Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:EXPT00568 (ascorbate)
 23,072 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glutathione dehydrogenase (EC 1.8.5.1) was partially purified from pea shoots. The pH optimum was 7.6. The K(m) values for GSH and dehydroascorbate were 4.4 and 0.44 millimolar, respectively. The enzyme was inhibited by iodoacetate and CuSO(4) but not significantly by ZnCl(2) or NaN(3). Part of the total enzyme activity was associated with isolated chloroplasts.Illuminated ruptured chloroplasts, in the presence of 50 micromolar NADP(H) and substrate concentrations of GSH or GSSG, catalyzed (dehydroascorbate plus glutathione)-dependent O(2) evolution with the concomitant reduction of dehydroascorbate to ascorbate. Oxidation of ascorbate by ascorbate oxidase activity associated with the chloroplasts was relatively insignificant. ZnCl(2) inhibited (dehydroascorbate plus glutathione)-dependent O(2) evolution but not ascorbate formation. The reaction was attributed to light-dependent reduction of GSSG (involving glutathione reductase) coupled to the reduction of dehydroascorbate (involving glutathione dehydrogenase). Light-dependent reduction of GSSG appears to be the rate-limiting step in this reaction sequence at physiological concentrations of GSH.
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PMID:Light-dependent reduction of dehydroascorbate by ruptured pea chloroplasts. 1666 43

The conversion of fructose-1,6-bisphosphate to glycerate-3-phosphate (PGA) was studied in a reconstituted spinach (Spinacia oleracea L.) chloroplast preparation to determine whether a chloroplast-localized NAB(P)H-oxidizing system (Kow, Smyth, Gibbs 1982 Plant Physiol 69: 72-76 with substrates of ascorbate, NAD(P)H, and H(2)O(2) could serve as a coupling enzyme in the recycling of NAD(P)H. The rate of PGA formation was monitored as an indicator of NAD(P) generation. With NAD as a cofactor, ascorbate enhanced PGA formation, and an additional increase resulted upon addition of glucose-glucose oxidase, a H(2)O(2)-generating enzyme. This increase in PGA formation due to H(2)O(2) was eliminated by the addition of catalase. With NADP and ferredoxin as cofactors, the recycling of NADP apparently was catalyzed both by ferredoxin-NADP reductase coupled to O(2) and by the NAD(P)H-oxidizing system.It was concluded that the oxidation of NAD(P)H by a system using ascorbate and H(2)O(2) can serve as a means of recycling NAD(P)H but that another reaction involving ascorbate and NAD(P)H may also function in the spinach chloroplast.
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PMID:Oxidation of NAD(P)H in a Reconstituted Spinach Chloroplast Preparation Using Ascorbate and Hydrogen Peroxide. 1666 86

Ruptured pea (Pisum sativum cv. Massey Gem) chloroplasts exhibited ascorbate peroxidase activity as determined by H(2)O(2)-dependent oxidation of ascorbate and ascorbate-dependent reduction of H(2)O(2). The ratio of ascorbate peroxidase to NADP-glyceraldehyde 3-phosphate dehydrogenase activity was constant during repeated washing of isolated chloroplasts. This indicates that the ascorbate peroxidase is a chloroplast enzyme. The pH optimum of ascorbate peroxidase activity was 8.2 and the K(m) value for ascorbate was 0.6 millimolar. Pyrogallol, glutathione, and NAD(P)H did not substitute for ascorbate in the enzyme catalyzed reaction. The enzyme was inhibited by NaN(3), KCN, and 8-hydroxyquinoline but not ZnCl(2) or iodoacetate. The ascorbate peroxidase activity of sonicated chloroplasts was inhibited by light but not in the presence of substrate concentrations of ascorbate.Illuminated ruptured chloroplasts, in the presence of 50 micromolar NADP(H), 2 millimolar l-ascorbate, and substrate concentrations of oxidized or reduced glutathione, catalyzed O(2) evolution when H(2)O(2) was added. Since the reaction was not inhibited by 0.1 millimolar NaN(3) and did not occur in the dark, it was concluded that catalase was not involved. Light-plus-H(2)O(2)-dependent O(2) evolution consisted of two distinct phases. The first phase was ascorbate-dependent and typically represented 10% of the total amount of O(2) evolved. The second phase was dependent on ascorbate and glutathione. The properties of the second phase were consistent with the operation of light-coupled glutathione reductase sequentially coupled to glutathione dehydrogenase and ascorbate peroxidase.
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PMID:Light-dependent reduction of hydrogen peroxide by ruptured pea chloroplasts. 1666 13

