Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

The structural comparison of copper-containing proteins has provided a new dimension to the relationships suggested by sequence similarities. Ryden (1988) summarized the putative relationships, suggesting that a primordial single-domain cupredoxin evolved into the multidomain copper oxidases. The structures have revealed the fact that the differences reside primarily in insertions and deletions at junctions between secondary-structure elements. The mechanism of evolution (e.g., integration of new sequences into regions not essential to the Greek key fold) remains unknown. Which of the properties of a cupredoxin fold are necessary for function is the subject of site-directed mutagenesis studies. Can two of the ligands be interchanged (e.g., the upstream histidine and partially answered by the multidomain copper oxidase structure. The Tyr-Cys-Thr sequence in plastocyanin (in which threonine is a member of the hydrogen-bonding pair) is homologous with the His-Cys-His sequence in ascorbate oxidase. In the latter electron transfer is believed to flow from the type I copper (bound by the cysteine) to the trinuclear cluster, probably via these histidine residues. Hence, one might infer that the tyrosine and threonine have some role in electron transfer. Tyr-83 has been previously implicated in NMR studies as a primary site of electron transfer. The multi-copper protein structures have revealed interesting new features. The extra coppers are bound at domain interfaces, and can be single metals or the novel trinuclear cluster, depending on the availability of liganding histidines. A structural model of ceruloplasmin suggests that it will have at least two type I sites and, possibly, a third type I site such as stellacyanin (no methionine ligand), as well as a binding site for a trinuclear cluster. The similarity of the sequences of N2O reductases and a domain of cytochrome oxidase to the sequences of proteins with known structures suggests that these, too, will have Greek key domains. Galactose oxidase and hemocyanin do not have Greek key folds in their functional domains, although each does have a Greek key domain. The need for a Greek key fold remains obscure. The apoproteins are clearly stable without metals; there are examples other than immunoglobulins of Greek key folds. So far copper seems to be found in a very limited subset of structures; other chapters in this volume show that zinc, for example, has a much wider variety of environments in proteins, as does iron. It may be that the copper-containing Greek key proteins represent a very small evolutionary niche.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Copper protein structures. 179 5

The definite structure and chemical stability of a new glucoside of L-ascorbic acid (AA) which was enzymatically glucosylated with rat intestinal and rice seed alpha-glucosidases were reported. The stability of this AA derivative in water under aerobic conditions was proved by its remarkable resistance against enhanced oxidative degradation by heat, Cu2+ ion or ascorbate oxidase, and it was found to have no reducing activity toward radicals. These properties were obviously distinguishable from those of AA. This glucoside was effectively hydrolyzed by alpha-glucosidases which possessed the ability to synthesize itself, resulting in the liberation of AA activity. The conjugate was composed of equimoles of AA and glucose. Nuclear magnetic resonance spectra, mass spectra, pH profiles of ultraviolet spectra and pK(a) value of 3.10 supported the coupling of alpha-glucose to the 2-position of AA. From these results, its structure was assigned 2-O-alpha-D-glucopyranosyl-L-ascorbic acid, being distinct from 6-O-alpha-D-glucopyranosyl-L-ascorbic acid formed with Aspergillus niger alpha-glucosidase. These findings indicate that the 2-O-glucoside formed by regioselective transglucosylation withstands oxidative degradation even in aqueous solutions and it can be used as an available active AA source for multicomponent liquid products.
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PMID:L-ascorbic acid alpha-glucoside formed by regioselective transglucosylation with rat intestinal and rice seed alpha-glucosidases: its improved stability and structure determination. 208 81

