    PubMed

    Degrand, L; Rakotozafy, L; Nicolas, J

    2015-08-15

    The carbohydrate oxidase (COXMn) from Microdochium nivale may well have desired functionalities as a dough and bread improver, similarly to Aspergillus niger glucose oxidase (GOX). COXMn catalyses the oxidation of various monosaccharides as well as maltooligosaccharides for which the best activity is obtained towards the maltooligosaccharides of polymerisation degrees 3 and 4. For the same activity towards glucose under air saturation, we show that COXMn exhibits a similar efficiency towards maltose as GOX towards glucose whatever the oxygen supply. Assays with COXMn show that no competition exists between carbohydrates naturally present in the wheat flour. We show that reaction products (d-glucono-δ-lactone and hydrogen peroxide) and the wheat flour dough component, ferulic acid, have no noticeable specific effect on the COXMn activity. The demonstrated differences in kinetics between COXMn and GOX allow predicting of differences in the functional behaviours of those enzymes during wheat flour dough formation. PMID:25794758

  17. [Effect of lectins from Azospirillum brasilense to peroxidase and oxalate oxidase activity regulation in wheat roots].

    PubMed

    Alen'kina, S A; Nikitina, V E

    2010-01-01

    Lectins were extracted from the surface of nitrogen-fixing soil bacteria Azospirillum brasilense Sp7 and from its mutant A. brasilense Sp7.2.3 defective in lectin activity. The ability oflectins to stimulate the rapid formation of hydrogen peroxide related to increase of oxalate oxidase and peroxidase activity in the roots of wheat seedlings has been demonstrated. The most rapid induced pathway of hydrogen peroxide formation in the roots of wheat seedlings was the oxalic acid oxidation by oxalate oxidase which is the effect oflectin in under 10 min in a concentration of 10 microg/ml. The obtained results show that lectins from Azospirillum are capable of inducing the adaptation processes in the roots of wheat seedlings.

  18. Activity of carbohydrate oxidases as influenced by wheat flour dough components.

    PubMed

    Degrand, L; Rakotozafy, L; Nicolas, J

    2015-08-15

    The carbohydrate oxidase (COXMn) from Microdochium nivale may well have desired functionalities as a dough and bread improver, similarly to Aspergillus niger glucose oxidase (GOX). COXMn catalyses the oxidation of various monosaccharides as well as maltooligosaccharides for which the best activity is obtained towards the maltooligosaccharides of polymerisation degrees 3 and 4. For the same activity towards glucose under air saturation, we show that COXMn exhibits a similar efficiency towards maltose as GOX towards glucose whatever the oxygen supply. Assays with COXMn show that no competition exists between carbohydrates naturally present in the wheat flour. We show that reaction products (d-glucono-δ-lactone and hydrogen peroxide) and the wheat flour dough component, ferulic acid, have no noticeable specific effect on the COXMn activity. The demonstrated differences in kinetics between COXMn and GOX allow predicting of differences in the functional behaviours of those enzymes during wheat flour dough formation.

  19. NADPH Oxidase Promotes Neutrophil Extracellular Trap Formation in Pulmonary Aspergillosis

    PubMed Central

    Röhm, Marc; Grimm, Melissa J.; D'Auria, Anthony C.; Almyroudis, Nikolaos G.

    2014-01-01

    NADPH oxidase is a crucial enzyme in antimicrobial host defense and in regulating inflammation. Chronic granulomatous disease (CGD) is an inherited disorder of NADPH oxidase in which phagocytes are defective in generation of reactive oxidant intermediates. Aspergillus species are ubiquitous, filamentous fungi, which can cause invasive aspergillosis, a major cause of morbidity and mortality in CGD, reflecting the critical role for NADPH oxidase in antifungal host defense. Activation of NADPH oxidase in neutrophils can be coupled to the release of proteins and chromatin that comingle in neutrophil extracellular traps (NETs), which can augment extracellular antimicrobial host defense. NETosis can be driven by NADPH oxidase-dependent and -independent pathways. We therefore undertook an analysis of whether NADPH oxidase was required for NETosis in Aspergillus fumigatus pneumonia. Oropharyngeal instillation of live Aspergillus hyphae induced neutrophilic pneumonitis in both wild-type and NADPH oxidase-deficient (p47phox−/−) mice which had resolved in wild-type mice by day 5 but progressed in p47phox−/− mice. NETs, identified by immunostaining, were observed in lungs of wild-type mice but were absent in p47phox−/− mice. Using bona fide NETs and nuclear chromatin decondensation as an early NETosis marker, we found that NETosis required a functional NADPH oxidase in vivo and ex vivo. In addition, NADPH oxidase increased the proportion of apoptotic neutrophils. Together, our results show that NADPH oxidase is required for pulmonary clearance of Aspergillus hyphae and generation of NETs in vivo. We speculate that dual modulation of NETosis and apoptosis by NADPH oxidase enhances antifungal host defense and promotes resolution of inflammation upon infection clearance. PMID:24549323

  20. Multicopper oxidase-1 orthologs from diverse insect species have ascorbate oxidase activity.

    PubMed

    Peng, Zeyu; Dittmer, Neal T; Lang, Minglin; Brummett, Lisa M; Braun, Caroline L; Davis, Lawrence C; Kanost, Michael R; Gorman, Maureen J