Preincubation of chloroplasts from pea leaves (Pisum sativum L. cv. Kelvedon) with 0.5 millimolar ferricyanide in the dark, caused a parallel inhibition of the rate of rise of the variable fluorescence and the rate of electron transport. Both reactions were inhibited to a similar extent by varying the time of preincubation, the concentration of ferricyanide during preincubation, and by raising the concentration of salts in the preincubation medium. Ferricyanide treatment of Tris-washed chloroplasts did not inhibit electron transport from the Photosystem II (PSII) electron donor 1,5-diphenylcarbazide to methylviologen. The inhibition of the variable fluorescence rise and of NADP reduction (caused by ferricyanide pretreatment) was bypassed by addition of the PSII electron donor couple hydroquinone/ascorbate. It was concluded that preincubation of chloroplasts with ferricyanide in the dark inhibited electron transport between water and PSII.
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PMID:Inhibition of oxygen evolution in chloroplasts by ferricyanide. 1666 85

1. A spectrophotometric assay of the rates of penetration of oxaloacetate and l-malate into mitochondria is described. The assay is based on the measurement of the oxidation of intramitochondrial NADH by oxaloacetate and of the reduction of intramitochondrial NAD(+) by malate. 2. The rate of entry of both oxaloacetate and l-malate into mitochondria is restricted, as shown by the fact that disruption of the mitochondrial structure can increase the rate of interaction between the dicarboxylic acids and intramitochondrial NAD(+) and NADH by between 100- and 1000-fold. 3. The rates of entry of oxaloacetate and malate into liver, kidney and heart mitochondria increased by up to 50-fold on addition of a source of energy, either ascorbate plus NNN'N'-tetramethyl-p-phenylenediamine aerobically, or ATP anaerobically. 4. In the absence of a source of energy the changes in the concentrations of intramitochondrial NAD(+) and NADH brought about by the addition of l-malate or oxaloacetate were followed by parallel changes in the concentrations of NADP(+) and NADPH, indicating the presence in the mitochondria of an energy-independent transhydrogenase system. 5. The results are discussed in relation to the hypothesis that malate acts as a carrier of reducing equivalents between mitochondria and cytoplasm.
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PMID:The permeability of mitochondria to oxaloacetate and malate. 1674 87

The symbiosis between legumes and rhizobia is characterised by the formation of dinitrogen-fixing root nodules. In natural conditions, nitrogen fixation is strongly impaired by abiotic stresses which generate over-production of reactive oxygen species. Since one of the nodule main antioxidant systems is the ascorbate-glutathione cycle, NADPH recycling that is involved in glutathione reduction is of great relevance under stress conditions. NADPH is mainly produced by glucose 6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) from the oxidative pentose phosphate pathway, and also by NADP(+)-dependent isocitrate dehydrogenase (ICDH; EC 1.1.1.42). In this work, 10 microM paraquat (PQ) was applied to pea roots in order to determine the in vivo relationship between oxidative stress and the activity of the NADPH-generating enzymes in nodules. Whereas G6PDH and 6PGDH activities remained unchanged, a remarkable induction of ICDH gene expression and a dramatic increase of the ICDH activity was observed during the PQ treatment. These results support that ICDH has a key role in NADPH recycling under oxidative stress conditions in pea root nodules.
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PMID:NADPH recycling systems in oxidative stressed pea nodules: a key role for the NADP+ -dependent isocitrate dehydrogenase. 1689 92

Treatment of E. coli extract with iron/ascorbate preferentially inactivated NADP-isocitrate dehydrogenase without affecting glucose-6-phosphate dehydrogenase. NADP-Isocitrate dehydrogenase required divalent metals such as Mg(2+), Mn(2+ )or Fe(2+) ion. Iron/ascorbate-dependent inactivation of the enzyme was accompanied with the protein fragmentation as judged by SDS-PAGE. Catalase protecting the enzyme from the inactivation suggests that hydroxyl radical is responsible for the inactivation with fragmentation. TOF-MS analysis showed that molecular masses of the enzyme fragments were 36 and 12, and 33 and 14 kDa as minor components. Based on the amino acid sequence analyses of the fragments, cleavage sites of the enzyme were identified as Asp307-Tyr308 and Ala282-Asp283, which are presumed to be the metal-binding sites. Ferrous ion bound to the metal-binding sites of the E. coli NADP-isocitrate dehydrogenase may generate superoxide radical that forms hydrogen peroxide and further hydroxyl radical, causing inactivation with peptide cleavage of the enzyme. Oxidative inactivation of NADP-isocitrate dehydrogenase without affecting glucose 6-phosphate dehydrogenase shows only a little influence on the antioxidant activity supplying NADPH for glutathione regeneration, but may facilitate flux through the glyoxylate bypass as the biosynthetic pathway with the inhibition of the citric acid cycle under aerobic growth conditions of E. coli.
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PMID:Oxidative inactivation of reduced NADP-generating enzymes in E. coli: iron-dependent inactivation with affinity cleavage of NADP-isocitrate dehydrogenase. 1689 33

