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Dehydroascorbic acid irreversibly inhibits hexokinase activity
Abstract
The oxidized form of vitamin C (dehydroascorbic acid, DHA) completely and irreversibly inactivates recombinant human hexokinase type I, in a pseudo-first order fashion. The inactivation reaction occurs without saturation, indicating that DHA does not form a reversible complex with hexokinase. Further characterization of this response revealed that the inactivation does not require oxygen and that dithiothreitol, while able to prevent the DHA-mediated loss of enzyme activity, failed to restore the activity of the DHA-inhibited enzyme. Inactivation was not associated with cleavage of the peptide chain or cross-linking. The decay in enzymatic activity was however both dependent on deprotonation of a residue with an alkaline pKa and associated with covalent binding of DHA to the protein. In addition, inactivation of hexokinase decreased or increased, respectively, in the presence of the substrates glucose or MgATP. Finally, amino acid analysis of the DHA-modified hexokinase revealed a decrease of cysteine residues.
Taken together, the above results are consistent with the possibility that covalent binding of the reagent with a thiol group of cysteine is a critical event for the DHA-mediated loss of hexokinase activity.
... Ascorbic acid (vitamin C) at pharmacological concentrations has pro-oxidant [22][23][24] and anti-cancer activities, as reported by us and others [25][26][27]. Ascorbate inhibits hexokinase activity [28], which produces glucose-6-phosphate to initiate two major metabolic pathways: glycolysis and the pentose phosphate pathway. Hexokinase 1 and 2 (HK1/HK2) are also associated with the mitochondrial membrane permeability transition pore (PTP) and prevents apoptosis, thus controlling reactive oxygen species (ROS) formation [29]. ...
... Based on these evidences, we targeted the metabolic peculiarity and plasticity of AML cells with an association of ascorbate to induce an oxidative stress and to interfere with hexokinase activity [28] and buformin to shut down the mitochondrial contribution in ATP production. Our data support the clinical evaluation of the ascorbate-buformin combination as a treatment option for refractory/relapsing AML patients and for older and unfit patients. ...
... From our previous experience in treating AML cells with ascorbate, we identified the 3 mM concentration as rapidly cytotoxic due to its pro-oxidant effect [25]. To investigate the metabolic effect of the drug, in this study AML cells were treated with 1 mM of ascorbate, which significantly inhibited the expression levels of Hexokinase II (HK2), a main glycolysis-initiating enzyme in the hemopoietic system ( Figure 3b) and in tumors [28]. Similar results were obtained using an antibody recognizing both Hexokinase I and HK2 (HK1/2) (Figure 3b). ...
In the present study, we characterized the metabolic background of different Acute Myeloid Leukemias’ (AMLs) cells and described a heterogeneous and highly flexible energetic metabolism. Using the Seahorse XF Agilent, we compared the metabolism of normal hematopoietic progenitors with that of primary AML blasts and five different AML cell lines. We assessed the efficacy and mechanism of action of the association of high doses of ascorbate, a powerful oxidant, with the metabolic inhibitor buformin, which inhibits mitochondrial complex I and completely shuts down mitochondrial contributions in ATP production. Primary blasts from seventeen AML patients, assayed for annexin V and live/dead exclusion by flow cytometry, showed an increase in the apoptotic effect using the drug combination, as compared with ascorbate alone. We show that ascorbate inhibits glycolysis through interfering with HK1/2 and GLUT1 functions in hematopoietic cells. Ascorbate combined with buformin decreases mitochondrial respiration and ATP production and downregulates glycolysis, enhancing the apoptotic effect of ascorbate in primary blasts from AMLs and sparing normal CD34+ bone marrow progenitors. In conclusion, our data have therapeutic implications especially in fragile patients since both agents have an excellent safety profile, and the data also support the clinical evaluation of ascorbate–buformin in association with different mechanism drugs for the treatment of refractory/relapsing AML patients with no other therapeutic options.
... Due to its redox potential (from +0.40 to +0.50 V) [47], ASC can quickly reduce the ROS, both indirectly contributing to the lipid radical-scavenging and then avoiding lipid peroxidation and preventing cell injury prior to activation of antioxidant enzymes, and directly by scavenging tocoferoxyl radicals, lipid peroxides, and oxidized metal ions [33,48]. Dehydroascorbate (DHA) was observed to accumulate in the apoplast after oxidative stress, and to inhibit the enzyme activity and root growth [37,49,50]. Additionally, DHA can be alternatively reduced by glutathione-ferredoxin-and NAD(P)Hdependent pathways demonstrating a metabolic link between the NAD(P)-dependent redox system and the low molecular weight antioxidants [51]. ...
... Due to its redox potential (from +0 +0.50 V) [47], ASC can quickly reduce the ROS, both indirectly contributing to the radical-scavenging and then avoiding lipid peroxidation and preventing cell injury to activation of antioxidant enzymes, and directly by scavenging tocoferoxyl radica pid peroxides, and oxidized metal ions [33,48]. Dehydroascorbate (DHA) was obs to accumulate in the apoplast after oxidative stress, and to inhibit the enzyme activit root growth [37,49,50]. Additionally, DHA can be alternatively reduced by glutath ferredoxin-and NAD(P)H-dependent pathways demonstrating a metabolic link bet the NAD(P)-dependent redox system and the low molecular weight antioxidants [5 The effects of InA on the content of ASC in L. esculentum and L. sativum are sho Figure 2. Seedlings of Lepidium sativum showed levels of ASC higher than Lycopersicon es tum both at 3 and at 6 days. ...