    2015-04-01

    Members of the multicopper oxidase (MCO) family of enzymes can be classified by their substrate specificity; for example, ferroxidases oxidize ferrous iron, ascorbate oxidases oxidize ascorbate, and laccases oxidize aromatic substrates such as diphenols. Our previous work on an insect multicopper oxidase, MCO1, suggested that it may function as a ferroxidase. This hypothesis was based on three lines of evidence: RNAi-mediated knock down of Drosophila melanogaster MCO1 (DmMCO1) affects iron homeostasis, DmMCO1 has ferroxidase activity, and DmMCO1 has predicted iron binding residues. In our current study, we expanded our focus to include MCO1 from Anopheles gambiae, Tribolium castaneum, and Manduca sexta. We verified that MCO1 orthologs have similar expression profiles, and that the MCO1 protein is located on the basal surface of cells where it is positioned to oxidize substrates in the hemolymph. In addition, we determined that RNAi-mediated knock down of MCO1 in A. gambiae affects iron homeostasis. To further characterize the enzymatic activity of MCO1 orthologs, we purified recombinant MCO1 from all four insect species and performed kinetic analyses using ferrous iron, ascorbate and two diphenols as substrates. We found that all of the MCO1 orthologs are much better at oxidizing ascorbate than they are at oxidizing ferrous iron or diphenols. This result is surprising because ascorbate oxidases are thought to be specific to plants and fungi. An analysis of three predicted iron binding residues in DmMCO1 revealed that they are not required for ferroxidase or laccase activity, but two of the residues (His374 and Asp380) influence oxidation of ascorbate. These two residues are conserved in MCO1 orthologs from insects and crustaceans; therefore, they are likely to be important for MCO1 function. The results of this study suggest that MCO1 orthologs function as ascorbate oxidases and influence iron homeostasis through an unknown mechanism. PMID:25701385

  1. Multicopper oxidase-1 orthologs from diverse insect species have ascorbate oxidase activity

    PubMed Central

    Peng, Zeyu; Dittmer, Neal T.; Lang, Minglin; Brummett, Lisa M.; Braun, Caroline L.; Davis, Lawrence C.; Kanost, Michael R.; Gorman, Maureen J.

    2015-01-01

    Members of the multicopper oxidase (MCO) family of enzymes can be classified by their substrate specificity; for example, ferroxidases oxidize ferrous iron, ascorbate oxidases oxidize ascorbate, and laccases oxidize aromatic substrates such as diphenols. Our previous work on an insect multicopper oxidase, MCO1, suggested that it may function as a ferroxidase. This hypothesis was based on three lines of evidence: RNAi-mediated knock down of Drosophila melanogaster MCO1 (DmMCO1) affects iron homeostasis, DmMCO1 has ferroxidase activity, and DmMCO1 has predicted iron binding residues. In our current study, we expanded our focus to include MCO1 from Anopheles gambiae, Tribolium castaneum, and Manduca sexta. We verified that MCO1 orthologs have similar expression profiles, and that the MCO1 protein is located on the basal surface of cells where it is positioned to oxidize substrates in the hemolymph. In addition, we determined that RNAi-mediated knock down of MCO1 in A. gambiae affects iron homeostasis. To further characterize the enzymatic activity of MCO1 orthologs, we purified recombinant MCO1 from all four insect species and performed kinetic analyses using ferrous iron, ascorbate and two diphenols as substrates. We found that all of the MCO1 orthologs are much better at oxidizing ascorbate than they are at oxidizing ferrous iron or diphenols. This result is surpring because ascorbate oxidases are thought to be specific to plants and fungi. An analysis of three predicted iron binding residues in DmMCO1 revealed that they are not required for ferroxidase or laccase activity, but two of the residues (His374 and Asp380) influence oxidation of ascorbate. These two residues are conserved in MCO1 orthologs from insects and crustaceans; therefore, they are likely to be important for MCO1 function. The results of this study suggest that MCO1 orthologs function as ascorbate oxidases and influence iron homeostasis through an unknown mechanism. PMID:25701385

  2. Variations in the alternative oxidase in chlamydomonas grown in air or high CO sub 2

    SciTech Connect

    Goyal, A.; Tolbert, N.E. )

    1989-03-01

    Chlamydomonas in the resting phase of growth has an equal capacity of about 15 micromole O{sub 2} uptake per hour per milligram of chlorophyll for both the cytochrome c, CN-sensitive respiration, and for the alternative, salicylhydroxamic acid-sensitive respiration. Alternative respiration capacity was measured as salicylhydroxamic acid inhibited O{sub 2} uptake in the presence of CN, and cytochrome c respiration capacity as CN inhibition of O{sub 2} uptake in the presence of salicylhydroxamic acid. Measured total respiration was considerably less than the combined capacities for respiration. During the log phase of growth on high (2-5%) CO{sub 2}, the alternative respiration capacity decreased about 90% but returned as the culture entered the lag phase. When the alternative oxidase capacity was low, addition of salicylic acid or cyanide induced its reappearance. When cells were grown on low (air-level) CO{sub 2}, which induced a CO{sub 2} concentrating mechanism, the alternative oxidase capacity did not decrease during the growth phase. Attempts to measure in vivo distribution of respiration between the two pathways with either CN or salicylhydroxamic acid alone were inconclusive.

  3. Endothelins and NADPH oxidases in the cardiovascular system.

    PubMed

    Dammanahalli, Karigowda J; Sun, Zhongjie

    2008-01-01

    1. The endothelin (ET) system and NADPH oxidase play important roles in the regulation of cardiovascular function, as well as in the pathogenesis of hypertension and other cardiovascular diseases. 2. Endothelins activate NADPH oxidases and thereby increase superoxide production, resulting in oxidative stress and cardiovascular dysfunction. Thus, NADPH oxidases may mediate the role of endothelins in some cardiovascular diseases. However, the role of reactive oxygen species (ROS) in mediating ET-induced vasoconstriction and cardiovascular disease remains under debate, as evidenced by conflicting reports from different research teams. Conversely, activation of NADPH oxidase can stimulate ET secretion via ROS generation, which further enhances the cardiovascular effects of NADPH oxidase. However, little is known about how ROS activate the endothelin system. It seems that the relationship between ET-1 and ROS may vary with cardiovascular disorders. 3. Endothelins activate NADPH oxidase via the ET receptor-proline-rich tyrosine kinase-2 (Pyk2)-Rac1 pathway. Rac1 is an important regulator of NADPH oxidase. There is ample evidence supporting direct stimulation by Rac1 of NADPH oxidase activity. In addition, Rac1-induced cardiomyocyte hypertrophy is mediated by the generation of ROS.

