Total ascorbate and dehydroascorbate concentrations in porcine ovarian stroma, follicles and corpora lutea throughout the estrous cycle and pregnancy

Ovarian tissues are thought to require ascorbate as an antioxidant and enzymatic cofactor for the processes of steroid and collagen synthesis. We measured the concentrations of total ascorbate and oxidized ascorbate (dehydroascorbate, DHA) in ovarian stroma, follicles and corpora lutea (CL) throughout the estrous cycle and pregnancy of the sow. Both total ascorbate and DHA concentrations were greatest in luteal tissue and lowest in ovarian stroma across all stages examined. Within the CL, total ascorbate levels were lowest during the early, early-mid, and late luteal phase and were elevated during the mid-luteal phase. Luteal total ascorbate concentrations were further elevated during early pregnancy and were comparable to mid-luteal phase concentrations during the remainder of gestation. Luteal DHA concentrations decreased from mid to late luteal phase, and were elevated throughout pregnancy. As the CL aged during the cycle, the DHA/total ascorbate ratio decreased and remained low throughout pregnancy. Total ascorbate concentrations in follicular tissue increased during the follicular phase and were lowest during the early luteal phase. The DHA concentrations and DHA/total ascorbate ratios in follicular tissue did not differ with stage. Total ascorbate and DHA concentrations in ovarian stroma were low and did not vary with stage. We conclude that periods of maximal luteal and follicular function are associated with increased concentrations of total ascorbate within the tissue. Furthermore, luteolysis appears to be associated with depletion of luteal ascorbate species.     

Ascorbate system in plant development

By using lycorine, a specific inhibitor of ascorbate biosynthesis, it was possible to demonstrate that plant cells consume a high quantity of ascorbate (AA). Thein vivo metabolic reactions utilizing ascorbate are the elimination of H₂O₂ by ascorbate peroxidase and the hydroxylation of proline residues present in the polypeptide chains by means of peptidyl-proline hydroxylase.Ascorbate acts in the cell metabolism as an electron donor, and consequently ascorbate free radical (AFR) is continuously produced. AFR can be reconverted to AA by means of AFR reductase or can undergo spontaneous disproportion, thus generating dehydroascorbic acid (DHA).During cell division and cell expansion ascorbate consumption is more or less the same; however, the AA/DHA ratio is 6–10 during cell division and 1–3 during cell expansion. This ratio depends essentially on the different AFR reductase activity in these cells. In meristematic cells AFR reductase is very high, and consequently a large amount of AFR is reduced to AA and a small amount of AFR undergoes disproportionation; in expanding cells the AFR reductase activity is lower, and therefore AFR is massively disproportionated, thus generating a large quantity of DHA. Since the transition from cell division to cell expansion is marked by a large drop of AFR reductase activity in the ER, it is suggested here that AFR formed in this compartment may be involved in the enlargement of the ER membranes and provacuole acidification.DHA is a toxic compound for the cell metabolism and as such the cell has various strategies to counteract its effects: (i) meristematic cells, having an elevated AFR reductase, prevent large DHA production, limiting the quantity of AFR undergoing disproportionation. (ii) Expanding cells, which contain a lower AFR reductase, are, however, provided with a developed vacuolar system and segregate the toxic DHA in the vacuole. (iii) Chloroplast strategy against DHA toxicity is efficient DHA reduction to AA using GSH as electron donor. This strategy is usually poorly utilized by the surrounding cytoplasm.DHA reduction does play an important role at one point in the life of the plant, that is, during the early stage of seed germination. The dry seed does not store ascorbate, but contains DHA, and several DHA-reducing proteins are detectable. In this condition, DHA reduction is necessary to form a limited AA pool in the seed for the metabolic requirements of the beginning of germination. After 30–40h ascorbateex novo synthesis starts, DHA reduction declines until a single isoform remains, as is typical in the roots, stem, and leaves of seedlings. Finally, DHA recycling also appears to be important under adverse environmental conditions and ascorbate deficiency.     