Allelochemicals are considered an environment-friendly and promising alternative for weed management, although much effort is still needed for understanding their mode of action and then promoting their use in plant allelopathy management practices. Here, we report that Inuloxin A (InA), an allelochemical isolated from Dittrichia viscosa, inhibited root elongation and growth of seedlings of Lycopersicon esculentum and Lepidium sativum at the highest concentrations tested. InA-induced antioxidant responses in the seedlings were investigated by analysing the contents of glutathione (GSH) and ascorbate (ASC), and their oxidized forms, dehydroascorbate (DHA), and glutathione disulphide (GSSG), as well as the redox state of thiol-containing proteins. An increase in ASC, DHA, and GSH levels at high concentrations of InA, after 3 and 6 days, were observed. Moreover, the ASC/DHA + ASC and GSH/GSSG + GSH ratios showed a shift towards the oxidized form. Our study provides the first insight into how the cell redox system responds and adapts to InA phytotoxicity, providing a framework for further molecular studies.
... Intracellular levels of DHA are therefore kept always very low, but nevertheless several reports proposed its involvement in various biological reactions. For example, it has been suggested that DHA is an inhibitor of the activities of enzymes [16][17][18] and transcription factors [19], although the information available is insufficient to define the biological relevance of these effects, in particular since the con-centrations employed in these studies were far greater than those reasonably achieved inside the cells. ...
... The presence of thiols in SVCT2 is well documented [36][37] and the activity of both the plasma membrane [38] and mitochondrial (Fig. 3B) SVCT2 is indeed susceptible to inhibition by thiol-reactive agents. In addition, DHA is known to react with -SH groups [28] and to inhibit the activity of enzymes containing critical cysteines [17][18]. ...
... It was suggested that DHA could be responsible for growth inhibition, since it inhibited the activity of pyridine nucleotide dependent-dehydrogenases measured in cell-free extracts . Recently, it has been shown that DHA at low concentrations inhibits the activity of several enzymes in vitro, including malate dehydrogenase, fructose 1,6-bisphosphatase (Morell et al. 1997) and hexokinase (Fiorani et al. 2000). Moreover, root growth inhibition has been observed in response to DHA administration in vivo (Cordoba-Pedregosa et al. 1996), whereas an increase in AA content stimulates growth (Cordoba-Pedregosa et al. 1996, Arrigoni et al. 1997. ...
... Furthermore, it has long been known that DHA, beside oxidising thiols, can form addition compounds as a consequence of the interaction of its carbonyl groups with amino acid residues (Drake et al. 1942). Recent results showed that DHA irreversibly inhibits human type I hexokinase, and the amino acid analysis of the DHA-modified enzyme reveals a decrease in the number of cysteine residues (Fiorani et al. 2000). ...
Administration of 1 mM dehydroascorbate (DHA) results in a rapid and large increase in cellular ascorbate (AA) content in both Lupinus albus L. and Allium cepa L. root tips. Uptake of DHA from the medium occurs at a high rate within 10–12 h of incubation, and is slowed down thereafter. In the first few h, DHA reduction to AA is apparently correlated to GSH depletion and slightly higher DHA reductase activity. DHA incubation also seems to induce new GSH synthesis. Longer DHA incubation (24 h) affects root growth by inhibiting cell proliferation. At this stage, an apparently generalised oxidation of SH-containing proteins is observed in DHA-treated roots. Treatment with 1 mM L-galactono-C C C C-lactone, the last precursor of AA bio-synthesis, results in an increase in AA content similar to that obtained with DHA, but stimulates growth and affects the redox state of SH-containing proteins in the opposite way. A possible multi-step mechanism of DHA reduction/ removal is suggested and the hypothesis that DHA inhibits cell cycle progression by affecting the redox state of SH-containing proteins is discussed.
... Because DOG is a HKphosphorylable glucose analog and OMG is not, it is possible that HK could be important in the mechanism of glucose transport inhibition, or rather in the inhibition of glucose use. These results are consistent with in vitro HK activity inhibition by dehydroascorbic acid, the oxidized form of ascorbic acid (Fiorani et al. 2000). If glucose use is inhibited the transport of other substrates may be stimulated. ...
... The mechanism of ascorbic acid effect remains to be understood. The differences in ascorbic acid effect on DOG and OMG uptake are consistent with HK inhibition by ascorbic acid in vitro (Fiorani et al. 2000). Thus, we can speculate that lactate stimulation is a consequence of glucose phosphorylation inhibition. ...
In this review, we discuss a novel function of ascorbic acid in brain energetics. It has been proposed that during glutamatergic synaptic activity neurons preferably consume lactate released from glia. The key to this energetic coupling is the metabolic activation that occurs in astrocytes by glutamate and an increase in extracellular [K(+)]. Neurons are cells well equipped to consume glucose because they express glucose transporters and glycolytic and tricarboxylic acid cycle enzymes. Moreover, neuronal cells express monocarboxylate transporters and lactate dehydrogenase isoenzyme 1, which is inhibited by pyruvate. As glycolysis produces an increase in pyruvate concentration and a decrease in NAD(+)/NADH, lactate and glucose consumption are not viable at the same time. In this context, we discuss ascorbic acid participation as a metabolic switch modulating neuronal metabolism between rest and activation periods. Ascorbic acid is highly concentrated in CNS. Glutamate stimulates ascorbic acid release from astrocytes. Ascorbic acid entry into neurons and within the cell can inhibit glucose consumption and stimulate lactate transport. For this switch to occur, an ascorbic acid flow is necessary between astrocytes and neurons, which is driven by neural activity and is part of vitamin C recycling. Here, we review the role of glucose and lactate as metabolic substrates and the modulation of neuronal metabolism by ascorbic acid.