  4. IgE binding to peanut allergens is inhibited by combined D-aspartic and D-glutamic acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    D-amino acids (D-aas) are reported to bind to IgE antibodies from people with allergy and asthma. The objectives of this study were to determine if D-aas bind or inhibit IgE binding to peanut allergens, and if they are more effective than L-amino acids (L-aas) in this respect. Several D-aa cocktails...

  5. The Use of Gel Electrophoresis to Study the Reactions of Activated Amino Acids with Oligonucleotides

    NASA Technical Reports Server (NTRS)

    Zieboll, Gerhard; Orgel, Leslie E.

    1994-01-01

    We have used gel electrophoresis to study the primary covalent addition of amino acids to oligonu-cleotides or their analogs and the subsequent addition of further molecules of the amino acids to generate peptides covalently linked to the oligonucleotides. We have surveyed the reactions of a variety of amino acids with the phosphoramidates derived from oligonucleotide 5 inches phosphates and ethylenediamine. We find that arginine and amino acids can interact with oligonucleotidesl through stacking interactions react most efficiently. D- and L-amino acids give indistinguishable families of products.

  6. The human lysyl oxidase-like 2 protein functions as an amine oxidase toward collagen and elastin.

    PubMed

    Kim, Young-Mi; Kim, Eun-Cheol; Kim, Youngho

    2011-01-01

    The lysyl oxidase-like 2 (LOXL2) protein is a human paralogue of lysyl oxidase (LOX) that functions as an amine oxidase for formation of lysine-derived cross-links found in collagen and elastin. In addition to the C-terminal domains characteristic to the LOX family members, LOXL2 contains four scavenger receptor cysteine-rich (SRCR) domains in the N-terminus. In order to assess the amine oxidase activity of LOXL2, we expressed a series of recombinant LOXL2 proteins with deletions in the SRCR domains, using an Escherichia coli expression system. All of the purified recombinant LOXL2 proteins, with or without the SRCR domains in the N-terminus, showed significant amine oxidase activity toward several different types of collagen and elastin in in vitro amine oxidase assays, indicating deletion of the SRCR domains does not interfere with amine oxidase activity of LOXL2. Further, amine oxidase activity of LOXL2 was not susceptible to inhibition by β-aminopropionitrile, an irreversible inhibitor of LOX, suggesting a different enzymatic mechanism between these two paralogues.

  7. D-Amino Acids in Living Higher Organisms

    NASA Astrophysics Data System (ADS)

    Fujii, Noriko

    2002-04-01

    The homochirality of biological amino acids (L-amino acids) and of the RNA/DNA backbone (D-ribose) might have become established before the origin of life. It has been considered that D-amino acids and L-sugars were eliminated on the primitive Earth. Therefore, the presence and function of D-amino acids in living organisms have not been studied except for D-amino acids in the cell walls of microorganisms. However, D-amino acids were recently found in various living higher organisms in the form of free amino acids, peptides, and proteins. Free D-aspartate and D-serine are present and may have important physiological functions in mammals. D-amino acids in peptides are well known as opioid peptides and neuropeptides. In protein, D-aspartate residues increase during aging. This review deals with recent advances in the study of D-amino acids in higher organisms.

  8. Apocyanin, a Microglial NADPH Oxidase Inhibitor Prevents Dopaminergic Neuronal Degeneration in Lipopolysaccharide-Induced Parkinson's Disease Model.

    PubMed

    Sharma, Neha; Nehru, Bimla

    2016-07-01

    Microglia-associated inflammatory processes have been strongly implicated in the development and progression of Parkinson's disease (PD). Specifically, microglia are activated in response to lipopolysaccharide (LPS) and become chronic source of cytokines and reactive oxygen species (ROS) production. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex is responsible for extracellular as well as intracellular production of ROS by microglia and its expression is upregulated in PD. Therefore, targeting NADPH oxidase complex activation using an NADPH oxidase inhibitor, i.e., apocyanin seems to be an effective approach. The aim of present study was to investigate the neuroprotective effects of apocyanin in a LPS-induced PD model. LPS (5 μg) was injected intranigral and apocyanin was administered daily at a dose of 10 mg/kg b.wt (i.p.) during the experiment. LPS when injected into the substantia nigra (SN) reproduced the characteristic hallmark features of PD in rats. It elicited an inflammatory response characterized by glial cell activation (Iba-1, GFAP). Furthermore, LPS upregulated the gene expression of nuclear factor-κB (NFκB), iNOS, and gp91PHOX and resulted in an elevated total ROS production as well as NADPH oxidase activity. Subsequently, this resulted in dopaminergic loss as depicted by decreased tyrosine hydroxylase (TH) expression with substantial loss in neurotransmitter dopamine and its metabolites, whereas treatment with apocyanin significantly reduced the number of glial fibrillary acidic protein (GFAP) and Iba-1-positive cells in LPS-treated animals. It also mitigated microglial activation-induced inflammatory response and elevation in NADPH oxidase activity, thus reducing the extracellular as well as intracellular ROS production. The present study indicated that targeting NADPH oxidase can inhibit microglial activation and reduce a broad spectrum of toxic factors generation (i.e., cytokines, ROS, and reactive nitrogen species [RNS

  9. Alternative oxidase and plastoquinol terminal oxidase in marine prokaryotes of the Sargasso Sea.

    PubMed

    McDonald, Allison E; Vanlerberghe, Greg C

    2005-04-11

    Alternative oxidase (AOX) represents a non-energy conserving branch in mitochondrial electron transport while plastoquinol terminal oxidase (PTOX) represents a potential branch in photosynthetic electron transport. Using a metagenomics dataset, we have uncovered numerous and diverse AOX and PTOX genes from the Sargasso Sea. Sequence similarity, synteny and phylogenetic analyses indicate that the large majority of these genes are from prokaryotes. AOX appears to be widely distributed among marine Eubacteria while PTOX is widespread among strains of cyanobacteria closely related to the high-light adapted Prochlorococcus marinus MED4, as well as Synechococcus. The wide distribution of AOX and PTOX in marine prokaryotes may have important implications for productivity in the world's oceans.

