Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.10.3.3 (ascorbate oxidase)
 778 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Since cytochrome c and acetylated cytochrome c disappear from the circulation with a half-life of 4 min, these proteins cannot be used for in vivo detection of superoxide radicals and related metabolites. To determine superoxide and other radicals in vivo, a cytochrome c derivative (SMAC) was synthesized by linking 1 mol of poly(styrene-co-maleic acid) butyl ester (SM) to cytochrome c, followed by acetylation of its lysyl amino groups. SMAC retained 8 and 80% of cytochrome c activity to react with ascorbyl and superoxide radicals, respectively. However, SMAC did not serve as a substrate for cytochrome c reductase and cytochrome c oxidase. When injected intravenously to the rat, SMAC circulated bound to albumin with a half-life of 130 min. SMAC was rapidly reduced in the circulation of intact animals. Treatment of animals with paraquat markedly enhanced the reduction of the circulating SMAC. We have synthesized an SM-conjugated superoxide dismutase (SOD) derivative (SM-SOD) that circulates bound to albumin with a half-life of 6 h. Kinetic analysis revealed that SM-SOD effectively inhibited the superoxide-dependent reduction of SMAC either in the presence or absence of 0.5 mM albumin. However, the reduction of the circulating SMAC was not inhibited by SM-SOD both in normal and paraquat-treated animals. Plasma samples from both animal groups also reduced cytochrome c and SMAC by an SOD-insensitive mechanism. However, after treatment with ascorbate oxidase, both plasma samples lost their activity to reduce cytochrome c and SMAC. These and other results suggest that ascorbyl radical might principally be responsible for the reduction of circulating SMAC and that plasma levels of ascorbyl radical might increase in paraquat-treated animals.
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PMID:Synthesis of a cytochrome c derivative with prolonged in vivo half-life and determination of ascorbyl radicals in the circulation of the rat. 131 36

Human colostrum contains several antioxidants which prevent the detection of human polymorphonuclear leukocyte (PMN) respiratory burst products. Using column chromatography to fractionate colostrum, two peaks of antioxidant activity were resolved away from colostral proteins and further characterized. One peak contained both cytochrome c-reducing activity and H2O2-depleting activity. This peak had the chromatographic, spectral, and antioxidant characteristics of ascorbate, and by high performance liquid chromatography (HPLC) methods, was shown to contain ascorbate as well as at least four other materials. The antioxidant activity in this peak was totally ascorbate oxidase sensitive and partially uricase sensitive. The other peak contained only H2O2-depleting activity and had the chromatographic, spectral, and antioxidant characteristics of uric acid. By HPLC, uric acid was the only component in this peak and its antioxidant activity was completely uricase sensitive and ascorbate oxidase resistant. Colostral uric acid levels were measured in eight postpartum women and found to be approximately one-third of simultaneously determined serum uric acid levels. Colostrum contains at least two separate antioxidants, one of which is ascorbate-like and the other is uric acid. We speculate that these antioxidants may function in human colostrum as traps for neutrophil-generated reactive oxygen metabolites.
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PMID:Colostral antioxidants: separation and characterization of two activities in human colostrum. 157 13

The rate of "in vivo" reduction of cytochrome c by ascorbic acid (AA) increases from 69 nmoles of cytochrome c for minute, to 202 nanomoles when ascorbate oxidase is added. Since the AA oxidation by AA oxidase is a system to generate ascorbic free radical (AFR), data suggest that AFR is a better reducing compound than ascorbate in cytochrome c reduction. Since the addition of oxidized glutathione and human immunoglobulins (-S-S- bridge containing compounds) in the medium produces a remarkable decrease in cytochrome c reduction, it is suggested that AFR could also reduce -S-S- groups.
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PMID:Reduction of cytochrome C by ascorbic free radical. 165 79

The reduction of ferricytochrome c within the perfusate in isolated lung perfusion systems has been demonstrated previously. We carried out the present study 1) to determine what reducing agents might be responsible for this reduction and 2) to determine whether the cytochrome c (cyto c) reduction within the recirculating perfusion system can be accounted for by relatively stable reducing agents released into the perfusate or whether some of the reduction is dependent on short-lived agents and/or proximity to the source of the agents within the lungs. Experiments were carried out with the use of isolated rabbit lungs perfused for 1 h in a recirculating system. In one group of experiments, ferricytochrome c was included in the recirculating perfusion system. In another group, the cyto c was added to produce the same concentration in samples after they were removed from a cyto c-free recirculating system. The recirculating cyto c was reduced at a rate of approximately 1.76 mumol/h, and approximately 22% was inhibitable by superoxide dismutase. Most of the rest could be inhibited by ascorbate oxidase within the recirculating perfusate. When the ferricytochrome c was added to the samples removed from the cyto c-free perfusion system, virtually the entire cyto c reducing capacity was inhibitable by ascorbate oxidase. Although reduced glutathione did accumulate in the recirculating perfusate, the quantity was not sufficient to have an important role in the cyto c reduction. We conclude that most of the cyto c reducing capacity within the lung perfusate could be accounted for by ascorbate released from the lungs.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Perfusate cytochrome c reduction in isolated rabbit lungs. 166 94