... It was suggested that DHA could be responsible for growth inhibition, since it inhibited the activity of pyridine nucleotide dependent-dehydrogenases measured in cell-free extracts (). Recently, it has been shown that DHA at low concentrations inhibits the activity of several enzymes in vitro, including malate dehydrogenase , fructose 1,6-bisphosphatase (Morell et al. 1997) and hexokinase (Fiorani et al. 2000 ). Moreover, root growth inhibition has been observed in response to DHA administration in vivo (Cordoba-Pedregosa et al. 1996), whereas an increase in AA content stimulates growth (Cordoba-Pedregosa et al. 1996, Arrigoni et al. 1997). ...
... Furthermore, it has long been known that DHA, beside oxidising thiols, can form addition compounds as a consequence of the interaction of its carbonyl groups with amino acid residues (Drake et al. 1942). Recent results showed that DHA irreversibly inhibits human type I hexokinase, and the amino acid analysis of the DHA-modified enzyme reveals a decrease in the number of cysteine residues (Fiorani et al. 2000). It is conceivable that protein-thiol oxidation does not quantitatively contribute to the observed rate of DHA reduction to AA. ...
Administration of 1 mM dehydroascorbate (DHA) results in a rapid and large increase in cellular ascorbate (AA) content in both Lupinus albus L. and Allium cepa L. root tips. Uptake of DHA from the medium occurs at a high rate within 10-12 h of incubation, and is slowed down thereafter. In the first few h, DHA reduction to AA is apparently correlated to GSH depletion and slightly higher DHA reductase activity. DHA incubation also seems to induce new GSH synthesis. Longer DHA incubation (24 h) affects root growth by inhibiting cell proliferation. At this stage, an apparently generalised oxidation of SH-containing proteins is observed in DHA-treated roots. Treatment with 1 mM L-galactono-gamma-lactone, the last precursor of AA biosynthesis, results in an increase in AA content similar to that obtained with DHA, but stimulates growth and affects the redox state of SH-containing proteins in the opposite way. A possible multi-step mechanism of DHA reduction/removal is suggested and the hypothesis that DHA inhibits cell cycle progression by affecting the redox state of SH-containing proteins is discussed.
... Consequently, this event could have a negative impact on the protein folding due to the interaction of carbonyl groups of the DHA with amino acid residues. Indeed, DHA irreversibly inhibits some enzymes, such as human type I hexokinase, that shows a smaller number of cysteine residues [49,50]. ...
Aflatoxins (AFs) are toxic secondary metabolites produced by Aspergillus spp. and are found in food and feed as contaminants worldwide. Due to climate change, AFs occurrence is expected to increase also in western Europe. Therefore, to ensure food and feed safety, it is mandatory to develop green technologies for AFs reduction in contaminated matrices. With this regard, enzymatic degradation is an effective and environmentally friendly approach under mild operational conditions and with minor impact on the food and feed matrix. In this work, Ery4 laccase, acetosyringone, ascorbic acid, and dehydroascorbic acid were investigated in vitro, then applied in artificially contaminated corn for AFB1 reduction. AFB1 (0.1 µg/mL) was completely removed in vitro and reduced by 26% in corn. Several degradation products were detected in vitro by UHPLC-HRMS and likely corresponded to AFQ1, epi-AFQ1, AFB1-diol, or AFB1dialehyde, AFB2a, and AFM1. Protein content was not altered by the enzymatic treatment, while slightly higher levels of lipid peroxidation and H2O2 were detected. Although further studies are needed to improve AFB1 reduction and reduce the impact of this treatment in corn, the results of this study are promising and suggest that Ery4 laccase can be effectively applied for the reduction in AFB1 in corn.
... In this context, the deletion of MLKL in the neuron leads to a change from neuronal death dependent on RIPK1-MLKL to RIPK1-and MLKL-independent neuronal death. We speculate that the accumulation of DHA in the absence of MLKL could cause metabolic crisis given that DHA is a potent inhibitor of hexokinase [53] and a glycolysis inhibitor in neurons [3]. Thus, the accumulation of DHA in cells deficient in MLKL could generate a change from necroptosis to other nonapoptotic types of death, such as autosis, because DHA can also act as an autophagy stimulator [54]. ...
Under physiological conditions, vitamin C is the main antioxidant found in the central nervous system and is
found in two states: reduced as ascorbic acid (AA) and oxidized as dehydroascorbic acid (DHA). However, under
pathophysiological conditions, AA is oxidized to DHA. The oxidation of AA and subsequent production of DHA
in neurons are associated with a decrease in GSH concentrations, alterations in glucose metabolism and neuronal
death. To date, the endogenous molecules that act as intrinsic regulators of neuronal necroptosis under conditions
of oxidative stress are unknown. Here, we show that treatment with AA regulates the expression of proand
antiapoptotic genes. Vitamin C also regulates the expression of RIPK1/MLKL, whereas the oxidation of AA
in neurons induces morphological alterations consistent with necroptosis and MLKL activation. The activation of
necroptosis by AA oxidation in neurons results in bubble formation, loss of membrane integrity, and ultimately,
cellular explosion. These data suggest that necroptosis is a target for cell death induced by vitamin C.