Ascorbate- and dehydroascorbic acid-mediated reduction of free radicals in the human erythrocyte

Nitroxides were used as models of persistent free radicals to study the antioxidant function of ascorbic acid in the human erythrocyte. It was concluded that: 1) ascorbate and other reductant(s) derived from dehydroascorbic acid (DHA) in the presence of thiols are the only significant reducing agents for nitroxides, 2) glutathione and DHA reduce nitroxides by a process that cannot be inhibited by ascorbic acid oxidase, 3) erythrocytes can be depleted of ascorbic acid by exhaustive washing in the presence of membrane-permeable cationic nitroxides such as N,N-dimethylamino-Tempo, 4) ascorbate-depleted cells do not reduce nitroxides; however, nitroxide reduction is restored when the cells are incubated with DHA, 5) reduction of nitroxides in ascorbate-depleted, DHA-treated cells is significantly faster than in buffered solutions of DHA and glutathione, 6) several equivalents of nitroxide are reduced relative to the intracellular ascorbate pool, 7) sustained nitroxide reduction is observed even when most of the intracellular ascorbate is oxidized, 8) spin trapping of oxyradicals in tert-butyl hydroperoxide-treated cells is accelerated with ascorbate depletion and inhibited with ascorbate loading, 9) ascorbate can be quantified within intact cells by analyzing the initial reduction rates of membrane-permeable cationic nitroxides, and 10) DHA-stimulated reduction of cationic nitroxides is slower and less extensive in erythrocytes deficient in glucose-6-phosphate dehydrogenase than in normal erythrocytes.     

Quantitative data on the contribution of GSH and Complex II dependent ascorbate recycling in plant mitochondria

The reduction of dehydroascorbate, the oxidized form of ascorbate plays important role in the maintenance of sufficient level of ascorbate. In plant mitochondria two DHA reducing mechanisms, the GSH-dependent and the mitochondrial electron transfer chain dependent ascorbate recycling have been characterized. Although both pathways have been extensively studied quantitative information about the electron fluxes from one or another direction for the reduction of DHA is not known. The cellular, mitochondrial glutathione pools and mitochondrial DHA reducing capacity was measured in BSO treated and control tobacco cells. While BSO caused dramatic decrease of cellular GSH content the difference was much smoother at mitochondrial level. The difference in DHA reduction capacity was even smoother affirming the existence of alternative, non-GSH dependent DHA reducing mechanism(s) in plant mitochondria. On the base of the parallel determination of mitochondrial GSH content and ascorbate production upon DHA addition, GSH (consumption) is responsible for the ~ 20 % of ascorbate production. Almost 90 % enhancement of ascorbate production could be provoked by the addition of Complex II substrate succinate which could be almost totally prevented by the concomitant addition of malonate or TTFA. On the base of these results, the importance of mitochondrial Complex II compared to GSH-dependent mechanisms in mitochondrial ascorbate recycling has been underestimated so far.     

Are diabetic neuropathy,retinopathy and nephropathy caused by hyperglycemic exclusion of dehydroascorbate uptake by glucose transporters?