Human colostrum manifests antioxidant properties, being capable of spontaneous reduction of cytochrome c, depletion of polymorphonuclear leukocyte-produced H2O2 and protection of epithelial cells from PMN-mediated detachment. These activities can be electrophoretically concentrated at either 3.5 kD or 50 kD dialysis membranes at mildly alkaline pH. They are progressively lost under increasingly alkaline conditions. They are resistant to 1-mM N-ethylmaleimide. Examination of a series of antioxidant compounds showed that ascorbate manifests several characteristics of colostrum, being able to reduce cytochrome c and deplete H2O2 but not altering PMN-mediated HEp2 cell detachment. Addition of ascorbate oxidase to colostrum decreased its cytochrome c-reducing activity by more than 85%, decreased its H2O2-depleting activity by nearly 50%, but did not alter its ability to protect HEp2 cells, all suggesting heterogeneity of colostral antioxidant activities. Treatment of colostrum with an enzymatic system (xanthine + xanthine oxidase) known to destroy ascorbate's cytochrome c-reducing activity yielded paradoxical results, decreasing colostral cytochrome c reduction in a dose-related manner, while increasing its H2O2-depleting activity. These studies demonstrate that a colostral component similar to ascorbate, a known antioxidant compound is responsible for the majority of colostral cytochrome c-reducing activity, for about half of its H2O2-depleting activity, and little, if any, of its protective effect on HEp2 cells. Thus colostral antioxidant activity is heterogeneous.
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PMID:Further characterization of human colostral antioxidants: identification of an ascorbate-like element as an antioxidant component and demonstration of antioxidant heterogeneity. 253 79

A direct spectrophotometric method was used for detection of the ascorbate free radical formed during enzyme catalysis with dopamine beta-monooxygenase and with ascorbate oxidase. The optical absorption spectra in the range of 330-390 nm for the free radical formed by either of these enzymes were quite similar to the previously reported spectrum from pulse radiolysis experiments. The second order rate constant for dismutation of the radical generated by dopamine beta-monooxygenase at 23 degrees C was estimated from the levels of radical in the steady state, and the values of 2.4 .10(-6) M-1 . s-1 at pH 7.0 and 9.7 . 10(-6) M-1 . s-1 at pH 6.0 were in close agreement with reported values from experiments in which the radical had been generated with ascorbate oxidase or with pulse radiolysis. Moreover, the steady state radical levels at different levels of dopamine beta-monooxygenase or its substrate tyramine were also those predicted by a mechanism of nonenzymic dismutation of the radical. We conclude, in agreement with our earlier report with the cytochrome c scavenger method, that the radical is not an enzyme-bound intermediate, but a product of dopamine beta-monooxygenase catalysis.
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PMID:Direct spectrophotometric detection of ascorbate free radical formed by dopamine beta-monooxygenase and by ascorbate oxidase. 738 45

Helicobacter pylori sonicate was shown to oxidize ascorbic acid. Ascorbic acid oxidation was determined by chromatography combined with electrochemical detection. Water soluble ascorbic acid oxidase activity was rather independent of pH with a pH optimum around 2. By gel filtration the oxidizing activity co-eluted with an absorbency peak at 408 nm. The relative molecular mass (Mr) was approximately 14,000. It is suggested that this oxidating activity was caused by a cytochrome c-like molecule. Ascorbic acid oxidating activity could also be extracted from bacterial membranes by detergents. Gel filtration showed several forms, the major one with a Mr = 19,000. pH optimum was 6-7. Other oxidase-positive bacterial strains like Campylobacter coli, Enterobacter cloacae and Pseudomonas aeruginosa could degrade ascorbic acid. Since ascorbic acid oxidation by Helicobacter pylori whole bacterial lysates has a pH optimum in the acidic range corresponding to pH in gastric fluid, the activity of the cytochrome c-like water soluble oxidant of Helicobacter pylori seems to be primarily important for the destruction of ascorbic acid in the gastric juice of infected patients.
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PMID:Investigation of Helicobacter pylori ascorbic acid oxidating activity. 777 46