... In this context, the deletion of MLKL in the neuron leads to a change from neuronal death dependent on RIPK1-MLKL to RIPK1-and MLKL-independent neuronal death. We speculate that the accumulation of DHA in the absence of MLKL could cause metabolic crisis given that DHA is a potent inhibitor of hexokinase [53] and a glycolysis inhibitor in neurons [3]. Thus, the accumulation of DHA in cells deficient in MLKL could generate a change from necroptosis to other nonapoptotic types of death, such as autosis, because DHA can also act as an autophagy stimulator [54]. ...
... In human erythrocytes incubated with 6 mM DHA, 13 C-NMR analysis of the chemical species present at 20 min posttreatment showed that 46% was DHA, 32% was AA, and only 17% was diketogulonic acid (DKG; Himmelreich et al., 1998). Therefore, DHA may be stable to fulfill physiological functions, including as an inhibitor of hexokinase (Fiorani et al., 2000;Fiorani et al., 1996), proteolysis (Lockwood, 1997), I-kappa B-alpha kinase-beta (Carcamo et al., 2004), SVCT2 (Fiorani et al., 2015), and STAT phosphorylation (Thon, Hosoi, & Ozawa, 2016) and able to conjugate with glutathione (GSH), forming a GSH-DHA complex (Regulus, Desilets, Klarskov, & Wagner, 2010). ...
For a long time, the effect of vitamin C on cancer cells has been a controversial concept. From Linus Pauling's studies in 1976, it was proposed that ascorbic acid (AA) could selectively kill tumor cells. However, further research suggested that vitamin C has no effect on tumor survival. In the last decade, new and emerging functions for vitamin C have been discovered using the reduced form, AA, and the oxidized form, dehydroascorbic acid (DHA), independently. In this review, we summarized the latest findings related to the effects of DHA on the survival and metabolism of tumor cells. At the same time, we put special emphasis on the bystander effect and the recycling capacity of vitamin C in various cellular models, and how these concepts can affect the experimentation with vitamin C and its therapeutic application in the treatment against cancer. We summarized the latest findings related to the effects of dehydroascorbic acid (DHA) on the survival and metabolism of tumor cells. DHA alters glucose metabolism and induces cell death in normal and tumor cells. The complex relationship between ascorbic acid and DHA will continue to be a subject of controversy with respect to vitamin C use as an antitumor drug.
... DHA has been shown to inhibit the phosphorylation of other important signaltransduction and transcription factors, including NF-κB and p38 MAPK by preventing the binding of ATP [369][370][371]. Specifically, NF-κB is of great interest because it is constitutively expressed in tumor cells and promotes tumor progression while inhibiting tumor-cell death. ...
A pro‐oxidant treatment encompassing a combined administration of ascorbate (VC) and menadione (VK3) on a series of carcinoma cell lines and xenotransplants resulted in specific cytotoxic activities that allowed us to describe a new mode of cell death named autoschizis. Observed in human prostate, bladder, and ovarian carcinoma cells as well as in a transgenic murine prostate cancer model, as well as in vivo, this mode of cell death process differs from apoptosis or necrosis according to the morphological and biochemical investigations collected, using morphological and biochemical techniques.
Comparing the untreated tumor cells with injured ones it was found that, in vitro, this autoschizis cell death is initiated by pro‐oxidant stress in which tumor cells deteriorate through irreversible cell damages caused by reactivation of nucleases. The initial damages consist in superficial and deep cytoplasmic changes involving membranes and the cytoskeleton resulting in dramatic cell size reduction through auto‐ or self‐excisions. Followed by a series of sequential injuries caused by hydrogen peroxide and reactive oxidative species on endomembranes,mitochondria and lysosomes and other organelle's injuries contribute to autophagic activities that also involve sequential nucleus defects. These injuries include karyolysis with nucleollus degradation ruled by reactivated nucleases and cathepsins K and L leaking from lysosomes verified by immunocytochemistry, DNA gel electrophoretic smear pattern, similar to Necrosis. Additionally, flow cytometry disclosed cell cycle blocks in G1/S and G2/M phases in all the treated tumor cells. Biochemical data from in vitro and in vivo tests showed that this mode of cell death is independent from caspase‐3 activation, while inhibiting the repair mechanisms of the cell through depletion of ATP, thiols, impeding protein, nucleic acid synthesis or repairs while starving energetically the tumor cells, i.e. trying to survive through autophagocytosis of reserves. Based on some data from biochemistry, this new mode of cell death verified by morphology and described in 1998 would encompass necroptosis, pathanos, pyroptosis and oxytosis.
In xenotransplanted carcinomas, this pro‐oxidant treatment induces major cell demise by autoschizis with significant reduction of the tumors' size and high survival rate of the host animals. Because this mode of killing specifically targets cancer cells and, based on preliminary clinical data, it has been suggested that this treatment would be a useful, safe and inexpensive strategy to be implemented synergistically with radiation and/or chemotherapy as an adjuvant or treatments in oncology.