Vitamin C exists in two major forms. The charged form, ascorbic acid (AA), is taken up into cells via sodium-dependent facilitated transport. The uncharged form, dehydroascorbate (DHA), enters cells via glucose transporters (GLUT) and is then converted back to AA within these cells. Cell types such as certain endothelial and epithelial cells as well as neurons that are particularly prone to damage during diabetes tend to be those that appear to be dependent on GLUT transport of DHA rather than sodium-dependent AA uptake. We hypothesize that diabetic neuropathies, nephropathies and retinopathies develop in part by exclusion of DHA uptake by GLUT transporters when blood glucose levels rise above normal. AA plays a central role in the antioxidant defense system. Exclusion of DHA from cells by hyperglycemia would deprive the cells of the central antioxidant, worsening the hyperglycemia-induced oxidative stress level. Moreover, AA participates in many cellular oxidation-reduction reactions including hydroxylation of polypeptide lysine and proline residues and dopamine that are required for collagen production and metabolism and storage of catecholamines in neurons. Increase in the oxidative stress level and metabolic perturbations can be expected in any tissue or cell type that relies exclusively or mainly on GLUT for co-transport of glucose and DHA including neurons, epithelial cells, and vascular tissues. On the other hand, since DHA represents a significant proportion of total serum ascorbate, by increasing total plasma ascorbate concentrations during hyperglycemia, it should be possible to correct the increase in the oxidative stress level and metabolic perturbations, thereby sparing diabetic patients many of their complications.     

Stimulation of prolyl hydroxylase activity by chelating agents.

The activity of purified prolyl hydroxylase was enhanced several fold by addition of some chelating agents to the assay medium. Chelating agents could be classified into three groups. The chelating agents of Group I such as α, α′-dipyridyl were inactive until they reached equimolar concentration with ferrous ion in the assay mixture. The Group II agents, EDTA, diethylenetriaminepentaacetic acid, etc., stimulated the enzymatic activity 1.5- to 3-fold at equimolar concentration with ferrous ion. But the agents of both groups precipitously inhibited the enzymatic activity at concentrations greater than ferrous ion. On the other hand, Group III chelating agents, such as nitrilotriacetic acid, enhanced the enzymatic activity 5- to 10-fold at concentrations greater than ferrous ion. Nucleoside triphosphates, which also stimulate the enzymatic activity several fold and whose optimal concentrations are 1–3 × 10^?m, may be analogous to nitrilotriacetic acid of Group III.     

Modulation of Dcytb (Cybrd 1) expression and function by iron,dehydroascorbate and Hif-2α in cultured cells

Background

Duodenal cytochrome b (Dcytb) is a mammalian plasma ferric reductase enzyme that catalyses the reduction of ferric to ferrous ion in the process of iron absorption. The current study investigates the relationship between Dcytb, iron, dehydroascorbate (DHA) and Hif-2α in cultured cell lines.

Methods

Dcytb and Hif-2α protein expression was analysed by Western blot technique while gene regulation was determined by quantitative PCR. Functional analyses were carried out by ferric reductase and ⁵⁹Fe uptake assays.

Results

Iron and dehydroascorbic acid treatment of cells inhibited Dcytb mRNA and protein expression. Desferrioxamine also enhanced Dcytb mRNA level after cells were treated overnight. Dcytb knockdown in HuTu cells resulted in reduced mRNA expression and lowered reductase activity. Preloading cells with DHA (to enhance intracellular ascorbate levels) did not stimulate reductase activity fully in Dcytb-silenced cells, implying a Dcytb-dependence of ascorbate-mediated ferrireduction. Moreover, Hif-2α knockdown in HuTu cells led to a reduction in reductase activity and iron uptake.

Conclusions

Taken together, this study shows the functional regulation of Dcytb reductase activity by DHA and Hif-2α.

General significance

Dcytb is a plasma membrane protein that accepts electrons intracellularly from DHA/ascorbic acid for ferrireduction at the apical surface of cultured cells and enterocytes.     

Dehydroascorbate reduction in plant mitochondria is coupled to the respiratory electron transfer chain