Ascorbate oxidase from the fungus Acremonium sp. HI-25 is a copper-containing glycoprotein that catalyzes the oxidation of ascorbic acid to dehydroascorbic acid. Monosaccharide composition analysis showed that the enzyme contains exclusively N-linked oligosaccharide chains. Following liberation by hydrazinolysis/re-N-acetylation, and fractionation by HPLC on anion exchange. Amide-80 and/or octadecyl silica columns after derivatization with p-aminobenzoic ethyl ester, the structures of the twelve major neutral oligosaccharides were identified by FAB-MS, 400 MHz 1H-NMR, methylation analysis, mild acid hydrolysis, and/or sequential exoglycosidase digestions. Acremonium sp. ascorbate oxidase was found to consist of high-mannose type oligosaccharides (76.3%) having 4 to 9 mannose residues and a series of novel D-galactofuranose-containing high-mannose type oligosaccharides (18.6%) with the following structure.
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PMID:Novel beta-D-galactofuranose-containing high-mannose type oligosaccharides in ascorbate oxidase from Acremonium sp. HI-25. 878 8

Ascorbic acid is concentrated in granulosa cells of the follicle, and ascorbate deficiency causes follicular atresia. Dehydroascorbic acid (DHAA), the oxidized form of ascorbic acid, serves as an important source for the recycling of ascorbate. As we previously demonstrated endocrine up-regulation of ascorbic acid transport by granulosa cells, we investigated DHAA as an alternate source of ascorbate in the follicle. Granulosa cells were cultured for 24 h, and DHAA uptake was initiated by the addition of 14C-labeled ascorbic acid (300 microM) in the presence of ascorbic acid oxidase (2 U/ml), which catalyzes DHAA production. Almost 90% of accumulated DHAA was present as ascorbic acid within 2 h. Preculture of cells for 24 h with FSH (50 ng/ml) and IGF-I (30 ng/ml) significantly stimulated DHAA uptake compared with the control (158 +/- 16 vs. 43 +/- 8 pmol/10(6) cells, respectively). DHAA uptake by granulosa cells was inhibited by D-glucose (ID50, approximately 2.5 mM) and by the glucose transport inhibitors phloretin (200 microM) and cytochalasin B (10 microM), which reduced uptake to 13 +/- 2% and 8 +/- 3% of the control, respectively. Northern and Western analysis of GLUT1 in granulosa cells following 24 h coincubation with FSH and IGF-I revealed up-regulation of GLUT1 at both the messenger RNA and protein levels (1.6- and 1.3-fold of control, respectively), suggesting that the stimulatory effects of FSH and IGF-I on DHAA transport are mediated by the induction of GLUT1. GLUT4 protein was not detectable by Western analysis. Endocrine-regulated DHAA transport may represent an important mechanism for maintaining adequate antioxidant tone within the developing follicle.
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PMID:Hormone-regulated and glucose-sensitive transport of dehydroascorbic acid in immature rat granulosa cells. 1043 24

Ascorbic acid (vitamin C) is an abundant component of plants. It reaches a concentration of over 20 mM in chloroplasts and occurs in all cell compartments, including the cell wall. It has proposed functions in photosynthesis as an enzyme cofactor (including synthesis of ethylene, gibberellins and anthocyanins) and in control of cell growth. A biosynthetic pathway via GDP-mannose, GDP-L-galactose, L-galactose, and L-galactono-1,4-lactone has been proposed only recently and is supported by molecular genetic evidence from the ascorbate-deficient vtc 1 mutant of Arabidopsis thaliana. Other pathways via uronic acids could provide minor sources of ascorbate. Ascorbate, at least in some species, is a precursor of tartrate and oxalate. It has a major role in photosynthesis, acting in the Mehler peroxidase reaction with ascorbate peroxidase to regulate the redox state of photosynthetic electron carriers and as a cofactor for violaxanthin de-epoxidase, an enzyme involved in xanthophyll cycle-mediated photoprotection. The hypersensitivity of some of the vtc mutants to ozone and UV-B radiation, the rapid response of ascorbate peroxidase expression to (photo)-oxidative stress, and the properties of transgenic plants with altered ascorbate peroxidase activity all support an important antioxidative role for ascorbate. In relation to cell growth, ascorbate is a cofactor for prolyl hydroxylase that posttranslationally hydroxylates proline residues in cell wall hydroxyproline-rich glycoproteins required for cell division and expansion. Additionally, high ascorbate oxidase activity in the cell wall is correlated with areas of rapid cell expansion. It remains to be determined if this is a causal relationship and, if so, what is the mechanism. Identification of the biosynthetic pathway now opens the way to manipulating ascorbate biosynthesis in plants, and, along with the vtc mutants, this should contribute to a deeper understanding of the proposed functions of this multifaceted molecule.
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PMID:Ascorbic acid in plants: biosynthesis and function. 1100 3

