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PubMed2000

The control of ascorbic acid synthesis and turnover in pea seedlings.

The rate of ascorbate synthesis and turnover in pea seedling embryonic axes was investigated in relation to its pool size. Ascorbate accumulated in embryonic axes of germinating pea seeds which has been supplied with ascorbate. Incorporation of [U-14C]glucose into ascorbate after a 2 h labelling period was reduced by ascorbate loading for 3 h and 20 h, providing evidence that ascorbate biosynthesis is inhibited by endogenous ascorbate. Ascorbate turnover was estimated by following the metabolism of [1-14C]ascorbate over 2 h after ascorbate loading and by the rate of decrease of the ascorbate pool size after ascorbate loading. Ascorbate turnover rate, determined by [1-14C]ascorbate metabolism, increased as a linear function of pool size. The absolute turnover rate was higher in ascorbate-loaded embryonic axes but was always about 13% of the pool per hour. The initial rate of ascorbate turnover, estimated from the net decrease in pool size after ascorbate loading, also showed a similar turnover rate to that estimated from [1-14C]ascorbate metabolism. Ascorbate loading had no effect on ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase or glutathione reductase activity. Ascorbate oxidase activity decreased after ascorbate loading.

Ascorbic Acid↗
PubMed1992

18O isotopic 13C NMR shift as proof that bifunctional peptidylglycine alpha-amidating enzyme is a monooxygenase.

The biosynthesis of C-terminal alpha-amidated peptides from their corresponding C-terminal glycine-extended precursors is catalyzed by peptidylglycine alpha-amidating enzyme (alpha-AE) in a reaction that requires copper, ascorbate, and molecular oxygen. Using bifunctional type A rat alpha-AE, we have shown that O2 is the source of the alpha-carbonyl oxygen of pyruvate produced during the amidation of dansyl-Tyr-Val-[alpha-13C]-D-Ala, as demonstrated by the 18O isotopic shift in the 13C NMR spectrum of [alpha-13C]lactate generated from [alpha-13C]pyruvate in the presence of lactate dehydrogenase and NADH. In addition, one-to-one stoichiometries have been determined for glyoxylate formed/dansyl-Tyr-Val-Gly consumed, pyruvate formed/dansyl-Tyr-Val-D-Ala consumed, dansyl-Tyr-Val-NH2 formed/ascorbate oxidized, and dansyl-Tyr-Val-NH2 formed/O2 consumed. Quantitative coupling of NADH oxidation to dansyl-Tyr-Val-NH2 production using Neurospora crassa semidehydroascorbate reductase showed that two one-electron reductions by ascorbate occurred per alpha-AE turnover. The stoichiometry of approximately 1.0 dansyl-Tyr-Val-NH2 produced/ascorbate oxidized observed in the absence of a semidehydroascorbate trap resulted from the disproportionation of two semidehydroascorbate molecules to ascorbate and dehydroascorbate.

Amino Acid Sequence↗
PubMed2002

Characterization of an omega-class glutathione S-transferase from Schistosoma mansoni with glutaredoxin-like dehydroascorbate reductase and thiol transferase activities.

Glutathione S-transferases (EC 2.5.1.18) (GSTs), are a family of multifunctional enzymes present in all living organisms whose main function is the detoxification of electrophilic compounds. GSTs are considered the most prominent detoxifying class II enzymes in helminths. We describe here the characterization of novel dehydroascorbate reductase and thiol transferase activities that reside in the human parasite Schistosoma mansoni GSTx. Protein sequence analysis of this parasite product showed lower identity to known GSTs. However, phylogenic analysis placed SmGSTx among the recently described omega class GSTs (GSTO1-1). We report here that SmGSTO protein is a 28-kDa polypeptide, detected in all life stages of the parasite, being highly expressed in adult worms. Like other omega class GSTs, SmGSTO showed very low activity toward classical GSTs substrates as 1-chloro-2,4-dinitrobenzene, and no binding affinity to glutathione-agarose matrix but showed some biochemical characteristics related with thioredoxins/glutaredoxins. Interestingly, SmGSTO was able to bind S-hexyl glutathione matrix and displayed significant glutathione-dependent dehydroascorbate reductase and thiol transferase enzymatic activities.