The reduction of dehydroascorbate (DHA) was investigated in plant mitochondria. Mitochondria isolated from Bright Yellow-2 tobacco cells were incubated with 1 m M of DHA, and the ascorbate generation was followed by high-performance liquid chromatography. Mitochondria showed clear ability to reduce DHA and to maintain a significant level of ascorbate. Ascorbate generation could be stimulated by the respiratory substrate succinate. The complex I substrate malate and the complex I inhibitor rotenone had no effect on the ascorbate generation from DHA. Similarly, the complex III inhibitor antimycin A, the alternative oxidase inhibitor salicylhydroxamic acid, and the uncoupling agent 2,4-dinitrophenol were ineffective on mitochondrial ascorbate generation both in the absence and in the presence of succinate. However, the competitive succinate dehydrogenase inhibitor malonate almost completely abolished the succinate-dependent increase in ascorbate production. The complex IV inhibitor KCN strongly stimulated ascorbate accumulation. These results together suggest that the mitochondrial respiratory chain of plant cells – presumably complex II – plays important role in the regeneration of ascorbate from its oxidized form, DHA.     

Steady-state kinetics of substrate binding and iron release in tomato ACC oxidase

1-Aminocyclopropane-1-carboxylate oxidase (ACC oxidase) catalyzes the last step in the biosynthetic pathway of the plant hormone, ethylene. This unusual reaction results in the oxidative ring cleavage of 1-aminocyclopropane carboxylate (ACC) into ethylene, cyanide, and CO2 and requires ferrous ion, ascorbate, and molecular oxygen for catalysis. A new purification procedure and assay method have been developed for tomato ACC oxidase that result in greatly increased enzymatic activity. This method allowed us to determine the rate of iron release from the enzyme and the effect of the activator, CO2, on this rate. Initial velocity studies support an ordered kinetic mechanism where ACC binds first followed by O2; ascorbate can bind after O2 or possibly before ACC. This kinetic mechanism differs from one recently proposed for the ACC oxidase from avocado.     

Ascorbate transport from the apoplast to the symplast in intact leaves

Infiltration of reduced ascorbate (ASC) into the leaves of Betula pendula Roth and subsequent measurement of its loss therein after incubation allowed us to follow ascorbate transport from apoplast to symplast in intact leaves. All of the ascorbate extracted from the native apoplast was in fully oxidized form, dehydroascorbate (DHA). When 5 m M of ASC was infiltrated into the leaves, its intense decay occurred, but only 55% of ASC lost was recovered in apoplast as DHA. When ASC was added to the freshly extracted intercellular washing fluid (IWF), ASC oxidation occurred as well. However, all oxidized ASC was recovered as DHA, indicating that further decomposition of DHA did not occur. Similarly, all of the ASC infiltrated into the leaves was found therein either as ASC or DHA after incubation of leaves for up to 60 min. On this base the ascorbate infiltrated into the leaves and not recovered in the IWF was interpreted as ascorbate taken up into the symplast. The calculated uptake rates of ascorbate at different ASC concentrations followed saturation kinetics with the maximum uptake rate of 300 nmol m⁻² plasma membrane (PM) area min⁻¹ and Michaelis constant of 12.8 m M . The uptake of ascorbate was significantly inhibited by the addition of dithiothreitol or by PM H⁺ ATPase inhibitor erythrosin B. Thus, our results support the previous observations that DHA is preferably transported from the apoplastic to the cytoplasmic side of the membrane and show that this process is dependent upon PM proton gradient.     

Dihydroartemisinin increases gemcitabine therapeutic efficacy in ovarian cancer by inducing reactive oxygen species

Ovarian cancer is the major cause of death in women gynecological malignancy and gemcitabine (GEM) is commonly used in related chemotherapy. However, more than 90% GEM is catalyzed into an inactive metabolite 2′-deoxy-2′,2′-difluorouridine by stromal and cellular cytidine deaminase (CDA). Dihydroartemisinin (DHA), which possesses an intramolecular endoperoxide bridge, could be activated by heme or ferrous iron to produce reactive oxygen species (ROS). The excess ROS generation will excite expression of heme oxygenase-1 and suppress CDA expression. Under low CDA expression, the inactivation of GEM is decreased in turn to exert excellent therapeutic efficiency. Herein, we first studied the ROS generation by DHA in vitro with A2780 cells by means of flow cytometry and confocal laser scanning microscopy. Furthermore, cytotoxicity assay in vitro showed that DHA + GEM had synergistic effect, with molar ratio of DHA and GEM at 10. Eventually, in A2780 ovarian cancer xenograft tumor model, DHA + GEM exhibited significant antitumor efficiency with lower blood toxicity than GEM alone. Noteworthy, the combination treatment group completely eliminated the tumors on day 14.     