Carbohydrate epitopes are capable of binding human IgE from allergic subjects and these epitopes play a role in the cross-reactivity between allergens from unrelated sources. A monoclonal antibody (5E6), specific for a carbohydrate epitope detectable on components of Cupressus arizonica pollen extract, has been produced and characterized. To study the relationship between the epitopes recognized by the monoclonal antibody and by IgE from allergic subjects. To investigate the presence of such carbohydrate IgE determinant in extracts from 21 pollen species belonging to 16 taxonomically related and unrelated families, by means of the monoclonal antibody. IgG-depleted fraction from protein G-purified human allergic serum was obtained. The monoclonal antibody and the IgE from the purified fraction were tested on two glycoproteins, polyamine oxidase and ascorbate oxidase, adsorbed on the ELISA plates. The relationship between the monoclonal- and the IgE-recognized epitopes was investigated by ELISA-competition experiments. Analysis of the distribution of this carbohydrate epitope was performed by direct binding of the monoclonal antibody onto the various extracts. The monoclonal antibody and the IgE were able to bind carbohydrate epitopes on the two plant glycoproteins, ascorbate oxidase and polyamine oxidase. Polyamine oxidase shows only one N-glycosilation site whose carbohydrate moiety seems to be composed of a branched chain of seven ordered sugars, i.e. two N-acetyl-D-glucosamine-, three mannose-, one fucose- and one xylose-residues. This structure bears the epitope recognized by mAb 5E6. Human IgE from the IgG-depleted fraction were found capable of inhibiting the monoclonal antibody binding. The allergenic epitope identified was shared by a large number of extracts with different levels of reactivity (OD490 ranging from 0.110 to 2.060). Our data support the finding that a monoclonal antibody specific for a carbohydrate epitope of Cupressus arizonica pollen extract detects an epitope which is also recognized by IgE from allergic subjects. This characterized reagent could be a useful tool for studying distribution of cross-reactive carbohydrate determinants in allergenic pollen extracts and their components.
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PMID:A monoclonal antibody specific for a carbohydrate epitope recognizes an IgE-binding determinant shared by taxonomically unrelated allergenic pollens. 1126 Jan 59

Individual enzyme-based biosensors involving three-electrode systems were developed for the detection of analytes comprising markers of the stage of maturity and quality in selected fruits of economic importance to tropical countries. Importantly, a common fabrication format has been developed to simplify manufacture and allow future integration of the individual sensors into a single multi-sensor array. Specifically, sensors for beta-D-glucose, total D-glucose, sucrose and ascorbic acid have been developed. Pectin, a natural polysaccharide present in plant cells, was used as a novel matrix to enhance enzyme entrapment and stabilisation in the sensors. Except for ascorbic acid, all the sensors function via the detection of enzymatically generated H2O2 at rhodinised carbon electrodes. Since ascorbic acid is electrochemically active at the working potential chosen (+350 mV vs. Ag/AgCl), it was measured directly. Enzyme sensors demonstrated expected response with respect to their substrates, typically 0-0.8 microA/20 mm2 electrode area response over analyte ranges of 0-7 mM. Interferences related to electrochemically active compounds present in fruits under study were significantly reduced by inclusion of a suitable cellulose acetate (CA) membrane or by enzymatic inactivation with ascorbate oxidase. Initial development was carried out into production of biosensor arrays. CA membranes were used to improve the linear range of the sensors, producing up to a fivefold improvement in the detection range compared to sensors without an additional diffusion barrier.
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PMID:Development of a common biosensor format for an enzyme based biosensor array to monitor fruit quality. 1294 57