... Dehydroascorbate has been shown to have a role in neuronal energy metabolism via activation of glucose-6-phosphate dehydrogenase thus increasing flux through the pentose phosphate pathway (Puskas et al., 2000;Cisternas et al., 2014). Furthermore, ascorbate has been identified as a kinase inhibitor (Carcamo et al., 2004) as well as an inhibitor of hexokinase (Fiorani et al., 2000) and its oxidized form can inhibit glyceraldehyde 3-phosphate dehydrogenase leading to an energetic crisis and cell death in cancer cells which uptake more dehydroascorbate via glucose transporters (Yun et al., 2015). These examples have not been shown in plants: the study of ascorbate in non-photosynthetic tissues may be key to revealing additional functions for the molecule; fruit is an ideal tissue, particularly in advanced ripening stages when photosynthetic activity is low. ...
Changing the balance between ascorbate, monodehydroascorbate, and dehydroascorbate in plant cells by manipulating the activity of enzymes involved in ascorbate synthesis or recycling of oxidized and reduced forms leads to multiple phenotypes. A systems biology approach including network analysis of the transcriptome, proteome and metabolites of RNAi lines for ascorbate oxidase, monodehydroascorbate reductase and galactonolactone dehydrogenase has been carried out in orange fruit pericarp of tomato (Solanum lycopersicum). The transcriptome of the RNAi ascorbate oxidase lines is inversed compared to the monodehydroascorbate reductase and galactonolactone dehydrogenase lines. Differentially expressed genes are involved in ribosome biogenesis and translation. This transcriptome inversion is also seen in response to different stresses in Arabidopsis. The transcriptome response is not well correlated with the proteome which, with the metabolites, are correlated to the activity of the ascorbate redox enzymes—ascorbate oxidase and monodehydroascorbate reductase. Differentially accumulated proteins include metacaspase, protein disulphide isomerase, chaperone DnaK and carbonic anhydrase and the metabolites chlorogenic acid, dehydroascorbate and alanine. The hub genes identified from the network analysis are involved in signaling, the heat-shock response and ribosome biogenesis. The results from this study therefore reveal one or several putative signals from the ascorbate pool which modify the transcriptional response and elements downstream.
... A low DHAR activity determines accumulation of DHA, which is considered to be toxic for plant cells. It has been reported that DHA inhibits the activity of several enzymes in vitro, including malate dehydrogenase, fructose 1,6-bisphosphatase (Morell et al., 1997), and hexokinase (Fiorani et al., 2000). A low APX activity in plants implies a low H 2 O 2 detoxification capacity, as APX is the key enzyme in H 2 O 2 removal (Asada, 1992). ...
During their life cycle, plants can undergo simultaneous attack by different pathogens that produce various toxins. It is well known that in some plant-fungal interactions, mycotoxins play an important role in pathogenesis and induce a reactive oxygen species increase. Plants counteract the overaccumulation of reactive oxygen species by reinforcing their defence systems. The mycotoxins T-2 toxin (T-2) and beauvericin (BEA) are produced by some Fusarium species and have different chemical structures, mechanisms of action and biological activities. In this study, the individual and combined effects of these two toxins on defence systems, such as the ascorbate-glutathione cycle and peroxidases, were evaluated in cherry tomato shoots. Hydrogen peroxide content as an index of oxidative stress was also measured. Inhibitory effects on ascorbate peroxidase, dehydroascorbate reductase and ascorbate, and stimulatory effects on glutathione reductase, monodehydroascorbate reductase and reduced glutathione were observed when tomato plants were simultaneously treated with BEA and T-2. The trend of these biochemical parameters highlight the presence of a range of defence mechanisms activated by plants in response to mycotoxins. The interaction between BEA and T-2 resulting in synergistic and/or antagonistic effects on the studied defence systems is also discussed. It is concluded that the effects of these mycotoxins alone are not predictive of their combined effects.
... Therefore, the changes in the AsA/DHA ratio are considered as a redox status indicator in plants (Yoshida et al. 2006;Foyer and Noctor 2011). At low concentrations, DHA has been shown to inhibit the activity of several enzymes in vitro, including malate dehydrogenase, fructose 1,6bisphosphatase (Morell et al. 1997), and hexokinase (Fiorani et al. 2000). Moreover, DHA administration in vivo was reported to inhibit root growth (Cordoba-Pedregosa et al. 1996), whereas enhanced growth was reported due to an increase in AsA content (Cordoba-Pedregosa et al. 1996;Arrigoni et al. 1997). ...
The enhanced generation of reactive oxygen species (ROS) under metal/metalloid stress is most common in plants, and the elevated ROS must be successfully metabolized in order to maintain plant growth, development, and productivity. Ascorbate (AsA) is a highly abundant metabolite and a water-soluble antioxidant, which besides positively influencing various aspects in plants acts also as an enigmatic component of plant defense armory. As a significant component of the ascorbate-glutathione (AsA-GSH) pathway, it performs multiple vital functions in plants including growth and development by either directly or indirectly metabolizing ROS and its products. Enzymes such as monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) and dehydroascorbate reductase (DHAR, EC 1.8.5.1) maintain the reduced form of AsA pool besides metabolically controlling the ratio of AsA with its oxidized form (dehydroascorbate, DHA). Ascorbate peroxidase (APX, EC 1.11.1.11) utilizes the reduced AsA pool as the specific electron donor during ROS metabolism. Thus, AsA, its redox couple (AsA/DHA), and related enzymes (MDHAR, DHAR, and APX) cumulatively form an AsA redox system to efficiently protect plants particularly against potential anomalies caused by ROS and its products. Here we present a critical assessment of the recent research reports available on metal/metalloid-accrued modulation of reduced AsA pool, AsA/DHA redox couple and AsA-related major enzymes, and the cumulative significance of these antioxidant system components in plant metal/metalloid stress tolerance.