Amino Acid Sequence↗
PubMed2003

Cooperative interaction between ascorbate and glutathione during mitochondrial impairment in mesencephalic cultures.

A decrease in total glutathione, and aberrant mitochondrial bioenergetics have been implicated in the pathogenesis of Parkinson's disease. Our previous work exemplified the importance of glutathione (GSH) in the protection of mesencephalic neurons exposed to malonate, a reversible inhibitor of mitochondrial succinate dehydrogenase/complex II. Additionally, reactive oxygen species (ROS) generation was an early, contributing event in malonate toxicity. Protection by ascorbate was found to correlate with a stimulated increase in protein-glutathione mixed disulfide (Pr-SSG) levels. The present study further examined ascorbate-glutathione interactions during mitochondrial impairment. Depletion of GSH in mesencephalic cells with buthionine sulfoximine potentiated both the malonate-induced toxicity and generation of ROS as monitored by dichlorofluorescein diacetate (DCF) fluorescence. Ascorbate completely ameliorated the increase in DCF fluorescence and toxicity in normal and GSH-depleted cultures, suggesting that protection by ascorbate was due in part to upstream removal of free radicals. Ascorbate stimulated Pr-SSG formation during mitochondrial impairment in normal and GSH-depleted cultures to a similar extent when expressed as a proportion of total GSH incorporated into mixed disulfides. Malonate increased the efflux of GSH and GSSG over time in cultures treated for 4, 6 or 8 h. The addition of ascorbate to malonate-treated cells prevented the efflux of GSH, attenuated the efflux of GSSG and regulated the intracellular GSSG/GSH ratio. Maintenance of GSSG/GSH with ascorbate plus malonate was accompanied by a stimulation of Pr-SSG formation. These findings indicate that ascorbate contributes to the maintenance of GSSG/GSH status during oxidative stress through scavenging of radical species, attenuation of GSH efflux and redistribution of GSSG to the formation of mixed disulfides. It is speculated that these events are linked by glutaredoxin, an enzyme shown to contain both dehydroascorbate reductase as well as glutathione thioltransferase activities.

Animals↗
PubMedDate not supplied

[Glutathione:dehydroascorbate oxidoreductase activity in rabbit eye tissues].

Glutathione: dehydroascorbate oxidoreductase was detected in rabbit eye tissues, activity of which was decreased as compared with that of glutathione reductase. The ratio between glutathione reductase activity and the total activity of the enzymes catalyzing glutathione oxidation (glutathione: dehydroascorbate oxidoreductase and glutathione: H2O2 peroxidase) was above one unit in cornea, ciliary body, sclera vascular tunic and iris of the eye; it was lower than one unit in vitreous body, lens, optic nerve and aqueous humor. Glutathione: dehydroascorbate oxidoreductase is one of the key component in the eye antioxidative system; it regulates the ratio of oxidized and reduced forms of ascorbic acid and glutathione.

Animals↗
PubMed1992

Glycation of MP26 and MP22 in bovine lens membranes.

Alkali treated membranes were isolated from mature bovine lenses and incubated with different sugars for 3 weeks to study the effect of glycation on the lens intrinsic membrane proteins, MP26 and MP22. The obtained results show that a) [1-14C] ascorbic acid (ASA) was able to glycate the intrinsic membrane proteins as rapidly as soluble lens proteins; b) on 15% acrylamide gels in SDS, glucose, fructose, galactose and ribose exhibited low activity for crosslinking membrane proteins; whereas ASA, dehydroascorbate (DHA), diketogulonate (DKG), xylosone and threose, all showed not only the formation of protein multimers, but also highly crosslinked products, which did not enter the spacer gel; c) except glycated MP22, all of the crosslinks of MP26 or MP22, and also the glycated MP26, showed cross reactivity with polyclonal MP26 antibody; d) the extent of crosslinking correlated with an equal loss of lysine and arginine contents by amino acid analysis.

