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

An amperometric method suitable for the continuous on-line measurement of cerebral hydrogen peroxide from a microdialysate has been successfully performed for the first time by using an enzyme-modified ring-disk plastic carbon film electrode (PCFE) in a thin-layer radial flow cell. PCFE consists of a ring electrode modified with horseradish peroxidase to detect H2O2 at 0.0 V (vs Ag/ AgCl) and a disk electrode coated with ascorbate oxidase (AOx) to preoxidize ascorbic acid (AA) and thus suppress interference via direct oxidation. Analytes in solution (brain dialysates or standards) are mixed on-line with a phosphate-buffered solution containing dissolved oxygen and chelating agent, EDTA. The buffered solution is used to provide the O2 necessary for the AOx catalytic reaction, stabilize the changes in dialysate pH that are associated with the in vivo formation of H2O2, and remove heavy metal ion impurities and thus suppress reactions between AA and H2O2. This procedure enables trace levels of H2O2 to be readily monitored, virtually interference-free from physiological levels of AA, uric acid, electroactive neurotransmitters and their principle metabolites, in a continuous-flow system.
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PMID:Continuous on-line measurement of cerebral hydrogen peroxide using enzyme-modified ring-disk plastic carbon film electrode. 1217 54

The activity of ascorbate oxidase (AOX) in mustard (Sinapis alba L.) cotyledons was markedly increased by irradiation with continuous far-red light. The involvement of phytochrome in this light-mediated response was demonstrated by red/far-red reversibility experiments. To determine immunochemically the contents of AOX in cotyledons, the antibody against the enzyme was raised in a rabbit. However, the antiserum was not monospecific to AOX; it also recognized glycoproteins. To remove antibodies that are specific to a carbohydrate moiety of glycoproteins, the anti-AOX antiserum was applied to a horseradish peroxidase-conjugated Sepharose column. By using the antibodies that were not retained in the column, the changes in the content of AOX were followed. Western immunoblot profiles revealed that the content of AOX protein in cotyledons notably increased after continuous far-red light treatment. Pulse-labeling experiments indicated that the synthesis of AOX protein occurred in the cotyledons. These results are in good agreement with the hypothesis that phytochrome-mediated increase in AOX activity is accompanied by the synthesis of the enzyme.
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PMID:Phytochrome Control of the Development of Ascorbate Oxidase Activity in Mustard (Sinapis alba L.) Cotyledons. 1223 99

A picoliter-volume electrochemical analytical chamber has been developed for detecting the metabolic flux resulting from the stress responses of a single plant cell. Electrochemical cells, with volumes as small as 100 pL, were fabricated by controlled electrochemical dissolution of a gold wire sealed in glass (the back-etching of the metal realizing an ultralow-volume titer chamber). In the first instance, the electrode contained within the chamber was characterized by the microinjection of standard aliquots of either ascorbic acid or hydrogen peroxide. In all cases, experimental currents obtained correlated well with theoretical calculations. Subsequently, single plant cells were micromanipulated into the chambers and were exposed to amounts of the detergent SDS (which permeabilized the cell membrane and released the intracellular contents). The flux of metabolite released from a single cell was estimated by using electrochemical-linked assays based upon the enzymes catalase, ascorbate oxidase, and horseradish peroxidase (in each case), in the presence of a mediator. In so doing, we investigated the activity of the cellular protection mechanisms through the determination of peroxides, while the individual cell was "stressed". The technique was found to provide a reliable and reproducible method for making single-cell measurements, using fabrication procedures that are both simple and do not require photolithographic methods.
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PMID:Electroanalysis of metabolic flux from single cells in simple picoliter-volume microsystems. 1238 Aug 23

A microfluidic device integrated with a nanoliter volume enzyme pre-reactor and an enzyme-modified electrode was developed for the highly selective continuous measurement of glutamate (Glu). The device consists mainly of two glass plates. One plate incorporates an electrochemical cell that consists of working electrode (WE), reference electrode (RE) and counter electrode (CE). The WE is modified with a bilayer film of Os-polyvinylpyrridine-based mediator containing horseradish peroxidase (Os-gel-HRP). The WE was operated at -50 mV versus Ag. The other plate has a thin layer flow channel integrated with a pre-reactor. The reactor has a number of micropillars (20 microm in diameter, 20 microm high and separated from each other by a 20 microm gap) modified with ascorbate oxidase (AAOx) to eliminate L-ascorbic acid (AA). The enzymatic oxidation of AA is superior to that obtained with our previously reported pre-electrolysis type micro-reactor since electrochemically reversible transmitters such as catecholamines do not provide a cathodic current at the WE. In addition, the high operation potential of the pre-reactor causes unknown electroactive species, which also cause interference at the detection electrode. As a result, we were able to detect 1 microM Glu continuously at a low flow rate even when AA concentration was 100 microM.
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PMID:Selective detection of L-glutamate using a microfluidic device integrated with an enzyme-modified pre-reactor and an electrochemical detector. 1283 43