Ascorbate, dehydroascorbate, and glucose transport was investigated in plant mitochondria and mitoplasts prepared from cultured BY2 tobacco cells. Using a rapid filtration method with radiolabeled ligands, we observed a specific glucose and dehydroascorbate transport, which was temperature and time dependent and saturable. Inhibition of mitochondrial respiration by KCN and the uncoupler 2,4-dinitrophenol did not influence the transport of the investigated compounds. Dehydroascorbate transport was inhibited by glucose and genistein, while glucose uptake was decreased upon 3-O-methyl-glucose, D-mannose, cytochalasin B or genistein addition. On the other hand, a low affinity low capacity ascorbate transport was found. Oxidizing agents (potassium ferricyanide or ascorbate oxidase) increased ascorbate uptake. The results demonstrate the presence of dehydroascorbate and glucose transport in plant mitochondria and suggest that it is mediated by the same or closely related transporter(s).
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PMID:Facilitated glucose and dehydroascorbate transport in plant mitochondria. 1523 71

Citrus fruit is widely consumed and provides ascorbate for human health. The ascorbate content in pulp is generally higher in orange (Citrus sinensis Osb.) than in Satsuma mandarin (Citrus unshiu Marc.). However, what contributes to such difference is still unknown. In the present study, ascorbate accumulation, expression profiles of genes involved in L-galactose pathway and activity changes of enzymes related with L-ascorbic acid (AA) oxidation and recycling were investigated during fruit development and ripening in fruit pulp of Satsuma mandarin and orange. As fruit ripens, total ascorbate (T-ASC) or AA content increased in mandarin whereas fluctuated on a relatively high level in orange. Concentrations of T-ASC or AA in pulp of orange were over 1.5-fold higher than that in pulp of Satsuma mandarin during fruit ripening. Further analysis showed that each transcript of four genes (encoding GDP-D-mannose-3',5'-epimerase, GDP-L-galactose-pyrophosphatase, L-galactose dehydrogenase and L-galactono-1,4-lactone dehydrogenase respectively) in orange was almost on a higher level and the activities of oxidation enzymes (ascorbate oxidase and ascorbate peroxidase) were lower during fruit ripening as compared with Satsuma mandarin. As ascorbate pool size is decided by the combination of biosynthesis, oxidation and recycling, therefore, higher expression of four genes along with lower activity of oxidation enzymes should contribute at least partially to the higher ASC accumulation in orange pulp.
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PMID:Comparison of ascorbate metabolism in fruits of two citrus species with obvious difference in ascorbate content in pulp. 2192 61

Ascorbic acid (L-AsA) is an important antioxidant in plants and humans. Vegetables are one of the main sources of ascorbic acid for humans. For instance, non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) is considered as one of the most important vegetables in south China. To elucidate the mechanism by which AsA accumulates, we systematically investigated the expression profiles of D-mannose/L-galactose pathway-related genes. We also investigated the recycling-related genes and AsA contents in different tissues of three non-heading Chinese cabbage cultivars, 'Suzhouqing', 'Wutacai' and 'Erqing' containing different amounts of AsA. Our results showed that six genes [D-mannose-6-phosphate isomerase 1 (PMI1), GDP-L-galactose phosphorylase 1 (GGP1), GGP2, GGP4, GDP-mannose-3', 5'-epimerase1 (GME1), and GME2] were expressed at high level and ascorbate oxidase (AAO) was expressed at low level. This expression pattern contributes, at least partially, to higher AsA accumulation in the leaves and petioles than in the roots. Eight genes (PMI1, GME, GGP, L-galactose-1-phosphate phosphatase, L-galactose dehydrogenase, L-galactono-1, 4-lactone dehydrogenase, monodehydroascorbate reductase 1, and glutathione reductase1) were also expressed at high level; AAO and ascorbate peroxidase (APX) were expressed at low level. This expression pattern may similarly contribute to higher AsA accumulation in 'Wutacai' and 'Suzhouqing' than in 'Erqing'. Therefore, the high expression levels of PMI, GME, and GGP and the low expression level of AAO contributed to the high AsA accumulation in non-heading Chinese cabbage.
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PMID:Comparison of ascorbic acid biosynthesis in different tissues of three non-heading Chinese cabbage cultivars. 2415 1


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