Animals↗
PubMed1993

Thioltransferases.

A family of small molecular weight proteins with thiol-disulfide exchange activity have been discovered, widely distributed from E. coli to mammalian systems, called thioltransferases or glutaredoxins. There are no substantiated reports of thioltransferases-glutaredoxins in plants; however, partially purified dehydroascorbate reductase from peas had thiol-disulfide exchange catalytic activity using glutathione as reductant and S-sulfocysteine as thiosulfate cosubstrate (unpublished data). Thus, this class of proteins is universally distributed. Based on mutagenesis studies, a sequence of Cys-Pro-Tyr(Phe)-Cys- followed by Arg-Lys- or Lys alone is critical for both the thiol-disulfide exchange reaction and the dehydroascorbate reductase activity. The dithiol-disulfide loop represented by this structure is unique since the cystine closer to the N-terminus has a highly acidic thiol pKa (3.8 as determined for the pig liver enzyme) that contributes to the protein's high S- nucleophilicity. Compared with the microbial enzyme, the mammalian thioltransferases (glutaredoxins) are extended at both N and C termini by 10-12 amino acid residues, including a second pair of cysteines toward the C-terminus with no known special function. Yeast thioltransferase is more like mammalian enzymes in length (106 amino acids) but more like E. coli glutaredoxin in being unblocked at the N-terminus and having only one set of cysteines; that is, at the active center. The three mammalian enzymes, for which sequences are available, are blocked at the N-terminus by an acetyl group linked to alanine with no known special function other than possibly to impart greater cellular turnover stability. A report of carbohydrate (8.6%) content in rat liver thioltransferase has not been verified by more sensitive methods of carbohydrate analysis, nor has carbohydrate been identified in samples of purified glutaredoxin from any source. Thiol transferase and glutaredoxin are two names for the same protein based on similarity of amino acid sequence, immunochemical cross-reactivity, and other enzyme properties. The inability of thioltransferase from some mammalian sources to act as an electron carrier in ribonucleotide reductase systems, whether homologous or heterologous in origin, remains to be explained in future studies.

Amino Acid Sequence↗
PubMed1999

Interaction of antioxidants and their implication in genetic anemia.

The generation of reactive oxygen species (ROS) is a steady-state cellular event in respiring cells. Their production can be grossly amplified in response to a variety of pathophysiological conditions such as inflammation, immunologic disorders, hypoxia, hyperoxia, metabolism of drug or alcohol, exposure to UV or therapeutic radiation, and deficiency in antioxidant vitamins. Uncontrolled production of ROS often leads to damage of cellular macromolecules (DNA, protein, and lipids) and other small antioxidant molecules. A number of major cellular defense mechanisms exist to neutralize and combat the damaging effects of these reactive substances. The enzymic system functions by direct or sequential removal of ROS (superoxide dismutase, catalase, and glutathione peroxidase), thereby terminating their activities. Metal binding proteins, targeted to bind iron and copper ions, ensure that these Fenton metals are cryptic. Nonenzymic defense consists of scavenging molecules that are endogenously produced (GSH, ubiquinols, uric acid) or those derived from the diet (vitamins C and E, lipoic acid, selenium, riboflavin, zinc, and the carotenoids). These antioxidant nutrients occupy distinct cellular compartments and among them, there are active recycling. For example, oxidized vitamin E (tocopheroxy radical) has been shown to be regenerated by ascorbate, GSH, lipoic acid, or ubiquinols. GSH disulfides (GSSG) can be regenerated by GSSG reductase (a riboflavin-dependent protein), and enzymic pathways have been identified for the recycling of ascorbate radical and dehydroascorbate. The electrons that are used to fuel these recycling reactions (NADH and NADPH) are ultimately derived from the oxidation of foods. Sickle cell anemia, thalassemia, and glucose-6-phosphate-dehydrogenase deficiency are all hereditary disorders with higher potential for oxidative damage due to chronic redox imbalance in red cells that often results in clinical manifestation of mild to serve hemolysis in patients with these disorders. The release of hemoglobin during hemolysis and the subsequent therapeutic transfusion in some cases lead to systemic iron overloading that further potentiates the generation of ROS. Antioxidant status in anemia will be examined, and the potential application of antioxidant treatment as an adjunct therapy under these conditions will be discussed.