The ascorbate content declined rapidly in broccoli (Brassica oleracea L. var. italica) florets, but not in the stem tissue, during post-harvest senescence. Ascorbate peroxidase (APX), ascorbate oxidase (AO), l-galactono-1,4-lactone dehydrogenase (GLDH), monodehydroascorbate reductase (MDAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) were investigated in gene expression after harvest in both florets and the stem tissue of broccoli. Cytosolic gene expressions (BO-APX 1, BO-APX 2, BO-AO, BO-MDAR 2, and BO-GR) were stimulated actively in broccoli florets after harvest. By contrast, it was observed that mRNA levels of chloroplastic APX, BO-sAPX and BO-tbAPX, had decreased by 12 h after harvest in broccoli florets, suggesting that the active oxygen species (AOS) scavenging system in chloroplasts was largely abolished in florets during the early hours of the post-harvest period. In addition, gene expressions in GLDH and other chloroplastic enzymes such as BO-MDAR 1 and BO-DHAR decreased rapidly within 24 h after harvest. Ethylene treatment had no effect on the ascorbate level and the expression of all genes investigated. The expressions of BO-GLDH and chloroplastic genes (BO-sAPX, BO-tbAPX, BO-MDAR 1, and BO-DHAR) mRNA were suppressed by treatment with methyl jasmonate (MJ) and abscisic acid (ABA) and were accompanied by the acceleration of ascorbate degradation. These data suggest that ascorbate metabolism tends to be inactivated in chloroplasts by transcriptional regulation, but not in the cytosol, when ascorbate decreases under stress conditions.
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PMID:Ascorbate metabolism in harvested broccoli. 1451 88

An effective microseparation system integrated with ring-disc electrodes and two microfluidic devices was fabricated for in vivo determination using a microdialysis pump. The major interference of ascorbic acid (AA) was excluded by direct oxidation with ascorbate oxidase. Glucose, glutamate, and choline were successfully determined simultaneously through the biosensors modified with a bilayer of osmium-poly(4-vinylpyridine)gel-horseradish peroxidase (Os-gel-HRP)/glucose oxidase (GOD), glutamate oxidase (GlutaOD) or choline oxidase (ChOD). To stabilize the biosensors, 0.2% polyethylenimine (PEI) was mixed with the oxidases. The cathodic currents of glucose, glutamate, and choline biosensors started to increase after the standard solutions were injected into the microseparation system. The on-line biosensors show a wide calibration range (10(-7)-10(-5) mol/L) with a detection limit of 10(-8) mol/L at the working potential of -50 mV. The variations of glucose, glutamate, and choline were determined simultaneously in a free moving rat when we perfused the medial frontal cortex with 100 micro mol/L N-methyl-D-aspartate (NMDA) solution, which is the agonist of the NMDA receptor.
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PMID:On-line biosensors for simultaneous determination of glucose, choline, and glutamate integrated with a microseparation system. 1451 55

Hydrogel-coated microsensors based on carbon fiber electrodes (CFEs) are promising tools for in vivo analysis of endogeneous compounds such as glutamate. However, their construction generally depends on manual fabrication, which often results in poor reproducibility. The aim of this study was to improve the reproducibility and performance of glutamate microsensors. CFEs (10 microm diameter, 300-500 microm long) were coated with a cross-linked redox-polymer hydrogel containing l-glutamate oxidase, horseradish peroxidase and ascorbate oxidase. Various parameters that are likely to influence the reproducibility of the glutamate microsensors were studied. It appeared that the most crucial step in determining the microsensor performance is the manual hydrogel-application procedure. To control this procedure an automated dipcoater was constructed, which allowed mechanical application of the hydrogel on the CFE under standardized conditions. Significant improvements in performance were seen when the CFEs were dipcoated for 10 min at 37 degrees C. Further improvements were obtained when the automated hydrogel application was combined with other cross-link methods, such as electrodeposition and electrostatic complexation. A crucial factor in determining the microsensor performance is the hydrogel thickness. Microscopic observations revealed that, despite the use of an automated dipcoater, the layer thickness was not constant. By combining the automated dipcoat technique with amperometry, the layer thickness could be indirectly monitored and controlled, which resulted in significant improvements of the reproducibility of the sensors.
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PMID:Improving the reproducibility of hydrogel-coated glutamate microsensors by using an automated dipcoater. 1558 41