  10. 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. PMID:27610153

  11. Structural and kinetic studies on the Ser101Ala variant of choline oxidase: Catalysis by compromise

    SciTech Connect

    Finnegan, S.; Orville, A.; Yuan, H.; Wang, Y.-F.; Weber, I. T.; Gadda, G.

    2010-09-15

    The oxidation of choline catalyzed by choline oxidase includes two reductive half-reactions where FAD is reduced by the alcohol substrate and by an aldehyde intermediate transiently formed in the reaction. Each reductive half-reaction is followed by an oxidative half-reaction where the reduced flavin is oxidized by oxygen. Here, we have used mutagenesis to prepare the Ser101Ala mutant of choline oxidase and have investigated the impact of this mutation on the structural and kinetic properties of the enzyme. The crystallographic structure of the Ser101Ala enzyme indicates that the only differences between the mutant and wild-type enzymes are the lack of a hydroxyl group on residue 101 and a more planar configuration of the flavin in the mutant enzyme. Kinetics established that replacement of Ser101 with alanine yields a mutant enzyme with increased efficiencies in the oxidative half-reactions and decreased efficiencies in the reductive half-reactions. This is accompanied by a significant decrease in the overall rate of turnover with choline. Thus, this mutation has revealed the importance of a specific residue for the optimization of the overall turnover of choline oxidase, which requires fine-tuning of four consecutive half-reactions for the conversion of an alcohol to a carboxylic acid.

  12. A Peroxidase-linked Spectrophotometric Assay for the Detection of Monoamine Oxidase Inhibitors

    PubMed Central

    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. PMID:27610153

  13. A low perfusion rate microreactor for continuous monitoring of enzyme characteristics: application to glucose oxidase.

    PubMed

    Posthuma-Trumpie, G A; Venema, K; van Berkel, W J H; Korf, J

    2007-11-01

    This report describes a versatile and robust microreactor for bioactive proteins physically immobilized on a polyether sulfone filter. The potential of the reactor is illustrated with glucose oxidase immobilized on a filter with a cut-off value of 30 kDa. A flow-injection system was used to deliver the reactants and the device was linked on-line to an electrochemical detector. The microreactor was used for on-line preparation of apoglucose oxidase in strong acid and its subsequent reactivation with flavin adenine dinucleotide. In addition we describe a miniaturized version of the microreactor used to assess several characteristics of femtomole to attomole amounts of glucose oxidase. A low negative potential over the electrodes was used when ferrocene was the mediator in combination with horseradish peroxidase, ensuring the absence of oxidation of electro-active compounds in biological fluids. A low backpressure at very low flow rates is an advantage, which increases the sensitivity. A variety of further applications of the microreactor are suggested.

  14. An amperometric biosensor for fish freshness detection from xanthine oxidase immobilized in polypyrrole-polyvinylsulphonate film.

    PubMed

    Dolmaci, Nezaket; Çete, Servet; Arslan, Fatma; Yaşar, Ahmet

    2012-08-01

    A new amperometric biosensor was developed for determining hypoxanthine in fish meat. Xanthine oxidase with pyrrole and polyvinylsulphonate was immobilized on the surface of a platinum electrode by electropolymerization. The determination of xanthine-hypoxanthine was performed by means of oxidation of uric acid liberated during the enzyme reaction on the surface of the enzyme electrode at + 0.30V (SCE). The effects of pH, substrate concentration, and temperature on the response of the xanthine-hypoxanthine biosensor were investigated. The linear working range of the enzyme electrode was 1.0 × 10(-7) -1.0 × 10(-3) M of the hypoxanthine concentration, and the detection limit was 1.0 × 10(-7)M. The apparent K(m(app)) and I(max) of the immobilized xanthine oxidase were found to be 0.0154 mM and 1.203 μA/mM, respectively. The best pH and temperature value for xanthine oxidase were selected as 7.75 and 25°C, respectively. The sensor was used for the determination of hypoxhantine in fish meat. Results show that the fish degraded very rapidly after seven days and the hypoxanthine amount was found to increase over days of storage.

  15. Enzymatic polymerization of dihydroquercetin using bilirubin oxidase.

    PubMed

    Khlupova, M E; Vasil'eva, I S; Shumakovich, G P; Morozova, O V; Chertkov, V A; Shestakova, A K; Kisin, A V; Yaropolov, A I

    2015-02-01

    Dihydroquercetin (or taxifolin) is one of the most famous flavonoids and is abundant in Siberian larch (Larix sibirica). The oxidative polymerization of dihydroquercetin (DHQ) using bilirubin oxidase as a biocatalyst was investigated and some physicochemical properties of the products were studied. DHQ oligomers (oligoDHQ) with molecular mass of 2800 and polydispersity of 8.6 were obtained by enzymatic reaction under optimal conditions. The oligomers appeared to be soluble in dimethylsulfoxide, dimethylformamide, and methanol. UV-visible spectra of oligoDHQ in dimethylsulfoxide indicated the presence of highly conjugated bonds. The synthesized oligoDHQ was also characterized by FTIR and (1)H and (13)C NMR spectroscopy. Comparison of NMR spectra of oligoDHQ with DHQ monomer and the parent flavonoids revealed irregular structure of a polymer formed via the enzymatic oxidation of DHQ followed by nonselective radical polymerization. As compared with the monomer, oligoDHQ demonstrated higher thermal stability and high antioxidant activity.

