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PubMed1979

[Phenoloxidase and peroxidase activity in presens of different effectors (author's transl)].

Small gut of the white rat was sectioned by the cryostat in slides of 10 microns and following effectors were investigated: KCN, sodium-diethyl-dithiocarbamate (DDC), NaF, sodium acetate, sodium pyruvate, 0.1 N HCl, 0.1 N NaOH, NaN3, and copper sulfate. Phenoloxidase is completely inhibited by DDC and HCl, incompletely by pyruvate and NaN3. Peroxidase also is completely inactivated by HCl and incompletely by NaN3. DDC has no effect on peroxidase, because a precipitate is formed. Neither of both enzyme activities is inhibited by KCN. In presence of KCN and copper sulfate an unspezific colouring of the tissue resulted in the phenoloxidase proof. Non of the investigated effectors is suitable to differentiate between phenoloxidase and peroxidase in histochemical investigations.

Animals↗
PubMed1989

Different selectivities of oxidants during oxidation of methionine residues in the alpha-1-proteinase inhibitor.

Oxidation of the reactive site methionine (Met) in alpha-1-proteinase inhibitor (alpha-1-PI) to methionine sulfoxide (Met(O] is known to cause depletion of its elastase inhibitory activity. To estimate the selectivity of different oxidants in converting Met to Met(O) in alpha-1-PI, we measured the molar ratio Met(O)/alpha-1-PI at total inactivation. This ratio was determined to be 1.2 for both the myeloperoxidase/H2O2/chloride system and the related compound NH2Cl. With taurine monochloramine, another myeloperoxidase-related oxidant, 1.05 mol Met(O) were generated per mol alpha-1-PI during inactivation. These oxidants attack preferentially one Met residue in alpha-1-PI, which is identical with Met 358, as concluded from the parallelism of loss of elastase inhibitory activity and oxidation of Met. A similar high specificity for Met oxidation was determined for the xanthine oxidase-derived oxidants. In contrast, the ratio found for ozone and m-chloroperoxybenzoic acid was 6.0 and 5.0, respectively, indicating oxidation of additional Met residues besides the relative site Met in alpha-1-PI, i.e. unselective action of these oxidants. Further studies were performed on the efficiency of oxidants for total depletion of the elastase inhibitory capacity of alpha-1-PI. Ozone and m-chloroperoxybenzoic acid were 10-fold less effective and the superoxide anion/hydroxyl radicals were 30-50-fold less effective to inactivate the elastase inhibitory activity as compared to the myeloperoxidase-derived oxidants. The myeloperoxidase-related oxidants are discussed as important regulators of alpha-1-PI activity in vivo.

Animals↗
PubMed1989

Simultaneous assay of hypoxanthine, xanthine and allopurinol by high-performance liquid chromatography and activation of immobilized xanthine oxidase as an enzyme reactor.

A selective and sensitive assay of substrates (hypoxanthine, xanthine and allopurinol) of xanthine oxidase by reversed-phase liquid chromatography coupled with the use of immobilized enzyme reactors is described. These compounds were oxidized by immobilized xanthine oxidase and produced hydrogen peroxide, which was determined fluorometrically using immobilized peroxidase and p-hydroxyphenylacetic acid. The detection limits of hypoxanthine, xanthine and allopurinol were approximately 50, 120 and 130 pg per injection, respectively. Immobilized xanthine oxidase inhibited by oxipurinol during the assay was reactivated by 2,6-dichlorophenolindophenol and could be used for a long period without a significant activity loss. These methods were applied to plasma and urine samples.

Allopurinol↗
PubMed2000

Reagentless biosensors based on self-deposited redox polyelectrolyte-oxidoreductases architectures.