Influence of Drought-Induced Water Stress on Soybean and Spinach Leaf Ascorbate-Dehydroascorbate level and Redox Status

We examined the influence of water stress (water deficit) induced by drought on the steady state levels of ascorbic acid (ASC), dehydroascorbate (DHA), and the ASC&rcolon;DHA redox status in leaflets of Glycine max (soybean) and leaves of Spinacia oleracea (spinach). Two soybean cultivars (cv. Essex and cv. Forrest) and one spinach cultivar (cv. Nordic) were grown in high-light growth chambers ( approximately 1000-1200 μmol m-2 s-1) or in the greenhouse during May, June, and July 1999. The cultivars were supplied with water until approximately 25-29 d postemergence, at which time one-half of the plants were not watered for a period of from 4.5 to 7.5 d; the other half of the plants were provided water daily and served as controls. On designated days, leaf water potential (PsiLeaf) was measured, and leaf disks of constant area were excised in the period between approximately 1230 and 1330 hours. Leaf disk samples were immediately frozen in liquid N2, samples were extracted, and ASC and DHA levels were measured and expressed as μmol per gram dry mass per time point. For the soybean cultivars, low PsiLeaf values ( approximately -3.00 to -3.95 MPa) were accompanied by slight decreases in ASC levels and slight increases in DHA levels per gram dry mass. In some cases, leaflet ASC levels of water-stressed soybeans were similar to controls or were even increased by as much as 1.2 times. In soybeans, the mole fraction of ASC remained at 93-99 mol% of the total ascorbate (ASC+DHA), indicating that most of the total ascorbate remained in the reduced form even at low water potential. In spinach plants subjected to water stress (-1.8 to -2.6 MPa), leaf ASC decreased as much as 38%, but the ASC remained at 96-99 mol% of the total ascorbate. It is concluded that during water stress, enzymes of the ascorbate-glutathione cycle in leaf mesophyll cells, as well as in the system that generates reductant to support DHA to ASC recycling, e.g., photosynthetic electron transport in chloroplasts, is able to remain active enough to maintain reduction of DHA to ASC.     

Mechanisms of ascorbic acid recycling in human erythrocytes.

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.     

Free radical involvement in the generation of trans‐root potential

The identity of the naturally occurring compounds that accept electrons from plasma membrane-bound redox systems in vivo is obscure. We analysed the effect of ascorbate, oxygen, iron, as well as their free radical forms, and also the free radical-generating and -quenching systems on the trans-root electrical potential, which had previously been shown to be coupled to plasma membrane-bound redox systems. The material was the primary root of 8-day-old maize (Zea mays L.) seedlings. Trans-root electrical potential difference was measured across excised roots. Different ascorbate (ascorbate, dehydroascorbate and ascorbate free radical) and oxygen redox forms (superoxide and hydroxide radicals and hydrogen peroxide), as well as scavenging agents of oxygen species (superoxide dismutase, catalase, mannitol), and ferric and ferrous ions were added to the solution flowing around the root. Ascorbate free radical induced the greatest depolarization of the trans-root potential when compared to other ascorbate redox forms, which is consistent with its suggested role as a natural electron acceptor. Addition of xanthine oxidase, with or without xanthine, also produced depolarizing effects. The presence of SOD magnified this effect both with ascorbate free radical and xanthine oxidase. When ferric or ferrous chloride and ferric EDTA were applied to the bathing medium, only free ferric ion produced a very pronounced depolarization. The magnitude and kinetics of trans-root potential depolarization, induced by the ascorbate redox forms and systems for the generation and scavenging of oxygen species, argue in favour of the mutually competing electron transfer role of ascorbate free radicals and superoxide radicals in the extracellular space of the root. These results provide evidence that at least a part of the electrical potential difference occurring across plant roots arises from current flow from the symplast, via the plasma membrane-bound redox systems, to naturally occurring compounds in the apoplast, and that this transfer is achieved through the mediation of their free radical forms.     