  16. [NADPH oxidases, Nox: new isoenzymes family].

    PubMed

    Chuong Nguyen, Minh Vu; Lardy, Bernard; Paclet, Marie-Hélène; Rousset, Francis; Berthier, Sylvie; Baillet, Athan; Grange, Laurent; Gaudin, Philippe; Morel, Françoise

    2015-01-01

    NADPH oxidases, Nox, are a family of isoenzymes, composed of seven members, whose sole function is to produce reactive oxygen species (ROS). Although Nox catalyze the same enzymatic reaction, they acquired from a common ancestor during evolution, specificities related to their tissue expression, subcellular localization, activation mechanisms and regulation. Their functions could vary depending on the pathophysiological state of the tissues. Indeed, ROS are not only bactericidal weapons in phagocytes but also essential cellular signaling molecules and their overproduction is involved in chronic diseases and diseases of aging. The understanding of the mechanisms involved in the function of Nox and the emergence of Nox inhibitors, require a thorough knowledge of their nature and structure. The objectives of this review are to highlight, in a structure/function approach, the main similar and differentiated properties shared by the human Nox isoenzymes.

  17. Visualization of monoamine oxidase in human brain

    SciTech Connect

    Fowler, J.S.; Volkow, N.D.; Wang, G.J.; Pappas, N.; Shea, C.; MacGregor, R.R.; Logan, J.

    1996-12-31

    Monoamine oxidase is a flavin enzyme which exists in two subtypes, MAO A and MAO B. In human brain MAO B predominates and is largely compartmentalized in cell bodies of serotonergic neurons and glia. Regional distribution of MAO B was determined by positron computed tomography with volunteers after the administration of deuterium substituted [11C]L-deprenyl. The basal ganglia and thalamus exhibited the greatest concentrations of MAO B with intermediate levels in the frontal cortex and cingulate gyrus while lowest levels were observed in the parietal and temporal cortices and cerebellum. We observed that brain MAO B increases with are in health normal subjects, however the increases were generally smaller than those revealed with post-mortem studies.

  18. Drugs related to monoamine oxidase activity.

    PubMed

    Fišar, Zdeněk

    2016-08-01

    Progress in understanding the role of monoamine neurotransmission in pathophysiology of neuropsychiatric disorders was made after the discovery of the mechanisms of action of psychoactive drugs, including monoamine oxidase (MAO) inhibitors. The increase in monoamine neurotransmitter availability, decrease in hydrogen peroxide production, and neuroprotective effects evoked by MAO inhibitors represent an important approach in the development of new drugs for the treatment of mental disorders and neurodegenerative diseases. New drugs are synthesized by acting as multitarget-directed ligands, with MAO, acetylcholinesterase, and iron chelation as targets. Basic information is summarized in this paper about the drug-induced regulation of monoaminergic systems in the brain, with a focus on MAO inhibition. Desirable effects of MAO inhibition include increased availability of monoamine neurotransmitters, decreased oxidative stress, decreased formation of neurotoxins, induction of pro-survival genes and antiapoptotic factors, and improved mitochondrial functions.

  19. NADPH Oxidases in Lung Health and Disease

    PubMed Central

    Bernard, Karen; Hecker, Louise; Luckhardt, Tracy R.; Cheng, Guangjie

    2014-01-01

    Abstract Significance: The evolution of the lungs and circulatory systems in vertebrates ensured the availability of molecular oxygen (O2; dioxygen) for aerobic cellular metabolism of internal organs in large animals. O2 serves as the physiologic terminal acceptor of mitochondrial electron transfer and of the NADPH oxidase (Nox) family of oxidoreductases to generate primarily water and reactive oxygen species (ROS), respectively. Recent advances: The purposeful generation of ROS by Nox family enzymes suggests important roles in normal physiology and adaptation, most notably in host defense against invading pathogens and in cellular signaling. Critical issues: However, there is emerging evidence that, in the context of chronic stress and/or aging, Nox enzymes contribute to the pathogenesis of a number of lung diseases. Future Directions: Here, we review evolving functions of Nox enzymes in normal lung physiology and emerging pathophysiologic roles in lung disease. Antioxid. Redox Signal. 20, 2838–2853. PMID:24093231

  20. ROS signalling, NADPH oxidases and cancer.

    PubMed

    Landry, William D; Cotter, Thomas G

    2014-08-01

    ROS (reactive oxygen species) have long been regarded as a series of destructive molecules that have a detrimental effect on cell homoeostasis. In support of this are the myriad antioxidant defence systems nearly all eukaryotic cells have that are designed to keep the levels of ROS in check. However, research data emerging over the last decade have demonstrated that ROS can influence a range of cellular events in a manner similar to that seen for traditional second messenger molecules such as cAMP. Hydrogen peroxide (H2O2) appears to be the main ROS with such signalling properties, and this molecule has been shown to affect a wide range of cellular functions. Its localized synthesis by the Nox (NADPH oxidase) family of enzymes and how these enzymes are regulated is of particular interest to those who work in the field of tumour biology.

  1. Auxin-activated NADH oxidase activity of soybean plasma membranes is distinct from the constitutive plasma membrane NADH oxidase and exhibits prion-like properties

    NASA Technical Reports Server (NTRS)

    Morre, D. James; Morre, Dorothy M.; Ternes, Philipp

    2003-01-01

    The hormone-stimulated and growth-related cell surface hydroquinone (NADH) oxidase activity of etiolated hypocotyls of soybeans oscillates with a period of about 24 min or 60 times per 24-h day. Plasma membranes of soybean hypocotyls contain two such NADH oxidase activities that have been resolved by purification on concanavalin A columns. One in the apparent molecular weight range of 14-17 kDa is stimulated by the auxin herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The other is larger and unaffected by 2,4-D. The 2,4-D-stimulated activity absolutely requires 2,4-D for activity and exhibits a period length of about 24 min. Also exhibiting 24-min oscillations is the rate of cell enlargement induced by the addition of 2,4-D or the natural auxin indole-3-acetic acid (IAA). Immediately following 2,4-D or IAA addition, a very complex pattern of oscillations is frequently observed. However, after several hours a dominant 24-min period emerges at the expense of the constitutive activity. A recruitment process analogous to that exhibited by prions is postulated to explain this behavior.