Reagentless fructose and alcohol biosensors have been produced with a versatile enzyme immobilisation technique which mimics natural interactions and flexibility of living systems. The electrode architecture is built up on electrostatic interactions by the sequential adsorption of redox polyelectrolytes and redox enzymes giving rise to the efficient transformation of substrate fluxes into electrocatalytic currents. All investigated multilayer structures were self-deposited on 3-mercapto-1-propanesulfonic acid monolayers self-assembled on gold electrodes. Fructose dehydrogenase, horseradish peroxidase (HRP) and the couple HRP-alcohol oxidase were electrochemically connected with a cationic poly[(vinylpyridine)Os(bpy)2Cl] redox polymer (RP) interface in a layer-by-layer self-deposited architecture. The dependence of the distance on the electrochemical response of this interface was also studied showing a clear decrease in the Faradaic current when the distance to the electrode surface was increased. The sensitivities obtained for each biosensor were 19.3, 58.1 and 10.6 mA M(-1) cm(-1) for fructose, H2O2 and methanol, respectively. The sensitivity values can be easily controlled by a rational deposition and manipulation of the charge in the catalytic layers. The electrostatic assembly of the electrochemical interface and the catalytic layers resulted in integrated biochemical systems in which mass transfer diffusion and heterogeneous catalytic and electron transfer steps are efficiently coupled and can be easily manipulated.

Alcohol Oxidoreductases↗
PubMed1998

Mechanism of inhibition of electron transfer by amino acid replacement K362M in a proton channel of Rhodobacter sphaeroides cytochrome c oxidase.

The three-dimensional structure of cytochrome coxidase (COX) reveals two potential input proton channels connecting the redox core of the enzyme with the negatively charged (N-) aqueous phase. These are denoted as the K-channel (for the highly conserved lysine residue, K362 in Rhodobacter sphaeroides COX) and the D-channel (for the highly conserved aspartate gating the channel at the N-side, D132 in R. sphaeroides). In this paper, it is shown that the K362M mutant form of COX from R. sphaeroides, although unable to turnover with dioxygen as electron acceptor, can utilize hydrogen peroxide as an electron acceptor, with either cytochrome c or ferrocyanide as electron donors, with turnover that is close to that of the wild-type enzyme. The peroxidase activity is similar to that of the wild-type oxidase and is coupled to the generation of a membrane potential and to proton pumping. In contrast, no peroxidase activity is revealed in the D-channel mutants of COX, D132N, and E286Q. Reduction by dithionite of heme a3 in the fully oxidized oxidase is severely inhibited in the K362M mutant, but not in the D132N mutant. Apparently, mutations in the D-channel arrest COX turnover by inhibiting proton uptake associated with the proton-pumping peroxidase phase of the COX catalytic cycle. In contrast, the K-channel appears to be dispensable for the peroxidase phase of the catalytic cycle, but is required for the initial reduction of the heme-copper binuclear center in the first half of the catalytic cycle.

Amino Acid Substitution↗
PubMed2002

Effect of wash water temperature and chlorination on phenolic metabolism and browning of stored iceberg lettuce photosynthetic and vascular tissues.

Cut tissues from distinct anatomical locations in iceberg lettuce (Lactuca sativa L.) were subjected to washing in cold (4 degrees C) and warm (47 degrees C) water with or without chlorine to assess their propensity to discoloration during storage. Total protein (Bradford method) and phenolic (TPH; Folin-Ciocalteu method) contents and polyphenol oxidase (PPO; spectrophotometric method using catechol as a substrate), peroxidase (POD; guaiacol substrate), and phenylalanine ammonia-lyase (PAL; phenylalanine substrate) activities were determined in photosynthetic and vascular tissue from outer and inner leaves. Unprocessed photosynthetic and inner leaf tissues had significantly higher (P < 0.05) levels of protein and TPH and PPO and POD activities than vascular and outer leaf tissues. PAL activities (on a fresh weight basis) were similar in all tissues. Changes in browning (light reflectance measurement) and phenolic metabolism in all four tissue types were observed during aerobic storage at 5 degrees C over 10 days. PAL activity increased in all tissues after 1-2 days of storage and then gradually decreased. POD activity also increased steadily for the storage duration. Protein content and PPO activity remained constant. Edge browning (measured with a Minolta Chroma Meter) and TPH increased in all tissues, especially in outer vascular tissue. Cut photosynthetic and vascular tissues washed at 4 and 47 degrees C with and without 100 microg mL(-1) chlorine for 3 min were analyzed during 7 days in storage at 5 degrees C. Enzyme activities and accumulation of phenolics in all tissues washed at 47 degrees C were significantly (P < 0.05) lower compared to controls or tissues washed at 4 degrees C. Chlorine had no additional effect at 47 degrees C but significantly (P < 0.05) reduced browning and accumulation of phenolics in lettuce washed at 4 degrees C. These results showed that inherent differences between tissues affect phenolic metabolism and browning in stored, fresh-cut lettuce.