... Therefore, the changes in the AsA/DHA ratio are considered as a redox status indicator in plants (Yoshida et al. 2006;Foyer and Noctor 2011). At low concentrations, DHA has been shown to inhibit the activity of several enzymes in vitro, including malate dehydrogenase, fructose 1,6bisphosphatase (Morell et al. 1997), and hexokinase (Fiorani et al. 2000). Moreover, DHA administration in vivo was reported to inhibit root growth (Cordoba-Pedregosa et al. 1996), whereas enhanced growth was reported due to an increase in AsA content (Cordoba-Pedregosa et al. 1996;Arrigoni et al. 1997). ...
The enhanced generation of reactive oxygen species (ROS) under metal/metalloid stress is most common in plants, and the elevated ROS must be successfully metabolized in order to maintain the plant growth, development and productivity. Ascorbate (AsA) is a highly abundant metabolite and a water soluble antioxidant, which besides positively influencing various aspects in plants acts also as an enigmatic component of plant defense armory. As a significant component of ascorbate-glutathione (AsA-GSH) pathway it performs multiple vital functions in plants including growth and development by either directly or indirectly metabolizing ROS and its products. Enzymes such as monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) and dehydroascorbate reductase (DHAR, EC 1.8.5.1) maintain the reduced form of AsA pool besides metabolically controlling the ratio of AsA with its oxidized form (dehydroascorbate, DHA). Ascorbate peroxidadse (APX, EC 1.11.1.11) utilizes the reduced AsA pool as the specific electron donor during ROS metabolism. Thus, AsA, its redox couple (AsA/DHA) and related enzymes (MDHAR, DHAR and APX) cumulatively form an AsA redox system to efficiently protect plants particularly against potential anomalies caused by ROS and its products. Here we present a critical assessment of the recent research reports available on metal/metalloid-accrued modulation of reduced AsA pool, AsA/DHA redox couple and AsA-related major enzymes, and the cumulative significance of these antioxidant system components in plant metal/metalloid stress tolerance.
... the results shown in Figs. 4 and 5 reveal that dehydroascorbic acid interferes with the multiplication of hSV-1 after the completion of viral dna replication, probably at the stage of the envelopment of nucleocapsids (i.e., the assembly of progeny virus particles). dehydroascorbic acid has been reported to have the ability to bind to proteins (16,17) and to inhibit certain kinases and enzymes (18)(19)(20), suggesting that it may inhibit certain protein(s) necessary for virus-host interactions, and may thereby interfere with virus multiplication. ...
IN THE PRESENT STUDY, DEHYDROASCORBIC ACID INHIBITED THE MULTIPLICATION OF VIRUSES OF THREE DIFFERENT FAMILIES: herpes simplex virus type 1 (HSV-1), influenza virus type A and poliovirus type 1. Although dehydroascorbic acid showed some cytotoxicity at higher concentrations, the observed antiviral activity was not the secondary result of the cytotoxic effect of the reagent, as the inhibition of virus multiplication was observed at reagent concentrations significantly lower than those resulting in cytotoxicity. Characterization of the mode of the antiviral action of dehydroascorbic acid against HSV-1 revealed that the addition of reagent at any time post infection inhibited the formation of progeny infectious virus in the infected cells, and a one-step growth curve showed that the addition of reagent allowed formation for an additional 2 h, but then almost completely suppressed it. These results indicate that the reagent inhibits HSV-1 multiplication after the completion of viral DNA replication, probably at the step of the envelopment of viral nucleocapsids at the Golgi apparatus of infected cells.
... BrQ = bromoquinone; VC = vitamin C (simplified structure of ascorbic acid); GSH = glutathione; NADH = nicotinamide adenine dinucleotide; Q-SG = substitution product of the reaction between BrQ and GSH. nase (37). In addition to the high levels of ROS produced by BrQ/VC, we demonstrated that this combination, but not VK 3 /VC, can chemically deplete the coenzyme NADH, which could have direct implications for the glycolytic pathway. ...
Apatone™, a combination of menadione (2-methyl-1,4-naphthoquinone, VK3) and ascorbic acid (vitamin C, VC) is a new strategy for cancer treatment. Part of its effect on tumor cells is related to the cellular pro-oxidative imbalance provoked by the generation of hydrogen peroxide (H2O2) through naphthoquinone redox cycling. In this study, we attempted to find new naphthoquinone derivatives that would increase the efficiency of H2O2 production, thereby potentially increasing its efficacy for cancer treatment. The presence of an electron-withdrawing group in the naphthoquinone moiety had a direct effect on the efficiency of H2O2 production. The compound 2-bromo-1,4-naphthoquinone (BrQ), in which the bromine atom substituted the methyl group in VK3, was approximately 10- and 19-fold more efficient than VK3 in terms of oxygen consumption and H2O2 production, respectively. The ratio [H2O2]produced / [naphthoquinone]consumed was 68 ± 11 and 5.8 ± 0.2 (µM/µM) for BrQ and VK3, respectively, indicating a higher efficacy of BrQ as a catalyst for the autoxidation of ascorbic acid. Both VK3 and BrQ reacted with glutathione (GSH), but BrQ was the more effective substrate. Part of GSH was incorporated into the naphthoquinone, producing a nucleophilic substitution product (Q-SG). The depletion of BrQ by GSH did not prevent its redox capacity since Q-SG was also able to catalyze the production of reactive oxygen species. VK3/VC has already been submitted to clinical trials for the treatment of prostate cancer and has demonstrated promising results. However, replacement of VK3 with BrQ will open new lines of investigation regarding this approach to cancer treatment.
