Human Erythrocyte Membranes Contain a Cytochrome b561 That May Be Involved in Extracellular Ascorbate Recycling (original) (raw)
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Recycling of the ascorbate free radical by human erythrocyte membranes
Free Radical Biology and Medicine, 2001
Reduction of the ascorbate free radical (AFR) at the plasma membrane provides an efficient mechanism to preserve the vitamin in a location where it can recycle ␣-tocopherol and thus prevent lipid peroxidation. Erythrocyte ghost membranes have been shown to oxidize NADH in the presence of the AFR. We report that this activity derives from an AFR reductase because it spares ascorbate from oxidation by ascorbate oxidase, and because ghost membranes decrease steady-state concentrations of the AFR in a protein-and NADH-dependent manner. The AFR reductase has a high apparent affinity for both NADH and the AFR (Ͻ 2 M). When measured in open ghosts, the reductase is comprised of an inner membrane activity (both substrate sites on the cytosolic membrane face) and a trans-membrane activity that mediates extracellular AFR reduction using intracellular NADH. However, the trans-membrane activity constitutes only about 12% of the total measured in ghosts. Ghost AFR reductase activity can also be differentiated from NADH-dependent ferricyanide reductase(s) by its sensitivity to the detergent Triton X-100 and insensitivity to enzymatic digestion with cathepsin D. This NADH-dependent AFR reductase could serve to recycle ascorbic acid at a crucial site on the inner face of the plasma membrane.
Journal of Biological Chemistry, 2004
Recycling of ascorbic acid from its oxidized forms helps to maintain the vitamin in human erythrocytes. To determine the relative contributions of recycling from the ascorbate radical and dehydroascorbic acid, we studied erythrocytes exposed to a trans-membrane oxidant stress from ferricyanide. Ferricyanide was used both to induce oxidant stress across the cell membrane and to quantify ascorbate recycling. Erythrocytes reduced ferricyanide with generation of intracellular ascorbate radical, the concentrations of which saturated with increasing intracellular ascorbate and which were sustained over time in cells incubated with glucose. Ferricyanide also generated dehydroascorbic acid that accumulated in the cells and incubation medium to concentrations much higher than those of the radical, especially in the absence of glucose. Ferricyanide-stimulated ascorbate recycling from dehydroascorbic acid depended on intracellular GSH but was well maintained at the expense of intracellular ascorbate when GSH was severely depleted by diethylmaleate. This likely reflects continued radical reduction, which is not dependent on GSH. Erythrocyte hemolysates showed both NAD-and NADPH-dependent ascorbate radical reduction. The latter was partially due to thioredoxin reductase. GSH-dependent dehydroascorbate reduction in hemolysates, which was both direct and enzyme-dependent, was greater than that of the radical reductase activity but of lower apparent affinity. Together, these results suggest an efficient two-tiered system in which high affinity reduction of the ascorbate radical is sufficient to remove low concentrations of the radical that might be encountered by cells not under oxidant stress, with back-up by a high capacity system for reducing dehydroascorbate under conditions of more severe oxidant stress.