Rate constants have been determined for the electron-transfer reactions between reduced horse heart cytochrome c and resting cucumber ascorbate oxidase as functions of pH, ionic strength, and temperature. The second-order rate constant for the oxidation of reduced cytochrome c was determined to be k = 820 M-1 s-1 in 0.2 M phosphate buffer at pH 6.0 and 25 degrees C. The activation parameters were estimated to be delta H++ = 5 kJ mol-1 and delta S++ = -188 Jmol-1 K-1. The rate constants increased with decreasing buffer concentration, indicating that the electron-transfer from cytochrome c to ascorbate oxidase is realized by the local electrostatic interaction between them in spite of the reaction between positively charged proteins. Reactions of type 2 copper-depleted ascorbate oxidase whose type 3 coppers were in the reduced or oxidized form indicated that the type 1 copper site accepts an electron from cytochrome c. The reaction rate was remarkably increased with decreasing pH for both the native enzyme and derivatives. Further, on addition of hexametaphosphate anion the rate of the electron-transfer decreased because the association of both proteins to realize the electron-transfer was inhibited due to a change in distribution of the local charge on the protein surface(s).
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PMID:Electron-transfer from cytochrome c to ascorbate oxidase and its type 2 copper-depleted derivatives. 805 89

Quantitation of the superoxide radical and its related metabolites in vivo is practically difficult predominantly because of their short biological half-lives. Though oxidized cytochrome c (cyt c) has been used for determining superoxide radicals in vitro, it cannot be used for in vivo analysis because of its low specificity as an electron acceptor and rapid disappearance from the circulation. To measure superoxide radicals and related metabolites in normal and pathologic subjects, we have synthesized a cyt c derivative (SMAC) with prolonged half-life in the circulation (T1/2 = 130 min) by conjugating acetylated cyt c with poly(styreneco-maleic acid) butyl ester (SM). An SM-conjugated superoxide dismutase (SM-SOD) with prolonged in vivo half-life was also synthesized. When injected intravenously to the rat, SMAC was rapidly reduced in the circulation of normal rats. The rate of SMAC reduction was markedly increased by intravenous administration of menadione, a compound capable of redox cycling and generating superoxide. The rate of SMAC reduction was not inhibited by a large dose of SM-SOD (27,000 unit/kg) in both normal and menadione-treated animals. The rate of SMAC reduction also increased in animals which were administered alloxan, a diabetogenic agents. In contrast to the experiments with menadione, the alloxan-enhanced reduction of SMAC was significantly inhibited by SM-SOD. Kinetic analysis using ascorbate oxidase suggested that ascorbyl radical was principally responsible for the SM-SOD-insensitive reduction of SMAC. Streptozotocin, another diabetogenic agent, failed to increase the rate of SMAC reduction. Thus, the effect of streptozotocin on the redox state of animals and the mechanism of its diabetogenic action might differ from those of alloxan. Combined use of SMAC and SM-SOD might permit quantitative studies on the occurrence of ascorbyl and superoxide radicals in the circulation of animals challenged with oxidative stress.
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PMID:Determination of superoxide and ascorbyl radicals in the circulation of animals under oxidative stress. 813 44

Abstract The biochemical pathway and genetics of autotrophic ammonia oxidation have been studied almost exclusively in Nitrosomonas europaea. Terrestrial autotrophic ammonia-oxidizing bacteria (AAOs), however, comprise two distinct phylogenetic groups in the beta-Proteobacteria, the Nitrosomonas and Nitrosospira groups. Hybridization patterns were used to assess the potential of functional probes in non-PCR-based molecular analysis of natural AAO populations and their activity. The objective of this study was to obtain an overview of functional gene homologies by hybridizing probes derived from N. europaea gene sequences ranging in size from 0.45 to 4.5 kb, and labeled with 32P to Southern blots containing genomic DNA from four Nitrosospira representatives. Probes were specific for genes encoding ammonia monooxygenase (amoA and amoB), hydroxylamine oxidoreductase (hao), and cytochrome c-554 (hcy). These probes produced hybridization signals, at low stringency (30 degreesC), with DNA from each of the four representatives; signals at higher stringency (42 degreesC) were greatly reduced or absent. The hybridization signals at low stringency ranged from 20 to 76% of the total signal obtained with N. europaea DNA. These results indicate that all four functional genes in the ammonia oxidation pathway have diverged between the Nitrosomonas and Nitrosospira groups. The hao probe produced the most consistent hybridization intensities among the Nitrosospira representatives, suggesting that hao sequences would provide the best probes for non-PCR-based molecular analysis of terrestrial AAOs. Since N. europaea can also denitrify, an additional objective was to hybridize genomic DNA from AAOs with probes for Pseudomonas genes involved in denitrification. These probes were specific for genes encoding heme-type dissimilatory nitrite reductase (dNir), Cu-type dNir, and nitrous oxide reductase (nosz). No hybridization signals were observed from probes for the heme-type dNir or nosz, but Nitrosospira sp. NpAV and Nitrosolobus sp. 24-C hybridized, under low-stringency conditions, with the Cu-type dNir probe. These results indicate that AAOs may also differ in their mechanisms and capacities for denitrification.
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PMID:Functional Gene Hybridization Patterns of Terrestrial Ammonia-Oxidizing Bacteria. 985 9


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