Catechol Oxidase↗
PubMed1987

Neuromelanogenic and cytotoxic properties of canine brainstem peroxidase.

We have isolated a heme protein from canine midbrains that possesses potent peroxidase activity. This enzyme catalyzes the oxidation of dopamine to neuromelanin in the presence of H2O2. We have further shown that the isolated peroxidase possesses potent cytotoxic activity in the presence of superoxide or H2O2 and Cl-. The enzyme possesses an endogenous NAD(P)H oxidase activity that can promote the cytotoxic activity by virtue of its production of superoxide. Other enzymes such as dihydroorotate dehydrogenase and galactose oxidase, which produce O2- and H2O2, respectively, are also effective in promoting the cytotoxic activity of the brainstem peroxidase. Although rat erythrocytes were routinely used as the target cell, other cell types, including rat hepatoma and mouse neuroblastoma cells, are also susceptible to the toxic action of the peroxidase. The cytotoxic action of the brainstem peroxidase is dramatically enhanced by kainic acid and is significantly enhanced by Mn2+, whereas dopamine was found to be a potent inhibitor of the cytotoxic activity. Based on these findings, we postulate a central role for the brainstem peroxidase in dopamine metabolism as well as in the biochemical and anatomical changes associated with Parkinson's disease.

Animals↗
PubMed1984

A glucose oxidase immunoenzyme stain for the detection of viral antigen or antibody on nitrocellulose transfer blots.

Separation of mixtures of proteins by polyacrylamide gel electrophoresis followed by transfer of the proteins to support media such as nitrocellulose and detection by immunologic procedures provides a powerful analytic tool for assaying the antibody specificity of antisera or for following the purification of antigens. This technique requires fewer assumptions about antigenic solubility or antibody reactivity than immunoprecipitation methods. We present a glucose oxidase immunoenzyme staining procedure for protein blots and illustrate its use for detecting antibody to several viruses. The glucose oxidase immunoenzyme stain has a lower background than some peroxidase stains. We have detected as little as 1 microgram/ml of antiviral antibody using this stain.

Aleutian Mink Disease Virus↗
PubMed2005

Transgenic expression of human complement regulators reduces skeletal muscle ischaemia/reperfusion injury in mice.

This study aimed to explore the hypothesis that activated complement components contribute significantly to I/R (ischaemia/reperfusion) injury in skeletal muscle. After 50, 70 and 90 min of tourniquet ischaemia and 24 h of reperfusion, viability of the medial gastrocnemius muscle in CBA-C57BL/6 wild-type mice, assessed histochemically by reduction of NBT (Nitro Blue Tetrazolium) dye, was 60, 21 and 8% respectively. Skeletal muscle viability after 70 min of ischaemia and 24 h of reperfusion in transgenic mice expressing a combination of human CD46, CD55 and CD59, all inhibitors of complement activation, was 45% compared with 24% in ischaemic reperfused wild-type mice (P=0.008; n=6 per group). Muscle from sham-treated transgenic mice and wild-type littermates had no significant loss of viability relative to normal contralateral gastrocnemius muscle. A significant reduction in myeloperoxidase activity (a measure of neutrophil infiltration), xanthine oxidase activity (a source of free radicals) and water content (a measure of oedema) was observed in ischaemic reperfused muscle from transgenic mice compared with ischaemic reperfused wild-type muscle (P<0.05). Haematoxylin and eosin-stained histological sections also showed less damage and less apparent leucocyte infiltration in muscles from ischaemic reperfused transgenic mice than those from wild-type animals given the same degree of injury. Muscles from sham-treated transgenic and wild-type controls were almost identical with normal muscle. It is concluded that complement activation contributes to the pathogenesis of I/R injury in murine skeletal muscle, resulting in increased neutrophil infiltration into the injured muscle, increased free radical production and vascular permeability during reperfusion, and a net detrimental effect on muscle viability.

Animals↗
PubMed2006

Radical scavenging and anti-inflammatory properties of STW 5 (Iberogast) and its components.