... This supports the hypothesis that changes in the cellular redox balance are not a simple consequence of disease conditions but part of the transduction signalling pathway that triggers defence responses under the opportune stimuli. It has been recently reported that, similar to the GSH/GSSG pair, the level and redox state of ASC play a regulatory role in cell metabolism, both acting at the level of gene expression and altering enzymatic pathways [13,20,34,64]. Even if, at present, no data supporting this hypothesis are yet available, the imbalance in the ASC redox pair produced during HR programme could also supply an additional signal that contributes to triggering the required metabolic alterations. ...
Plant resistance to pathogens requires the activation of complex metabolic pathways in the infected cells, aimed at recognizing pathogen presence and hindering its propagation within plant tissues. In spite of this both compatible and incompatible responses induce alterations in plant metabolism, only in the latter the plant is able to efficiently block pathogen penetration without suffering excessive damage. One of the most studied incompatible responses is based on the hypersensitive response (HR), in which cells surrounding the site of pathogen penetration switch on genes encoding for phytoalexin synthesis and other pathogenesis related proteins before activating programmed cell death (PCD). The production of reactive oxygen species (ROS) is a key event in HR. Several enzymatic systems have been proposed to be responsible for the oxidative burst characterizing HR. In this review, the involvement of antioxidant redox systems, in particular those related to ascorbate (ASC) and glutathione (GSH), in activating both compatible and incompatible plant responses is analysed. Increasing lines of evidence indicate that alterations in the levels and/or redox state of ASC and/or GSH, as well as in the activity of their redox enzymes, occur during the HR programme. These alterations do not seem to be a mere consequence of the oxidative stress induced by the massive ROS production, but they are induced as part of the transduction pathways triggering defence responses and PCD. The possibility that ASC and GSH systems are links in a redox signalling chain activating defence strategies is also discussed.
... The purpose of this study was to radioactively label the reduced form of vitamin C with technetium-99m ( 99m Tc) in order to obtain the radioactive complex 99m Tc-AA. Several studies have been carried out in order to study the metabolism of AA by labelling this compound to different radionuclides ( 3 H, 18 F, 14 C) [19][20][21][22][23][24][25][26]. However, there is practically no information documenting the labelling of AA with 99m Tc. ...
Vitamin C exists in two forms: the reduced (ascorbic acid--AA) and oxidized form (dehydroascorbic acid--DHA). This is a nutrient whose benefits are long known and widely publicized, being most of them related to its antioxidant action. As an antioxidant, the main role of vitamin C is to neutralize free radicals, reducing oxidative stress. However, some controversial studies suggest that this nutrient may have a preventive and therapeutic role in cancer disease due to their possible pro-oxidant activity, promoting the formation of reactive oxygen species that can induce cell death in cancer cells. This factor, coupled with the decrease of antioxidant enzymes and increase of decompartmentalized transition metals in tumor cells may result in the selective cytotoxicity of vitamin C and the subsequent revelation of its therapeutic potential. In this way the first purpose of this work was radioactively label the reduced form of vitamin C with Tc-99m, its quality control by HPLC and the time stability. The second purpose was to use the radioactive complex 99mTc-AA in in vitro and in vivo studies in order to evaluate its uptake by colorectal cancer cells and biodistribution in mices, respectively. The results suggest that the pharmaceutical formulation developed, which was reproducible and stable over time, was residually taken up by colorectal cancer cells. Future studies are needed to deepen our understanding about the radioactive complex 99mTc-AA and clarify the mechanisms of action of vitamin C in oncologic disease.
... Oxidation of AsA by AO generates MDHA, which, in the presence of iron, can react with oxygen to generate H 2 O 2 . MDHA is also rapidly converted to DHA, which certain in vitro studies indicate can disrupt cell membranes, cause protein cross-linking, and inhibit enzymes involved in glycolysis and protein digestion [55,[63][64][65][66]. Therefore, it has been proposed that DHA may have toxic or anti-nutritive effects on insects [55], but this has not been tested in vivo. ...
Analysis of a diverse cross-sample of plant-insect interactions suggests that the abundance of vitamin C (L-ascorbic acid, ascorbate or AsA) in plants influences their susceptibility to insect feeding. These effects may be mediated by AsAs roles as an essential dietary nutrient, as an antioxidant in the insect midgut, or as a substrate for plant-derived ascorbate oxidase, which can lead to generation of toxic reactive oxygen species. Ascorbate can also influence the efficacy of plant defenses such as myrosinases and tannins, and alter insects' susceptibility to natural enemies. Conversely, herbivores appear to influence both de novo synthesis and redox cycling of AsA in their host plants, thereby potentially altering the nutritional value of crops and their susceptibility to pests. The recent development of genetically modified crops with enhanced AsA content provides both an impetus and a tool set for further studies on the role of AsA in plant-insect interactions.