Prooxidant action of xanthurenic acid and quinoline compounds: Role of transition metals in the generation of reactive oxygen species and enhanced formation of 8-hydroxy-2′-deoxyguanosine in DNA†

Xanthurenic acid, a product of tryptophan–NAD pathway, and quinoline compounds produced reactive oxygen species as a complex with iron. Aconitase, the most sensitive enzyme to oxidative stress was inactivated effectively by xanthurenic acid and to a lesser extent by 8-quinolinol in the presence of ferrous sulfate. The inactivation of aconitase was iron-dependent, and was prevented by TEMPOL, a scavenger of reactive oxygen species, suggesting that reduced iron bound to xanthurenic acid or 8-quinolinol can activate oxygen molecule to form superoxide radical. However, kynurenic acid and quinaldic acid without 8-hydroxyl group did not produce reactive oxygen species. Of the quinoline compounds tested, xanthurenic acid and 8-quinolinol with 8-hydroxyl group stimulated the autooxidation of ferrous ion, but kynurenic acid and quinaldic acid did not affect the oxidation of ferrous ion. Hydroxyl group at 8-positions of quinoline compounds was essential for the binding of iron causing the generation of reactive oxygen species. 8-Quinolinol effectively enhanced the ascorbate/copper-mediated formation of 8-hydroxy-2′-deoxyguanosine in DNA, suggesting the quinolinol/copper-dependent stimulation hydroxyl radical formation. Xanthurenic acid and 8-quinolinol as the metal–chelate complexes can show various cytotoxic effects by generating reactive oxygen species through the ferrous or cuprous ion-dependent activation of oxygen molecule. † This paper is dedicated to centennial of the birthday of the late Professor Emeritus Yahito Kotake, a pioneer of the xanthurenic acid research.     

Recycling of vitamin C from its oxidized forms by human endothelial cells

Endothelial cells encounter oxidant stress due to their location in the vascular wall, and because they generate reactive nitrogen species. Because ascorbic acid is likely involved in the antioxidant defenses of these cells, we studied the mechanisms by which cultures of EA.hy926 endothelial cells recycle the vitamin from its oxidized forms. Cell lysates reduced the ascorbate free radical (AFR) by both NADH- and NADPH-dependent mechanisms. Most NADH-dependent AFR reduction occurred in the particulate fraction of the cells. NADPH-dependent reduction resembled that due to NADH in having a high affinity for the AFR, but was mediated largely by thioredoxin reductase. Reduction of dehydroascorbic acid (DHA) required GSH and was both direct and enzyme dependent. The latter was saturable, half-maximal at 100 microM DHA, and comparable to rates of AFR reduction. Loading cells to ascorbate concentrations of 0.3-1.6 mM generated intracellular DHA concentrations of 20-30 microM, indicative of oxidant stress in culture. Whereas high-affinity AFR reduction is the initial and likely the preferred mechanism of ascorbate recycling, any DHA that accumulates during oxidant stress will be reduced by GSH-dependent mechanisms.     