A combination of ethanolic extracts from nine medicinal plants is successfully used in STW 5 (Iberogast((R))) for treatment of gastrointestinal disorders. To elucidate possible modes of action, the focus of this study is on antioxidant properties of the phytomedicine STW 5. In fact, functional gastrointestinal diseases, such as non-ulcer dyspepsia (NUD) and irritable bowel syndrome, are often initiated by or correlated to inflammatory processes, where oxidants such as reactive oxygen species (ROS) play a crucial role. Prominent in vivo sources of ROS generation are represented by the enzymes xanthine oxidase (XOD) or myeloperoxidase (MPO). Applying these enzymes in models in vitro, we show that STW 5 and its components possess strong antioxidant activities. Depending on the model investigated, even pro-oxidant activities of single components of STW 5 could be observed. Interestingly, these effects were absent in STW 5, indicating cooperation between the components. Moreover, if one of the component extracts of STW 5 is omitted, the antioxidant activity is reduced. Thus we conclude that all the single extracts combined in STW 5 are of importance for the therapeutic effect, working in concert. The component of STW 5 performing best in vitro differed with the model investigated, respectively, with ROS and ROS generators. In the XOD system, the extracts of lemon balm leaf and peppermint leaf showed the best antioxidant result, whereas concerning MPO driven chlorination reactions, bitter candy tuft extract was the most efficient antioxidant. Best protection against peroxynitrite induced oxidation of methionine like sulfur-compounds exhibited the STW 5 components lemon balm leaf, Matricaria flower and peppermint leaf.

Anti-Inflammatory Agents↗
PubMed2006

Congenital hypothyroidism caused by new mutations in the thyroid oxidase 2 (THOX2) gene.

OBJECTIVE: Congenital primary hypothyroidism (CH) occurs in one of 4000 births and in 20% of the cases CH is due to a defect in thyroid hormonogenesis. Candidate genes were examined to determine the precise aetiology of suspected dyshormonogenesis in CH. DESIGN: The genes that code for thyroid peroxidase (TPO), pendrin (PDS), sodium iodide symporter (NIS) and thyroid oxidase 2 (THOX2) were sequenced directly from genomic DNA. PATIENTS: Two girls found to have CH in the neonatal screening programme and suspected of having thyroid dyshormonogenesis were investigated to identify their molecular defect. RESULTS: Patient A had a novel heterozygous 1 bp insertion in the THOX2 gene (ins602g). This insertion results in a frameshift that predicts a premature stop at codon 300. Analysis of cDNA, transcribed from lymphocyte RNA, showed that this mutation causes skipping of exon 5, resulting in a frameshift and a premature stop at codon 254. The euthyroid mother was also a heterozygous carrier of the mutation whereas the father was homozygous for the wild-type THOX2 gene. In patient B, compound heterozygous mutations (ins602g-->fsX300 and D506N) were identified. D506N was present in one allele of the clinically unaffected mother and in a brother, whereas the euthyroid father was heterozygous for ins602g. Sixty normal individuals did not harbour the mutations. Sequencing of the TPO, PDS and NIS genes revealed no mutations. CONCLUSIONS: The identified THOX2 mutations, which have not been described previously, are the probable causes of CH in the patients. Mutations in the THOX2 gene should be considered as the molecular cause of CH in young patients with thyroid dyshormonogenesis.

Base Sequence↗
PubMed1993

Ubiquity of lignin-degrading peroxidases among various wood-degrading fungi.

Phanerochaete chrysosporium is rapidly becoming a model system for the study of lignin biodegradation. Numerous studies on the physiology, biochemistry, chemistry, and genetics of this system have been performed. However, P. chrysosporium is not the only fungus to have a lignin-degrading enzyme system. Many other ligninolytic species of fungi, as well as other distantly related organisms which are known to produce lignin peroxidases, are described in this paper. In this study, we demonstrated the presence of the peroxidative enzymes in nine species not previously investigated. The fungi studied produced significant manganese peroxidase activity when they were grown on an oak sawdust substrate supplemented with wheat bran, millet, and sucrose. Many of the fungi also exhibited laccase and/or glyoxal oxidase activity. Inhibitors present in the medium prevented measurement of lignin peroxidase activity. However, Western blots (immunoblots) revealed that several of the fungi produced lignin peroxidase proteins. We concluded from this work that lignin-degrading peroxidases are present in nearly all ligninolytic fungi, but may be expressed differentially in different species. Substantial variability exists in the levels and types of ligninolytic enzymes produced by different white not fungi.