Plant growth and development are driven by electron transfer reactions. Modifications of redox components are both monitored and induced by cells, and are integral to responses to environmental change. Key redox compounds in the soluble phase of the cell are NAD, NADP, glutathione and ascorbate--all of which interact strongly with reactive oxygen. This review takes an integrated view of the NAD(P)-glutathione-ascorbate network. These compounds are considered not as one-dimensional 'reductants' or 'antioxidants' but as redox couples that can act together to condition cellular redox tone or that can act independently to transmit specific information that tunes signalling pathways. Emphasis is placed on recent developments highlighting the complexity of redox-dependent defence reactions, and the importance of interactions between the reduction state of soluble redox couples and their concentration in mediating dynamic signalling in response to stress. Signalling roles are assessed within the context of interactions with reactive oxygen, phytohormones and calcium, and the biochemical reactions through which redox couples could be sensed are discussed.
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PMID:Metabolic signalling in defence and stress: the central roles of soluble redox couples. 1708 May 95

NADPH is an important molecule in the redox balance of the cell. In this paper, using olive tissue cultures as a model of the function of the NADPH-generating dehydrogenases in the mechanism of oxidative stress induced by severe salinity conditions was studied. When olive (Olea europaea) plants were grown with 200 mM NaCl, a 40% reduction in leaf fresh weight was produced. The content of non-enzymatic antioxidants such as ascorbate and glutathione was diminished between 20% to 39%, whereas the H2O2 content was increased threefold. In contrast, the analysis of the activity and protein contents of the main antioxidative enzymes showed a significant increase of catalase, superoxide dismutase and glutathione reductase. Overall, these changes strongly suggests that NaCl induces oxidative stress in olive plants. On the other hand, while the content of glucose-6-phosphate was increased almost eightfold in leaves of plants grown under salt stress, the content of NAD(P)H (reduced and oxided forms) did not show significant variations. Under salt stress conditions, the activity and protein contents of the main NADPH-recycling enzymes, glucose-6-phosphate dehydrogenase (G6PDH), isocitrate dehydrogenase (ICDH), malic enzyme (ME) and ferrodoxin-NADP reductase (FNR) showed an enhancement of 30-50%. In leaves of olive plants grown with 200 mM NaCl, analysis of G6PDH by immunocytochemistry and confocal laser scanning microscopy showed a general increase of this protein in epidermis, palisade and spongy mesophyll cells. These results indicate that in olive plants, salinity causes reactive oxygen species (ROS)-mediated oxidative stress, and plants respond to this situation by inducing different antioxidative enzymes, especially the NADPH-producing dehydrogenases in order to recycle NADPH necessary for the protection against oxidative damages. These NADP-dehydrogenases appear to be key antioxidative enzymes in olive plants under salt stress conditions.
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PMID:The dehydrogenase-mediated recycling of NADPH is a key antioxidant system against salt-induced oxidative stress in olive plants. 1708 Sep 66

D-Galacturonic acid reductase, a key enzyme in ascorbate biosynthesis, was purified to homogeneity from Euglena gracilis. The enzyme was a monomer with a molecular mass of 38-39 kDa, as judged by SDS-PAGE and gel filtration. Apparently it utilized NADPH with a Km value of 62.5+/-4.5 microM and uronic acids, such as D-galacturonic acid (Km=3.79+/-0.5 mM) and D-glucuronic acid (Km=4.67+/-0.6 mM). It failed to catalyze the reverse reaction with L-galactonic acid and NADP(+). The optimal pH for the reduction of D-galacturonic acid was 7.2. The enzyme was activated 45.6% by 0.1 mM H(2)O(2), suggesting that enzyme activity is regulated by cellular redox status. No feedback regulation of the enzyme activity by L-galactono-1,4-lactone or ascorbate was observed. N-terminal amino acid sequence analysis revealed that the enzyme is closely related to the malate dehydrogenase families.
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PMID:Functional characterization of D-galacturonic acid reductase, a key enzyme of the ascorbate biosynthesis pathway, from Euglena gracilis. 1709 Sep 24


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