  2. 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. PMID:27295021

  3. Plastid terminal oxidase 2 (PTOX2) is the major oxidase involved in chlororespiration in Chlamydomonas

    PubMed Central

    Houille-Vernes, Laura; Rappaport, Fabrice; Wollman, Francis-André; Alric, Jean; Johnson, Xenie

    2011-01-01

    By homology with the unique plastid terminal oxidase (PTOX) found in plants, two genes encoding oxidases have been found in the Chlamydomonas genome, PTOX1 and PTOX2. Here we report the identification of a knockout mutant of PTOX2. Its molecular and functional characterization demonstrates that it encodes the oxidase most predominantly involved in chlororespiration in this algal species. In this mutant, the plastoquinone pool is constitutively reduced under dark-aerobic conditions, resulting in the mobile light-harvesting complexes being mainly, but reversibly, associated with photosystem I. Accordingly, the ptox2 mutant shows lower fitness than wild type when grown under phototrophic conditions. Single and double mutants devoid of the cytochrome b6f complex and PTOX2 were used to measure the oxidation rates of plastoquinols via PTOX1 and PTOX2. Those lacking both the cytochrome b6f complex and PTOX2 were more sensitive to light than the single mutants lacking either the cytochrome b6f complex or PTOX2, which discloses the role of PTOX2 under extreme conditions where the plastoquinone pool is overreduced. A model for chlororespiration is proposed to relate the electron flow rate through these alternative pathways and the redox state of plastoquinones in the dark. This model suggests that, in green algae and plants, the redox poise results from the balanced accumulation of PTOX and NADPH dehydrogenase. PMID:22143777

  4. Polyphenol Oxidase Activity Expression in Ralstonia solanacearum

    PubMed Central

    Hernández-Romero, Diana; Solano, Francisco; Sanchez-Amat, Antonio

    2005-01-01

    Sequencing of the genome of Ralstonia solanacearum revealed several genes that putatively code for polyphenol oxidases (PPOs). To study the actual expression of these genes, we looked for and detected all kinds of PPO activities, including laccase, cresolase, and catechol oxidase activities, in cellular extracts of this microorganism. The conditions for the PPO assays were optimized for the phenolic substrate, pH, and sodium dodecyl sulfate concentration used. It was demonstrated that three different PPOs are expressed. The genes coding for the enzymes were unambiguously correlated with the enzymatic activities detected by generation of null mutations in the genes by using insertional mutagenesis with a suicide plasmid and estimating the changes in the levels of enzymatic activities compared to the levels in the wild-type strain. The protein encoded by the RSp1530 locus is a multicopper protein with laccase activity. Two other genes, RSc0337 and RSc1501, code for nonblue copper proteins exhibiting homology to tyrosinases. The product of RSc0337 has strong tyrosine hydroxylase activity, and it has been shown that this enzyme is involved in melanin synthesis by R. solanacearum. The product of the RSc1501 gene is an enzyme that shows a clear preference for oxidation of o-diphenols. Preliminary characterization of the mutants obtained indicated that PPOs expressed by R. solanacearum may participate in resistance to phenolic compounds since the mutants exhibited higher sensitivity to l-tyrosine than the wild-type strain. These results suggest a possible role in the pathogenic process to avoid plant resistance mechanisms involving the participation of phenolic compounds. PMID:16269713

  5. Nox family NADPH oxidases: Molecular mechanisms of activation.

    PubMed

    Brandes, Ralf P; Weissmann, Norbert; Schröder, Katrin

    2014-11-01

    NADPH oxidases of the Nox family are important enzymatic sources of reactive oxygen species (ROS). Numerous homologue-specific mechanisms control the activity of this enzyme family involving calcium, free fatty acids, protein-protein interactions, intracellular trafficking, and posttranslational modifications such as phosphorylation, acetylation, or sumoylation. After a brief review on the classic pathways of Nox activation, this article will focus on novel mechanisms of homologue-specific activity control and on cell-specific aspects which govern Nox activity. From these findings of the recent years it must be concluded that the activity control of Nox enzymes is much more complex than anticipated. Moreover, depending on the cellular activity state, Nox enzymes are selectively activated or inactivated. The complex upstream signaling aspects of these events make the development of "intelligent" Nox inhibitors plausible, which selectively attenuate disease-related Nox-mediated ROS formation without altering physiological signaling ROS. This approach might be of relevance for Nox-mediated tissue injury in ischemia-reperfusion and inflammation and also for chronic Nox overactivation as present in cancer initiation and cardiovascular disease.

  6. Reducing peanut allergens by high pressure combined with polyphenol oxidase

    NASA Astrophysics Data System (ADS)

    Chung, Si-Yin; Houska, Milan; Reed, Shawndrika

    2013-12-01

    Polyphenol oxidase (PPO) has been shown to reduce major peanut allergens. Since high pressure (HP) can increase enzyme activity, we postulated that further reduction of peanut allergens can be achieved through HP combined with PPO. Peanut extracts containing caffeic acid were treated with each of the following: (1) HP; (2) HP+PPO; (3) PPO; and (4) none. HP was conducted at 300 and 500 MPa, each for 3 and 10 min, 37 °C. After treatment, SDS-PAGE was performed and allergenic capacity (IgE binding) was determined colorimetrically in inhibition enzyme-linked immunosorbent assay and Western blots, using a pooled plasma from peanut-allergic patients. Data showed that HP alone had no effect on major peanut allergens. However, HP at 500 MPa combined with PPO (HP500/PPO) induced a higher (approximately twofold) reduction of major peanut allergens and IgE binding than PPO alone or HP300/PPO. There was no difference between treatment times. We concluded that HP500/PPO at 3-min enhanced a twofold reduction of the allergenic capacity of peanut extracts, as compared to PPO itself.