Lipid peroxidation of rabbit small intestinal microvillus membrane vesicles by iron complexes

Fe(II)- and Fe(III)-induced lipid peroxidation of rabbit small intestinal microvillus membrane vesicles was studied. Ferrous ammonium sulphate, ferrous ascorbate at a molar ratio of 10:1, and ferric citrate, at molar ratios of 1:1 and 1:20, did not stimulate lipid peroxidation. Ferrous ascorbate, 1:1, induced low stimulation, while ferrous ascorbate, 1:20 gave higher stimulation of lipid peroxidation. These results show that in our experimental system, ascorbate is a promotor rather than an inhibitor of lipid peroxidation. Ferric nitrilotriacetate (at molar ratios of 1:2 and 1:10), at an iron concentration of 200 microM, was by far the most effective in inducing lipid peroxidation. Superoxide dismutase, mannitol and glutathione had no effect, while catalase, thiourea and vitamin E markedly decreased ferrous ascorbate 1:20-induced lipid peroxidation. Ferric nitrilotriacetate-induced lipid peroxidation was slightly reduced by catalase and mannitol, significantly reduced by superoxide dismutase, and completely inhibited by thiourea. Glutathione caused a 100% increase in the ferric nitrilotriacetate-induced lipid peroxidation. These results suggest that Fe(II) in the presence of trace amounts of Fe(III), or an oxidizing agent and Fe(III) in the presence of Fe(II) or a reducing agent, are potent stimulators of lipid peroxidation of microvillus membrane vesicles. Addition of deferoxamine completely inhibited both ferrous ascorbate, 1:20 and ferric nitrilotriacetate-induced lipid peroxidation, demonstrating the requirement for iron for its stimulation. Iron-induced peroxidation of microvillus membrane may have physiological significance because it could already be demonstrated at 2 microM iron concentration.     

Proline hydroxylation by cell free extract of a streptomycete

Free L-proline was hydroxylated to free L-hydroxyproline by cell free extract of Streptomyces griseoviridus P8648. The hydroxylation reaction required ferrous ion, 2-ketoglutarate and ascorbate. Zinc ion, ethylenediaminetetraacetic acid and alpha,alpha'-dipyridyl inhibited the reaction. Optimum temperature and pH were 25.0 degrees C and 7.5, respectively.     

Cytosolic dehydroascorbate reductase is important for ozone tolerance in Arabidopsis thaliana

Dehydroascorbate reductase (DHAR) is a key component of the ascorbate recycling system. Three functional DHAR genes are encoded in the Arabidopsis genome. Ozone exposure increased the expression of the cytosolic DHAR (cytDHAR) gene alone. We characterized an Arabidopsis mutant with a deficient cytDHAR. The mutant completely lacked cytDHAR activity and was highly ozone sensitive. The amounts of total ascorbate and glutathione were similar in both lines, but the amount of apoplastic ascorbate in the mutant was 61.5% lower. These results indicate that the apoplastic ascorbate, which is generated through the reduction of DHA by cytDHAR, is important for ozone tolerance.     

gamma-Butyrobetaine hydroxylase and the protective role of glutathione peroxidase

The selenoenzyme glutathione peroxidase in the presence of GSH effectively replaced catalase in the in vitro assay for gamma-butyrobetaine hydroxylase. Quantitatively, glutathione peroxidase was an order of magnitude more efficient than catalase, with maximal activity at less than 0.1 microM glutathione peroxidase in a standard reaction. Glutathione peroxidase prevented the loss of gamma-butyrobetaine hydroxylase during preliminary incubation with ferrous ions but without other substrates as well as in the course of the reaction. Regardless of whether glutathione peroxidase or catalase was present in the assay, the ascorbate concentrations needed to achieve half-maximal rates were similar (about 1 mM). Phosphate stimulated the rate of L-carnitine synthesis. Ferrous ion saturation indicated a pronounced effect of phosphate on the maximal velocity of the enzyme-catalyzed reaction, but its mechanism of action remains to be elucidated. Based on the subcellular distribution of gamma-butyrobetaine hydroxylase, catalase, and glutathione peroxidase, the role of glutathione peroxidase assumes importance. However, initial studies indicated that the assayable activity of liver gamma-butyrobetaine hydroxylase and L-carnitine concentrations in liver, blood plasma, and muscle were not significantly altered in selenium-deficient rats.