Anemia↗
PubMed1997

Purification and characterization of glutaredoxin (thioltransferase) from rice (Oryza sativa L.).

We purified and characterized glutaredoxin (thioltransferase), which catalyzes thiol/disulfide exchange reaction, for the first time in plants. The purification procedure employed an immunoabsorbent, antiglutaredoxin-Sepharose. Glutaredoxin was purified about 2,200-fold from rice bran and it appeared to be homogeneous on SDS-PAGE. MALDI-TOF mass spectrometry revealed that the protein has a molecular mass of 11,097.9 Da. Rice glutaredoxin consists of 105 amino acid residues, containing the tetrapeptide -Cys-Phe-Pro (Tyr)-Cys-, which constitutes the active site of Escherichia coli and mammalian glutaredoxins. Inactivation assay also indicated that cysteine residues are responsible for enzyme activity. Kinetic analyses revealed that the enzyme did not exhibit normal Michaelis-Menten kinetics. The enzyme has an optimum pH of about 8.7 with 2-hydroxyethyl disulfide as a substrate. In addition, rice glutaredoxin has dehydroascorbate reductase activity, like mammalian glutaredoxin.

Amino Acid Sequence↗
PubMed1968

Sporicidal action of auto-oxidized ascorbic acid for Clostridium.

Neutralized ascorbic acid (AA), buffered or unbuffered and autoclaved or filter-sterilized, was sporicidal for Clostridium. A 0.2% concentration of AA was generally employed, and spore counts were made in a soft-agar modification of Wynne's medium in Prickett tubes. Spores of Clostridium botulinum 115B were less susceptible than those of C. sporogenes PA 3679, whereas C. bifermentans spores were by far the most sensitive. At 75 C, spores of PA 3679 were killed at a rate of about 9% at 0 min (warm-up) to 99+% at 100 min. The lower the temperature, the longer the time needed for a given lethality. The percentage of killing increased with increasing concentrations of AA, and the rate of killing was lower at a higher concentration of spores. At least two mechanisms were operative: a major mechanism involving a product(s) of AA auto-oxidation, and a minor mechanism involving copper-ascorbate toxicity. AA reduced in natural gas was not sporicidal after 18.5 hr at 25 C, whereas 92% of the spores were killed by oxidized AA. Although H(2)O(2) per se was sporicidal, catalase did not reverse lethality of fresh or oxidized AA. Dehydroascorbate was as sporicidal as any AA preparation. Added copper (0.00001%) increased the rate of lethality of freshly prepared AA from 66 to 83% but was not effective with thoroughly oxidized AA. Ethylenediaminetetraacetic acid, NH(4) (+), and phosphate partially reversed AA toxicity, deionized water had no effect, and complex media, as well as thioglycolate, eliminated AA lethality. Since the percentage of killing was affected by spore concentration, AA did not seem to stimulate "lethal germination."

Ascorbic Acid↗
PubMed1992

Glutathione deficiency increases hepatic ascorbic acid synthesis in adult mice.