Alcohol Oxidoreductases↗
PubMed1976

[Purification and characterization of peroxidase from Phellinus igniarius (author's transl)].

A Peroxidase (EC 1.11.1.7) of the basidiomycet Phellinus igniarius was derived from mycel and a medium containing glucose and extract of yeast by using various methods of preparation. The enzyme resists extreme conditions (pH, temperature salt concentration). Its optimum pH for activities is in the acid range. Two isoenzymes were found. The molecular weight, isoelectric point, Michaelis-Menten constant, indolacetic acid oxidase activity and spectral and analytical properties of this peroxidase were determined. It is assumed that the enzyme has an intracellular as well as an extracellular field of activity.

Amino Acids↗
PubMed2006

Sodium/iodide symporter (NIS) gene expression is the limiting step for the onset of thyroid function in the human fetus.

CONTEXT: Terminal differentiation of the human thyroid is characterized by the onset of follicle formation and thyroid hormone synthesis at 11 gestational weeks (GW). OBJECTIVE: This study aimed to investigate the ontogeny of thyroglobulin (Tg), thyroid peroxidase (TPO), sodium/iodide symporter (NIS), pendrin (PDS), dual oxidase 2 (DUOX2), thyroid-stimulating hormone receptor (TSHR), and thyroid transcription factor 1 (TITF1), forkhead box E1 (FOXE1), and paired box gene 8 (PAX8) in the developing human thyroid. DESIGN: Thyroid tissues from human embryos and fetuses (7-33 GW; n = 45) were analyzed by quantitative PCR to monitor mRNA expression for each gene and by immunohistochemistry to determine the cellular distribution of TITF1, TSHR, Tg, TPO, NIS, and the onset of T4 production. A broken line regression model was fitted for each gene to compare the loglinear increase in expression before and after the onset of T4 synthesis. RESULTS: TITF1, FOXE1, PAX8, TSHR, and DUOX2 were stably expressed from 7 to 33 GW. Tg, TPO, and PDS expression was detectable as early as 7 GW and was correlated with gestational age (all, P < 0.01), and the slope of the regression line was significantly different before and after the onset of T4 synthesis at 11 GW (all, P < 0.01). NIS expression appeared last and showed the highest fit by the broken line regression model of all genes (correlation age P < 0.0001, broken line regression P < 0.0001). Immunohistochemical studies detected TITF1, TSHR, and Tg in unpolarized thyrocytes before follicle formation. T(4) and NIS labeling were only found in developing follicles from 11 GW on. CONCLUSION: These results imply a key role of NIS for the onset of human thyroid function.

Female↗
PubMed1994

Dissociation of thyrotropin-dependent enzyme activities, reduced iodide transport, and preserved iodide organification in nonfunctioning thyroid adenoma and multinodular goiter.

Several biochemical and functional modifications demonstrated in goitrous tissues could reflect the effect of goitrogenic factors. Growth-enhancing agents, including TSH itself, have been involved in goitrogenesis. To study comparatively the variation patterns of some TSH-dependent enzymes within single goitrous tissues, we measured the activities of peroxidase (TPO), NADPH-cytochrome-c (cyt-c) reductase, and monoamine oxidase (MAO) in tissues from cold follicular adenoma and multinodular goiter. Iodide transport and organification were also evaluated. Perinodular and necropsy tissues were used as controls. The mean TPO activity measured by guaiacol as well as triiodide assays was significantly increased in multinodular goiter, whereas a nonsignificant increment was observed in cold adenoma. NADPH-cyt-c reductase and MAO were markedly increased in the two types of pathological tissues. The individual activities of the three enzymes showed dissimilar modifications within single samples and among different tissues. There was no correlation in the activities of the enzymes within single specimens from cold adenoma and multinodular goiter, except for MAO and NADPH-cyt-c reductase in multinodular goiter, for which a significant correlation was obtained. In this tissue, MAO and TPO measured by guaiacol assay were weakly correlated. TPO activity evaluated by guaiacol oxidation was correlated with that measured by triiodide formation in cold adenoma, but not in multinodular goiter. The mean iodide organification values assayed by iodotyrosine formation in the absence of exogenous H2O2 in particulate fractions from cold adenoma and multinodular goiter were within the normal range. A reduced iodide transport, evaluated as the thyroid/medium ratio, was observed in slices from these tissues. The dissociation of the three enzyme activities in single specimens from cold adenoma and multinodular goiter along with the reduced iodide transport in these tissues support the hypothesis that factors other than TSH or with TSH-like effects could be involved in the abnormal thyroid growth.