Mechanisms of ascorbic acid recycling in human erythrocytes
Biochimica et Biophysica Acta (BBA) - General Subjects, 2001
Vitamin C, or ascorbic acid, is efficiently recycled from its oxidized forms by human erythrocytes. In this work the dependence of this recycling on reduced glutathione (GSH) was evaluated with regard to activation of the pentose cycle and to changes in pyridine nucleotide concentrations. The two-electron-oxidized form of ascorbic acid, dehydroascorbic acid (DHA) was rapidly taken up by erythrocytes and reduced to ascorbate, which reached intracellular concentrations as high as 2 mM. In the absence of D-glucose, DHA caused dose-dependent decreases in erythrocyte GSH, NADPH, and NADH concentrations. In the presence of 5 mM D-glucose, GSH and NADH concentrations were maintained, but those of NADPH decreased. Reduction of extracellular ferricyanide by erythrocytes, which reflects intracellular ascorbate recycling, was also enhanced by D-glucose, and ferricyanide activated the pentose cycle. Diethylmaleate at concentrations up to 1 mM was found to specifically deplete erythrocyte GSH by 75^90% without causing oxidant stress in the cells. Such GSH-depleted erythrocytes showed parallel decreases in their ability to take up and reduce DHA to ascorbate, and to reduce extracellular ferricyanide. These results show that DHA reduction involves GSH-dependent activation of D-glucose metabolism in the pentose cycle, but that in the absence of D-glucose DHA reduction can also utilize NADH. ß
Erythrocyte defenses against hydrogen peroxide: the role of ascorbic acid
Biochimica et Biophysica Acta (BBA) - General Subjects, 1998
. Ascorbate has been reported to increase intracellular hydrogen peroxide H O generation in human erythrocytes. In the 2 2 present work, the basis for this prooxidant effect of the vitamin was investigated in the context of erythrocyte defenses against H O . Ascorbate added to erythrocytes caused a dose-dependent increase in intracellular H O , which was 2 2 2 2 Ž . measured as inactivation of endogenous catalase in the presence of 3-amino-1,2,4-triazole aminotriazole . Ascorbate-induced catalase inactivation was not observed when only the intracellular ascorbate concentration was increased, when cells were incubated with ascorbate in plasma, or when extracellular Fe 3q was chelated. Together, these results suggest that the observed ascorbate-induced H O generation is due to Fe 3q -catalyzed oxidation of extracellular, as opposed to intracellular, 2 2 ascorbate by molecular oxygen. Rather than generate an oxidant stress in erythrocytes, ascorbate was one of the most sensitive intracellular antioxidants to H O coming from outside the cells. On the other hand, intracellular ascorbate 2 2 contributed little to the detoxification of H O , which was found to be mediated by both catalase and by the GSH system. 2 2 q 1998 Elsevier Science B.V. Ž .
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2008
Duodenal cytochrome b (Dcytb or Cybrd1) is an iron-regulated protein, highly expressed in the duodenal brush border membrane. It has ferric reductase activity and is believed to play a physiological role in dietary iron absorption. Its sequence identifies it as a member of the cytochrome b 561 family. A His-tagged construct of human Dcytb was expressed in insect Sf9 cells and purified. Yields of protein were increased by supplementation of the cells with 5-aminolevulinic acid to stimulate heme biosynthesis. Quantitative analysis of the recombinant Dcytb indicated two heme groups per monomer. Site-directed mutagenesis of any of the four conserved histidine residues (His 50, 86, 120 and 159) to alanine resulted in much diminished levels of heme in the purified Dcytb, while mutation of the non-conserved histidine 33 had no effect on the heme content. This indicates that those conserved histidines are heme ligands, and that the protein cannot stably bind heme if any of them is absent. Recombinant Dcytb was reduced by ascorbate under anaerobic conditions, the extent of reduction being 67% of that produced by dithionite. It was readily reoxidized by ferricyanide. EPR spectroscopy showed signals from low-spin ferriheme, consistent with bis-histidine coordination. These comprised a signal at g max = 3.7 corresponding to a highly anisotropic species, and another at g max = 3.18; these species are similar to those observed in other cytochromes of the b 561 family, and were reducible by ascorbate. In addition another signal was observed in some preparations at g max = 2.95, but this was unreactive with ascorbate. Redox titrations indicated an average midpoint potential for the hemes in Dcytb of + 80 mV ± 30 mV; the data are consistent with either two hemes at the same potential, or differing in potential by up to 60 mV. These results indicate that Dcytb is similar to the ascorbate-reducible cytochrome b 561 of the adrenal chromaffin granule, though with some differences in midpoint potentials of the hemes.