  7. cDNA cloning and expression of potato polyphenol oxidase.

    PubMed

    Hunt, M D; Eannetta, N T; Yu, H; Newman, S M; Steffens, J C

    1993-01-01

    Polyphenol oxidases (PPOs) of plants are copper metalloproteins which catalyze the oxidation of mono- and o-diphenols to o-diquinones. Although PPOs are believed to be primarily responsible for the deleterious browning of many fruit and vegetable crops and are thought to be involved in plant-pest interactions, direct evidence for these roles is lacking. We report the cloning of two PPO cDNAs from Solanum tuberosum leaves. These cDNAs exhibit 97% and 98% sequence similarity at the DNA and deduced amino acid levels, respectively. Putative copper-binding regions of both cDNAs are very similar to those of mammalian, bacterial and Neurospora tyrosinases. Both leaf PPO cDNAs appear to encode polypeptides which are processed to a mature molecular weight of 57,000. In potato leaves, petioles, roots, and flowers, PPO is encoded by ca. 2 kb transcripts. Leaf PPO mRNA is developmentally regulated and only detectable in young foliage. In contrast, the protein profile of immunologically detectable PPO remains constant from the apical node through the eleventh leaf node. PMID:7678763

  8. Human sulfite oxidase electrochemistry on gold nanoparticles modified electrode.

    PubMed

    Frasca, Stefano; Rojas, Oscar; Salewski, Johannes; Neumann, Bettina; Stiba, Konstanze; Weidinger, Inez M; Tiersch, Brigitte; Leimkühler, Silke; Koetz, Joachim; Wollenberger, Ulla

    2012-10-01

    The present study reports a facile approach for sulfite biosensing, based on enhanced direct electron transfer of a human sulfite oxidase (hSO) immobilized on a gold nanoparticles modified electrode. The spherical core shell AuNPs were prepared via a new method by reduction of HAuCl(4) with branched poly(ethyleneimine) in an ionic liquids resulting particles with a diameter less than 10nm. These nanoparticles were covalently attached to a mercaptoundecanoic acid modified Au-electrode where then hSO was adsorbed and an enhanced interfacial electron transfer and electrocatalysis was achieved. UV/Vis and resonance Raman spectroscopy, in combination with direct protein voltammetry, are employed for the characterization of the system and reveal no perturbation of the structural integrity of the redox protein. The proposed biosensor exhibited a quick steady-state current response, within 2 s, a linear detection range between 0.5 and 5.4 μM with a high sensitivity (1.85 nA μM(-1)). The investigated system provides remarkable advantages in the possibility to work at low applied potential and at very high ionic strength. Therefore these properties could make the proposed system useful in the development of bioelectronic devices and its application in real samples.

  9. Oxidation reaction by xanthine oxidase: theoretical study of reaction mechanism.

    PubMed

    Amano, Tatsuo; Ochi, Noriaki; Sato, Hirofumi; Sakaki, Shigeyoshi

    2007-07-01

    The oxidation process by molybdenum-containing enzyme, xanthine oxidase, is theoretically studied with a model complex representing the reaction center and a typical benchmark substrate, formamide. Comparisons were systematically made among reaction mechanisms proposed previously. In the concerted and stepwise mechanisms that were theoretically discussed previously, the oxidation reaction takes place with a moderate activation barrier. However, the product is less stable than the reactant complex, which indicates that these mechanisms are unlikely. Moreover, the product of the concerted mechanism is not consistent with the isotope experimental result. In addition to those mechanisms, another mechanism initiated by the deprotonation of the active site was newly investigated here. In the transition state of this reaction, the carbon atom of formamide interacts with the oxo ligand of the Mo center and the hydrogen atom is moving from the carbon atom to the thioxo ligand. This reaction takes place with a moderate activation barrier and considerably large exothermicity. Furthermore, the product by this mechanism is consistent with the isotope experimental result. Also, our computations clearly show that the deprotonation of the active site occurs with considerable exothermicity in the presence of glutamic acid and substrate. The intermediate of the stepwise mechanism could not be optimized in the case of the deprotonated active site. From all these results, it should be concluded that the one-step mechanism with the deprotonated active site is the most plausible.

  10. Homology in the structure and the prosthetic groups between two different terminal ubiquinol oxidases, cytochrome a1 and cytochrome o, of Acetobacter aceti.

    PubMed

    Matsushita, K; Ebisuya, H; Adachi, O

    1992-12-01

    Acetobacter aceti produces two different terminal oxidases dependent on the culture conditions, shaking and static cultures. Cells grown on shaking culture contain cytochrome a1, while cytochrome o is present in cells grown on static culture. Cytochrome a1 and cytochrome o of A. aceti were compared especially with respect to the protein structure and the prosthetic groups. Cytochrome a1 exhibited lower CN sensitivity and higher affinity for O2 than cytochrome o. Both terminal oxidases consisted of four nonidentical polypeptides of which the molecular sizes were identical between both enzymes. Cytochrome a1 cross-reacted with an antibody raised against cytochrome o at the same level as cytochrome o did, and an antibody elicited against cytochrome a1 cross-reacted with both cytochrome o and cytochrome a1 at the same intensity, which indicates that both oxidases are indistinguishable immunochemically. Furthermore, almost the same peptide mapping pattern with chymotrypsin was observed in subunit I and in subunit II between both terminal oxidases, and the amino-terminal sequences in the subunit II of both oxidases were identical at least in their 10 amino acids. As for the prosthetic groups, both oxidases were shown to contain two heme-irons and one copper atom. Further, high performance liquid chromatography analysis of the heme moieties extracted from both the purified enzymes indicated that cytochrome a1 contains hemes b and a at a ratio of 1 to 1, whereas cytochrome o contains the same amounts of hemes b and o. Thus, data indicate that cytochrome a1 and cytochrome o of A. aceti are cytochrome ba and cytochrome bo ubiquinol oxidases, respectively, and that both oxidases have a closely similar protein structure and prosthetic groups, in which only heme a in the heme/copper binuclear center of cytochrome a1 is replaced by heme o in that of cytochrome o.