Adenoma↗
PubMed2006

Myeloperoxidase, xanthine oxidase and superoxide dismutase in the gastric mucosa of Helicobacter pylori positive and negative pediatric patients.

The aim of this study was determination and comparison of the levels of myeloperoxidase (MPO), xanthine oxidase (XO), and superoxide dismutase (SOD) in gastric mucosa of children who were infected and noninfected with Helicobacter pylori (HP). The MPO, and XO enzyme activities were detected via kinetic measurement, and the MPO, XO and SOD enzyme protein levels were detected via Western blot, in antral mucosa specimens of 43 patients who underwent upper gastrointestinal endoscopy with various indications. The diagnosis of HP infection was made with a positive rapid urease test and histopathologic detection. MPO activity and enzyme protein levels were measured in 14 [8 HP (+) and 6 HP (-)], and in 9 [5 HP (+) and 4 HP (-)] while XO activity and enzyme protein levels were measured in 16 [10 HP (+) and 6 HP (-)] and in 9 [5 HP (+) and 4 HP (-)] patients, respectively. SOD protein level was detected in 13 [7 HP (+) and 6 HP (-)] patients. Of 43 patients 25 were HP (+) and 18 were HP (-). MPO activities were 75.6 +/- 40.5 and 98.8 +/- 44.1 U/g. protein (p = 0.302) while XO activities were 0.5 +/- 0.3 and 0.4 +/- 0.2 U/g. protein in HP (+) and HP (-) patients, respectively (p = 0.625). Measured enzyme protein levels of MPO, XO and SOD were found statistically indifferent in HP (+) and HP (-) patients (p = 0.327, p = 0.086, and p = 0.775, respectively). The results of this study revealed that, MPO, XO and SOD conditions in gastric mucosa alone were not affected from HP presence. That's why MPO, XO, and SOD may not have important roles in the pathogenesis of HP related gastric disease in children.

Adolescent↗
PubMed1998

Biochemical and functional changes during the bovine fetal thyroid development.

To establish biochemical and functional relations during thyroid development, the activity of thyroid peroxidase (TPO), nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome c reductase and monoamine oxidase (MAO) in a particulate fraction and the iodide transport and organification in slices of bovine fetal thyroid were examined throughout gestation. The cytochemical localization of TPO, H2O2 generating sites and MAO was also studied. Fetal glands were grouped in stages I to V according to increasing developmental features; adult tissues were also analyzed. TPO activity in each of the fetal stages was higher than in the adult; a marked increase was observed in stages IV and V. Iodide transport (T/M) was similar in stages I to V and the adult. Iodide organification in fetal thyroids showed a similar pattern to that of TPO activity. When compared with the adult, at midgestation (stages II to III), a lower iodination coexisted with a higher TPO activity. The activity of NADPH-cytochrome c reductase and MAO, two enzymes previously proposed to participate in thyroid H2O2 generation, did not parallel the level of iodide organification. Cells from stages II to V exhibited a positive cytochemical reaction for TPO in the rough endoplasmic reticulum (RER) and the perinuclear cisternae (PC). In stages IV, V, and adult, TPO was occasionally found in apical vesicles and microvilli, whereas H2O2 was detected within the RER and the PC. MAO reaction was positive in adult, but not in fetal thyroid. These results indicate that a high TPO activity accompanied the onset of the organification process during fetal thyroid development. The level of iodination was associated with the presence of TPO at a proper site rather than to the level of TPO activity. Evidence against a role of NADPH-cytochrome c reductase and MAO in the iodide organification was obtained.

Animals↗