Glutathione deficiency, induced in adult mice by administering buthionine sulfoximine (an inhibitor of glutathione synthesis), led to a rapid and substantial increase in ascorbate in the liver. This effect was apparent 2-4 hr after giving the inhibitor; subsequently, the level of ascorbate decreased and that of dehydroascorbate increased markedly, supporting the conclusion that glutathione functions physiologically to keep ascorbate in its reduced form. In kidney and lung also, ascorbate levels decreased, and dehydroascorbate increased. Increased synthesis of ascorbate in glutathione-deficient adult mice seems to protect against tissue damage. In contrast, newborn rats, which (like guinea pigs and humans) apparently do not synthesize ascorbate, suffer severe damage to liver and other organs; previous studies showed that administration of ascorbate prevents such tissue damage. The findings support the view that the antioxidant actions of glutathione and ascorbate are closely linked and involve a mechanism in which decrease of the glutathione level, perhaps associated with an oxidative event, stimulates ascorbate synthesis.

Animals↗
PubMed1996

Identification of a second functional glutaredoxin encoded by the bacteriophage T4 genome.

Thioredoxins and glutaredoxins are small ubiquitous redox proteins that were discovered as hydrogen donors for ribonucleotide reductase, the key enzyme for deoxyribonucleotide biosynthesis. Some organisms encode more than one redox protein. In this study, we demonstrate that an open reading frame in the bacteriophage T4 genome, reported earlier and designated as Y55.7 (Tomaschewski, J., and Rüger, W. (1987) Nucleic Acids Res. 15, 3632-3633), encodes a second functional redox protein. Gene y55.7 was cloned and expressed in Escherichia coli. Purified Y55.7 protein had glutathione-dependent thioltransferase and dehydroascorbate reductase activities indicative of a functional glutaredoxin. The protein is expressed at all stages of the T4 infection cycle and can serve as a hydrogen donor for the phage ribonucleotide reductase in in vitro experiments.

Bacteriophage T4↗
PubMedDate not supplied

Ascorbic acid recycling in N-myc amplified human neuroblastoma cells.

The present study investigated the ability of two neuroblastoma cell lines (SK-N-SH, with one copy of N-myc, and SK-N-BE(2), with over 150 copies of N-myc) to recycle ascorbate by quantifying semidehydroascorbate reductase and dehydroascorbate reductase activities. Both cell lines expressed dehydroascorbate activity (SK-N-SH 28.4 +/- 9.8, SK-N-BE(2) 21.7 +/- 5.2 nmol/min/mg protein). Intracellular semidehydroascorbate activity was present only in SK-N-BE(2) cells (4.7 +/- 1.2 nmol/min/mg protein). Extracellular ascorbate was regenerated by semidehydroascorbate membrane activity, the activity of SK-N-BE(2) being twice that of SK-N-SH cells. The present data may explain the ability of the tumor to progress or regress through mechanisms involving both myc oncogene and apoptosis.

Ascorbic Acid↗
PubMed1998

Erythrocyte ascorbate recycling: antioxidant effects in blood.

Ascorbic acid is an important antioxidant in human plasma, but requires efficient recycling from its oxidized forms to avoid irreversible loss. Human erythrocytes prevented oxidation of ascorbate in autologous plasma, an effect that required recycling of ascorbate within the cells. Erythrocytes had a high capacity to take up dehydroascorbate, the two-electron oxidized product of ascorbate, and to reduce it to ascorbate. Uptake and conversion of dehydroascorbate to ascorbate was saturable, was half-maximal at 400 microM dehydroascorbate, and achieved a maximal intracellular ascorbate concentration of 1.5 mM. In the presence of 100 microM dehydroascorbate, erythrocytes had the capacity to regenerate a 35 microM ascorbate concentration in blood every 3 min. Ascorbate recycling from DHA required intracellular GSH. Depletion of erythrocyte GSH by more than 50% with diamide did not acutely affect the cellular ascorbate content, but did impair the subsequent ability of GSH-depleted cells to recycle dehydroascorbate to ascorbate. Whereas erythrocyte ascorbate recycling was coupled to GSH, an overwhelming extracellular oxidant stress depleted both ascorbate and alpha-tocopherol before the GSH content of cells fell appreciably. Recycled ascorbate was released from cells into plasma, but at a rate less than one tenth that of dehydroascorbate uptake and conversion to ascorbate. Nonetheless, ascorbate released from cells protected endogenous alpha-tocopherol in human LDL from oxidation by a water soluble free radical initiator. These results suggests that recycling of ascorbate in erythrocytes helps to maintain the antioxidant reserve of whole blood.