  11. Dephenolization of industrial wastewaters catalyzed by polyphenol oxidase

    SciTech Connect

    Atlow, S.C.; Bonadonna-Aparo, L.; Klibanov, A.M.

    1984-01-01

    A new enzymatic method for the removal of phenols from industrial aqueous effluents has been developed. The method uses the enzyme polyphenol oxidase which oxidizes phenols to the corresponding o-quinones; the latter then undergo a nonenzymatic polymerization to form water-insoluble aggregates. Therefore, the enzyme in effect precipitates phenols from water. Polyphenol oxidase has been found to nearly completely dephenolize solutions of phenol in the concentration range from 0.01 to 1.0 g/L. The enzymatic treatment is effective over a wide range of pH and temperature; a crude preparation of polyphenol oxidase (mushroom extract) is as effective as a purified, commercially obtained version. In addition to phenol itself, polyphenol oxidase is capable of precipitating from water a number of substituted phenols (cresols, chlorophenols, naphthol, etc.). Also, even pollutants which are unreactive towards polyphenol oxidase can be enzymatically coprecipitated with phenol. The polyphenol oxidase treatment has been successfully used to dephenolize two different real industrial wastewater samples, from a plant producing triarylphosphates and from a coke plant. The advantage of the polyphenol oxidase dephenolization over the peroxidase-catalyzed one previously elaborated by the authors is that the former enzyme uses molecular oxygen instead of costly hydrogen peroxide (used by peroxidase) as an oxidant.

  12. Iodide Oxidation by a Novel Multicopper Oxidase from the Alphaproteobacterium Strain Q-1

    PubMed Central

    Suzuki, Mio; Eda, Yoshifumi; Ohsawa, Shiaki; Kanesaki, Yu; Yoshikawa, Hirofumi; Tanaka, Kan; Muramatsu, Yasuyuki; Yoshikawa, Jun; Sato, Ikuo; Fujii, Takaaki

    2012-01-01

    Alphaproteobacterium strain Q-1 is able to oxidize iodide (I−) to molecular iodine (I2) by an oxidase-like enzyme. One of the two isoforms of the iodide-oxidizing enzyme (IOE-II) produced by this strain was excised from a native polyacrylamide gel, eluted, and purified. IOE-II appeared as a single band (51 kDa) and showed significant in-gel iodide-oxidizing activity in sodium dodecyl sulfate-polyacrylamide gel electrophoresis without heat treatment. However, at least two bands with much higher molecular masses (150 and 230 kDa) were observed with heat treatment (95°C, 3 min). IOE-II was inhibited by NaN3, KCN, EDTA, and a copper chelator, o-phenanthroline. In addition to iodide, IOE-II showed significant activities toward phenolic compounds such as syringaldazine, 2,6-dimethoxy phenol, and p-phenylenediamine. IOE-II contained copper atoms as prosthetic groups and had UV/VIS absorption peaks at 320 and 590 nm. Comparison of several internal amino acid sequences obtained from trypsin-digested IOE-II with a draft genome sequence of strain Q-1 revealed that the products of two open reading frames (IoxA and IoxC), with predicted molecular masses of 62 and 71 kDa, are involved in iodide oxidation. Furthermore, subsequent tandem mass spectrometric analysis repeatedly detected peptides from IoxA and IoxC with high sequence coverage (32 to 40%). IoxA showed homology with the family of multicopper oxidases and included four copper-binding regions that are highly conserved among various multicopper oxidases. These results suggest that IOE-II is a multicopper oxidase and that it may occur as a multimeric complex in which at least two proteins (IoxA and IoxC) are associated. PMID:22447601

  13. 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

  14. 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

  15. 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

  16. CotA, a multicopper oxidase from Bacillus pumilus WH4, exhibits manganese-oxidase activity.

    PubMed

    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

  17. 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

  18. 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

  19. NADPH oxidase deficiency in X-linked chronic granulomatous disease.

    PubMed Central

    Hohn, D C; Lehrer, R I

    1975-01-01

    We measured the cyanide-insensitive pyridine nucleotide oxidase activity of fractionated resting and phagocytic neutrophils from 11 normal donors, 1 patient with hereditary deficiency of myeloperoxidase, and 7 patients with X-linked chronic granulomatous disease (CGD). When measured under optimal conditions (at pH 5.5 and in the presence of 0.5 mM Mn++), NADPH oxidase activity increased fourfold with phagocytosis and was six-fold higher than with NADH. Phagocytic neutrophils from patients with CGD were markedly deficient in NADPH oxidase activity. Images PMID:235560

  20. Identification of yeasts from clinical specimens by oxidase test.

    PubMed

    Kumar, S; Arora, B S; Mathur, M D

    2000-10-01

    A total of 100 yeasts and yeast like fungi isolates from clinical specimens were negative for oxidase production on Sabouraud dextrose agar. When grown on Columbia agar, chocolate agar, tryptose agar, Mueller-Hinton agar, brain heart infusion and a medium resembling Sabouraud's dextrose agar but with starch instead of dextrose, all the isolate of Candida albicans (55), C. guilliermondii (6), C. parapsilosis (14), C. tropicalis (6), C. pseudotropicalis (6) and Crytococcus neoformans (2) were positive for oxidase producation. Torulopsis glabrata (2), Saccharomyces cervisiae (2) and two out of seven isolates of C. krusei were negative for oxidase test. PMID:11344606