Feruloyl-polysaccharides can be oxidatively coupled in isolated cell walls by peroxidase plus exogenous H(2)O(2) in vitro, but the extent to which similar reactions may occur in the apoplast in vivo was unclear. Numerous cellular factors potentially control feruloyl coupling in vivo, and their net controlling influence is not readily studied in vitro. Therefore, we have monitored apoplastic feruloyl coupling in cultured maize cells in vivo using a radiolabelled model substrate, 5-O-feruloyl-alpha-L: -arabinofuranosyl-(1-->3)-beta-D: -xylopyranosyl-(1-->4)-D: -xylose (FAXX). FAXX was expected to permeate the wall and to undergo reactions analogous to those normally exhibited by apoplastic feruloyl-polysaccharides in vivo. Little difference was found between the fates of [feruloyl-(14)C]FAXX and [pentosyl-(3)H]FAXX, indicating negligible apoplastic hydrolase or transferase activities. Very little radioactivity entered the protoplasm. Maize cells that had recently been washed in fresh medium were able to bind most of the FAXX (90%) in their cell walls, regardless of the age of the culture. During wall-binding, the [(14)C]feruloyl groups were converted to [(14)C]dehydrodiferulates and larger coupling products, as revealed by TLC after alkaline hydrolysis. As expected for an oxidative reaction, wall-binding was delayed by added anti-oxidants (ascorbate, ferulate, sinapate, chlorogenate or rutin). It was also completely inhibited by iodide, an H(2)O(2)-scavenger, indicating a role for peroxidase rather than oxidase. The observations indicate that oxidative coupling of feruloyl groups occurred within the cell wall, dependent on endogenous apoplastic H(2)O(2) and wall-localised peroxidase, in vivo. Cells that had not recently been washed in fresh medium were much less able to bind FAXX, indicating the presence in the apoplast of an endogenous inhibitor of oxidative coupling. This inhibitor was of low M(r), was destroyed by heating, and remained in the aqueous phase (pH approximately 3.5) when shaken with ethyl acetate. Its effectiveness was not altered by ascorbate oxidase. It is thus a small, heat-labile, hydrophilic inhibitor (not ascorbate) which we suggest plays a natural role in the control of wall cross-linking, and thus potentially in the control of cell growth.
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PMID:Oxidative coupling of a feruloyl-arabinoxylan trisaccharide (FAXX) in the walls of living maize cells requires endogenous hydrogen peroxide and is controlled by a low-Mr apoplastic inhibitor. 1604 78

Amperometric hydrogel-coated glutamate microsensors form a promising concept to detect glutamate levels directly in brain tissue. These microsensors are constructed by coating a carbon fiber electrode (CFE) (10 microm diameter; 300-500 microm long) with a five-component redox-hydrogel, in which L-glutamate oxidase, horseradish peroxidase, and ascorbate oxidase are wired via poly(ethylene glycol) diglycidyl ether to an osmium-containing redox polymer. Coating with a thin Nafion film completes the construction. Prior to use in vivo, a reliable and reproducible construction of microsensors with a high performance is required. For an optimal microsensor performance, the balance between the five individual hydrogel components is critical. However, due to their small size, hydrogel application to CFE's need to be performed by dip-coating. Dip-coating is a difficult procedure to control and does not allow individual application of hydrogel constituents. To improve the microsensor construction and to better control the dip-coating procedure, we have recently developed an automated device. Throughout this study, automatic dip-coating was performed with premixed solutions, in which the amount of a single component was varied. This allowed us to optimize the hydrogel composition, which resulted in a significant improvement of the microsensor properties in terms of sensitivity, current density, linearity, detection limit, and interference by ascorbic acid.
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PMID:Improving glutamate microsensors by optimizing the composition of the redox hydrogel. 1613 Oct 61

Cotton plants (Gossypium hirsutum. Linn. var. Sankar 4) were grown at normal and toxic levels of substrate manganese, and the altered metabolism of manganese toxic plants was studied. The tissues of plants exposed to toxic levels of manganese had higher activities of peroxidase and polyphenol oxidase, and the activities of catalase, ascorbic acid oxidase, glutathione oxidase and cytochrome c oxidase were lowered. In addition, the high manganese tissue had lower contents of ATP and glutathione but higher amounts of ascorbic acid. The respiration of the partially expanded leaves and the growing tips of toxic plants were depressed when compared to that of the normal tissues. The metabolic changes of manganese toxicity of cotton are placed in the following order: accumulation of manganese in the leaf tissue; a rise in respiration; stimulation of polyphenol oxidase; the appearance of initial toxicity symptoms; the evolution of ethylene and stimulation of peroxidase; the presence of severe toxicity symptoms; the depression of terminal oxidases and respiration; abscission of the growing tip and proliferation of the stem tissue. The early stimulation of polyphenol oxidase may be used to detect potential manganese toxicity.
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PMID:The manganese toxicity of cotton. 1665 24


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