Antioxidants↗
PubMed2006

Ascorbate transport and recycling by SH-SY5Y neuroblastoma cells: response to glutamate toxicity.

Neurons maintain relatively high intracellular concentrations of vitamin C, or ascorbic acid. In this work we studied the mechanisms by which neuronal cells in culture transport and maintain ascorbate, as well as how this system responds to oxidant stress induced by glutamate. Cultured SH-SY5Y neuroblastoma cells took up ascorbate, achieving steady-state intracellular concentrations of 6 mM and higher at extracellular concentrations of 200 microM and greater. This gradient was generated by relatively high affinity sodium-dependent ascorbate transport (Km of 113 microM). Ascorbate was also recycled from dehydroascorbate, the reduction of which was dependent on GSH, but not on D-glucose. Glutamate in concentrations up to 2 mM caused an acute concentration-dependent efflux of ascorbate from the cells, which was prevented by the anion channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. Intracellular ascorbate did not affect radiolabeled glutamate uptake, showing absence of heteroexchange.

Ascorbic Acid↗
PubMed2005

Response of ascorbate peroxidase isoenzymes and ascorbate regeneration system to abiotic stresses in Cucumis sativus L.

Ascorbate peroxidase (APX) isoenzymes, distributing in at least four distinct cell compartments, the chloroplastic stroma (sAPX) and thylakoid membrane (tAPX), microbody (mAPX) and cytosol (cAPX), catalyze the reduction of H(2)O(2) to water by using ascorbic acid (AsA) as specific electron donor. In order to better clarify the response of APX isoenzymes and AsA regeneration enzymes to abiotic stresses, the activities of APX isoenzymes as well as monodehydroascorbate reductase (MDAR), glutathione reductase (GR) and dehydroascorbate reductase (DHAR) were investigated in cucumber plants after heat, methyl viologen (MV) and H(2)O(2) treatments. The activities of cAPX, sAPX, mAPX increased after a slight decline throughout the experiment. Consistent closely with sAPX activity, the expression of sAPX followed a similar change pattern, indicating that sAPX was regulated at the transcriptional level. In contrast, constitutive expression was observed in tAPX activity and no significant changes in tAPX activity were found throughout the experiment. The increases in MDAR and GR were accompanied with enhanced level of AsA/DHA, implying that the AsA regeneration system plays an essential role in compensating AsA degradation.

Ascorbate Peroxidases↗
PubMed1999

Prooxidant effects of ascorbate in rat brain slices.

Ascorbate is a well-known reducing agent, but it can generate oxidative potential under appropriate condition. In rat cerebral cortex homogenate, 1 mM ascorbate decreased thiobarbituric acid-reactive substances (TBARS) content to 86% +/- 4% of control values, confirming that ascorbate is a reducing agent. However, ascorbate increased TBARS, in a dose-related manner, in slices prepared from cerebral cortex. Ferrous ion (Fe2+) had little effect on ascorbate-induced lipid oxidation in cortical slices, and EDTA did not have an influence on the ascorbate-induced oxidative action. Conversely, ascorbate plus Fe2+ elevated TBARS content to more than threefold over ascorbate alone in tissue homogenates. In summary, ascorbate is a reducing agent in the brain tissue homogenate but has an oxidizing effect in brain slices. A hypothesis is proposed to explain the oxidative effects of ascorbate in cortical slices, wherein extracellular ascorbate is oxidized to dehydroascorbate, which is rapidly carried into the cells via a glucose transporter (GLUT). The dehydroascorbate in cytosol is then reduced back to ascorbate, and, during the reduction process, cellular components are oxidized.

Androstadienes↗