Purification and Characterization of a Glutathione Dependent Dehydroascorbate Reductase from Human Erythrocytes

Xu, Dian; Washburn, Michael P.; Sun, Guo; Wells, William W.

doi:10.1006/bbrc.1996.0555

Cited by 57 publications

(23 citation statements)

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“…As expected, based on multiple dehydroascorbic acid-reducing mechanisms within RBCs [37], no dehydroascorbic acid was detected in RBCs (Supplementary Fig. 1, Supplementary Table 1).…”

Section: Resultssupporting

confidence: 80%

Vitamin C in mouse and human red blood cells: An HPLC assay

Wang

et al. 2012

Analytical Biochemistry

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Although vitamin C (ascorbate) is present in whole blood, measurements in red blood cells (RBCs) are problematic because of interference, instability, limited sensitivity, and sample volume requirements. We describe a new technique using HPLC with coulometric electrochemical detection for ascorbate measurement in RBCs of humans, wild-type mice, and mice unable to synthesize ascorbate. Exogenously added ascorbate was fully recovered even when endogenous RBC ascorbate was below the detection threshold (25 nM). Twenty microliters of whole blood or 10 μl of packed RBCs was sufficient for assay. RBC ascorbate was stable for 24 h from whole-blood samples at 4 °C. Processed, stored samples were stable for >1 month at −80 °C. Unlike other tissues, ascorbate concentrations in human and mouse RBCs were linear in relation to plasma concentrations (R = 0.8 and 0.9, respectively). In healthy humans, RBC ascorbate concentrations were 9–57 μM, corresponding to ascorbate plasma concentrations of 15–90 μM. Mouse data were similar. In human blood stored as if for transfusion, initial RBC ascorbate concentrations varied approximately sevenfold and decreased 50% after 6 weeks of storage under clinical conditions. With this assay, it becomes possible for the first time to characterize ascorbate function in relation to endogenous concentrations in RBCs.

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“…As expected, based on multiple dehydroascorbic acid-reducing mechanisms within RBCs [37], no dehydroascorbic acid was detected in RBCs (Supplementary Fig. 1, Supplementary Table 1).…”

Section: Resultssupporting

confidence: 80%

Vitamin C in mouse and human red blood cells: An HPLC assay

Wang

et al. 2012

Analytical Biochemistry

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“…It prevents tissue damage [30,31]. Vitamin C is routinely prescribed in the hospital to aid recovery from several ailments and diseases, such as cold, cough, influenza, sores, wounds, gingivitis, skin diseases, diarrhoea, malaria, and bacterial infections [32].…”

Section: The Vitamin C Content Of the Stored Probioticated Juice Samplesmentioning

confidence: 99%

Probiotic Viability, Physicochemical and Sensory Properties of Probiotic Pineapple Juice

Adebayo-Tayo¹,

Akpeji²

2016

Fermentation

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(1) Background: Probiotication is an important method in the food industry, and the use of probiotic dairy products has prevented lactose intolerant patients and vegetarians from their consumption. Hence, there is a need to incorporate probiotics in fruit juice without lactose; (2) Method: Probiotic viability, and physicochemical and sensory evaluation of stored probioticated pineapple juice using lactic acid bacteria (LAB) (Pediococcus pentosaceus LaG1, Lactobacillus rhamnosus GG, Pediococcus pentosaceus LBF2) as a single and mixed starter was investigated; (3) Results: There was an increase in the lactic acid production, and reduction in pH, vitamin C content, and colour during storage. At weeks 3 and 4, Propp2 and Pcontrol samples had the highest lactic acid content (317.9 mg/L and 160.34 mg/L). The vitamin C content ranged from 2.91-7.10 mg/100 g. There was a general reduction in total soluble solids during storage. The probiotic LAB were viable throughout the storage time (1.05-1.10 × 10 9 cfu/mL) in the juice samples. There was no significant difference in terms of taste, aroma, colour, or appearance during the time of storage; (4) Conclusion: The pineapple juice supported the viability, lactic acid production, vitamin C development, and the antagonistic potential of the probiotic candidate. This result is useful for the development of probiotic fruit juice as functional foods and nutraceuticals with health beneficial effect.