Ascorbate maintains a low plasma oxygen level
Scientific Reports, 2020
In human blood, oxygen is mainly transported by red blood cells. Accordingly, the dissolved oxygen level in plasma is expected to be limited, although it has not been quantified yet. Here, by developing dedicated methods and tools, we determined that human plasma pO2 = 8.4 mmHg (1.1% O2). Oxygen solubility in plasma was believed to be similar to water. Here we reveal that plasma has an additional ascorbate-dependent oxygen-reduction activity. Plasma experimental oxygenation oxidizes ascorbate (49.5 μM in fresh plasma vs < 2 μM in oxidized plasma) and abolishes this capacity, which is restored by ascorbate supplementation. We confirmed these results in vivo, showing that the plasma pO2 is significantly higher in ascorbate-deficient guinea pigs (Ascorbateplasma < 2 μM), compared to control (Ascorbateplasma > 15 μM). Plasma low oxygen level preserves the integrity of oxidation-sensitive components such as ubiquinol. Circulating leucocytes are well adapted to these conditions, ...
Free Radical Biology and Medicine, 2003
The combination of ascorbate, transition metal ions, and hydrogen peroxide (H 2 O 2 ) is an efficient hydroxyl radical generating system called "the Udenfriend system." Although the pro-oxidant role of ascorbate in this system has been well characterized in vitro, it is uncertain whether ascorbate also acts as a pro-oxidant under physiological conditions. To address this question, human plasma, used as a representative biological fluid, was either depleted of endogenous ascorbate with ascorbate oxidase, left untreated, or supplemented with 25 M-1 mM ascorbate. Subsequently, the plasma samples were incubated at 37°C with 50 M-1 mM iron (from ferrous ammonium sulfate), 60 or 100 M copper (from cupric sulfate), and/or 200 M or 1 mM H 2 O 2 . Although endogenous and added ascorbate was depleted rapidly in the presence of transition metal ions and H 2 O 2 , no cholesterol ester hydroperoxides or malondialdehyde were formed, i.e., ascorbate protected against, rather than promoted, lipid peroxidation. Conversely, depletion of endogenous ascorbate was sufficient to cause lipid peroxidation, the rate and extent of which were enhanced by the addition of metal ions but not H 2 O 2 . Ascorbate also did not enhance protein oxidation in plasma exposed to metal ions and H 2 O 2 , as assessed by protein carbonyl formation and depletion of reduced thiols. Interestingly, neither the rate nor the extent of endogenous ␣-tocopherol oxidation in plasma was affected by any of the treatments. Our data show that even in the presence of redox-active iron or copper and H 2 O 2 , ascorbate acts as an antioxidant that prevents lipid peroxidation and does not promote protein oxidation in human plasma in vitro.
Antioxidant Ascorbate Is Stabilized by NADH-Coenzyme Q 10 Reductase in the Plasma Membrane
Journal of Bioenergetics and Biomembranes, 1997
Plasma membranes isolated from K562 cells contain an NADH-ascorbate free radical reductase activity and intact cells show the capacity to reduce the rate of chemical oxidation of ascorbate leading to its stabilization at the extracellular space. Both activities are stimulated by CoQ10 and inhibited by capsaicin and dicumarol. A 34-kDa protein (p34) isolated from pig liver plasma membrane, displaying NADH-CoQ10
Ascorbate Recycling by Erythrocytes During Aging in Humans
Rejuvenation Research, 2009
Erythrocytes play a crucial role in recycling ascorbate in blood plasma. The erythrocyte ascorbate free radical (AFR) reductase is involved in the reduction of AFR to ascorbic acid (ASC) in the plasma. In the present study, we report an age-dependent increase in the activity of erythrocyte AFR reductase in humans that shows a significant positive correlation with the activity of plasma membrane redox system (PMRS). We explain the agedependent increase in erythrocyte ASC recycling on the basis of a compensatory/protective mechanism that operates to maintain the ASC level in plasma and thereby minimize oxidative stress during aging.