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“…The specific activity of spinach chloroplast DHAR was found to be seven times higher than that of DHAR from rice bran [8,15]. Moreover, spinach chloroplast DHAR has a 100-fold lower K m value for DHA and several-fold higher specific activity than porcine DHAR and other DHA-reducing enzymes [7,[11][12][13]15,16].DHA-reducing enzymes commonly include a C-X-X-C motif. It has been demonstrated by site-directed mutagenesis that the C22 residue in pig liver thioltransferase is essential for the reduction of DHA [17].…”

mentioning

confidence: 99%

“…Enzymes that reduce DHA are distributed not only in plant cells but also in mammalian cells [7][8][9][10][11][12][13][14]. However, the enzymatic properties of spinach chloroplast DHAR are different from those of other DHA-reducing enzymes.…”

mentioning

confidence: 99%

Mechanism of the reaction catalyzed by dehydroascorbate reductase from spinach chloroplasts

Shimaoka

Miyake

Yokota

2003

European Journal of Biochemistry

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Dehydroascorbate reductase (DHAR) reduces dehydroascorbate (DHA) to ascorbate with glutathione (GSH) as the electron donor. We analyzed the reaction mechanism of spinach chloroplast DHAR, which had a much higher reaction specificity for DHA than animal enzymes, using a recombinant enzyme expressed in Escherichia coli. Kinetic analysis suggested that the reaction proceeded by a bi-uni-uni-uni-ping-pong mechanism, in which binding of DHA to the free, reduced form of the enzyme was followed by binding of GSH. The K m value for DHA and the summed K m value for GSH were determined to be 53 ± 12 lM and 2.2 ± 1.0 mM, respectively, with a turnover rate of 490 ± 40 s )1. Incubation of 10 lM DHAR with 1 mM DHA and 10 lM GSH resulted in stable binding of GSH to the enzyme. Bound GSH was released upon reduction of the GSH-enzyme adduct by 2-mercaptoethanol, suggesting that the adduct is a reaction intermediate. Site-directed mutagenesis indicated that C23 in DHAR is indispensable for the reduction of DHA. The mechanism of catalysis of spinach chloroplast DHAR is proposed.Keywords: dehydroascorbate reductase; catalytic mechanism; ping-pong mechanism; oxidative stress; ascorbate.Ascorbate functions not only as an antioxidant but also as a substrate for ascorbate peroxidase (APX) and violaxanthin de-epoxidase in chloroplasts [1,2]. APX catalyzes the decomposition of hydrogen peroxide in the active oxygenscavenging system and the reaction catalyzed by violaxanthin de-epoxidase in the xanthophyll cycle is involved in the down-regulation of the activity of photosystem II. These enzymes are involved in the dissipation of excess light energy and protect plants from oxidative stress. In reactions catalyzed by APX and violaxanthin de-epoxidase, ascorbate is oxidized to monodehydroascorbate (MDA) and then dehydroascorbate (DHA) is produced via the spontaneous disproportionation of MDA. The regeneration of ascorbate is essential for the maintenance of the activity of the active oxygen-scavenging system and the xanthophyll cycle. MDA and DHA are reduced to ascorbate by MDA reductase and ferredoxin, and DHA reductase (DHAR) in chloroplasts, respectively [3][4][5][6].The reduction of DHA to ascorbate by DHAR (EC 1.8.5.1) involves GSH as the electron donor. Enzymes that reduce DHA are distributed not only in plant cells but also in mammalian cells [7][8][9][10][11][12][13][14]. However, the enzymatic properties of spinach chloroplast DHAR are different from those of other DHA-reducing enzymes. The specific activity of spinach chloroplast DHAR was found to be seven times higher than that of DHAR from rice bran [8,15]. Moreover, spinach chloroplast DHAR has a 100-fold lower K m value for DHA and several-fold higher specific activity than porcine DHAR and other DHA-reducing enzymes [7,[11][12][13]15,16].DHA-reducing enzymes commonly include a C-X-X-C motif. It has been demonstrated by site-directed mutagenesis that the C22 residue in pig liver thioltransferase is essential for the reduction of DHA [17]. Spinach chloroplast DHAR also has this ...

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Purification and Characterization of a Glutathione Dependent Dehydroascorbate Reductase from Human Erythrocytes

Cited by 57 publications

References 0 publications

Vitamin C in mouse and human red blood cells: An HPLC assay

Vitamin C in mouse and human red blood cells: An HPLC assay

Probiotic Viability, Physicochemical and Sensory Properties of Probiotic Pineapple Juice

Mechanism of the reaction catalyzed by dehydroascorbate reductase from spinach chloroplasts

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