... The VC:VK 3 interaction not only fosters single-electron reduction to produce the long-lived semiquinone and ascorbyl radicals and increases the rate of redox cycling of the quinone to form H 2 O 2 and other ROS [69], but also undergoes two-electron transfer, which ensures that AA and DHA will be present at pharmacologic levels for a protracted period of time. Three key glycolytic enzymes-hexokinase (HK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and glucose-6phosphate dehydrogenase (G6PD)-are known to exhibit redox sensitivity and can be inhibited by DHA when purified enzymes or erythrocyte lysates are employed [117][118][119]. While the inhibitory effect of DHA on all 3 enzymes can be abrogated by adding sufficiently high concentrations of the enzymes' specific substrates, the DHA-mediated inhibition of HK persists in intact erythrocytes because the critical concentration of its substrate (glucose) cannnot be achieved since glucose diffuses out of the erythrocytes. ...
A human bladder carcinoma cell line RT4 was sham-treated with buffer or treated with ascorbate (VC) alone, menadione alone (VK(3)), or a combination of ascorbate:menadione (VC+VK(3)) for 1, 2, and 4 h. Cytotoxic damage was found to be treatment-dependent in this sequence: VC+VK(3)>VC>VK(3)>sham. The combined treatment induced the greatest oxidative stress, with early tumor cell injury affecting the cytoskeletal architecture and contributing to the self-excisions of pieces of cytoplasm freed from organelles. Additional damage, including a reduction in cell size, organelle alterations, nuclear damage, and nucleic acid degradation as well as compromised lysosome integrity, is caused by reactivation of DNases and the redox cycling of VC or VC+VK(3). In addition, cell death caused by VC+VK(3) treatment as well as by prolonged VC treatment is consistent with cell demise by autoschizis, not apoptosis. This report confirms and complements previous observations about this new mode of tumor cell death. It supports the contention that a combination of VC+VK(3), also named Apatone, could be co-administered as a nontoxic adjuvant with radiation and/or chemotherapies to kill bladder tumor cells and other cancer cells without any supplementary risk or side effects for patients.
... The observed antiviral activities of dehydroascorbic acid clearly indicate that its effect is not due to an antioxidant mechanism since this reagent has no reducing potential. It has been shown that dehydroascorbic acid binds to proteins (15,16) and can modify lysine residues to form glycation adducts (17), or more specifically inhibits certain kinases and enzymes (18)(19)(20), although we do not know whether these mechanisms operate in the observed antiviral activity of dehydroascorbic acid at this moment. The observed weak activity of ascorbic acid in the absence of ferric ion and an enhanced effect in the presence of ferric ion may be due to free radicals or dehydroascorbic acid formed, or both. ...
In the present study, ascorbic acid weakly inhibited the multiplication of viruses of three different families: herpes simplex virus type 1 (HSV-1), influenza virus type A and poliovirus type 1. Dehydroascorbic acid, an oxidized form of ascorbic acid and hence without reducing ability, showed much stronger antiviral activity than ascorbic acid, indicating that the antiviral activity of ascorbic acid is due to factors other than an antioxidant mechanism. Moreover, addition of 1 mM Fe3+, which oxidizes ascorbic acid to dehydroascorbic acid and also enhances the formation of hydroxyl radicals by ascorbic acid in the culture media, strongly enhanced the antiviral activity of ascorbic acid to a level significantly stronger than that of dehydroascorbic acid. Although both ascorbic acid and dehydroascorbic acid showed some cytotoxicity, the degree of cytotoxicity of the former was 10-fold higher than the latter, suggesting that the observed antiviral activity of ascorbic acid with and without ferric ion is, at least in part, a secondary result of the cytotoxic effect of the reagent, most likely due to the free radicals. However, the possibility that oxidation of ascorbic acid also contributed to the antiviral effects of ascorbic acid exists, in particular in the presence of ferric ion, since dehydroascorbic acid exhibited a very strong antiviral activity. Characterization of the mode of antiviral action of dehydroascorbic acid revealed that the addition of the reagent even at 11 h post infection almost completely inhibited the formation of progeny infectious virus in the infected cells, indicating that the reagent inhibits HSV-1 multiplication probably at the assembly process of progeny virus particles after the completion of viral DNA replication.
... phosphorylable glucose analogue and OMG is not, we conclude that GLUT3 and hexokinase are important in the mechanism of glucose transport inhibition, or rather in the inhibition of glucose utilization. These results are consistent with in vitro hexokinase activity inhibition by dehydroascorbic acid, the oxidized form of ascorbic acid [17] . It is possible that ascorbic acid inside the cell is oxidized, and thus, hexokinase (and maybe GLUT3) is inhibited. ...
In this paper, we present a novel function for ascorbic acid. Ascorbic acid is an important water-soluble antioxidant and cofactor in various enzyme systems. We have previously demonstrated that an increase in neuronal intracellular ascorbic acid is able to inhibit glucose transport in cortical and hippocampal neurons. Because of the presence of sodium-dependent vitamin C transporters, ascorbic acid is highly concentrated in brain, testis, lung, and adrenal glands. In this work, we explored how ascorbic acid affects glucose and lactate uptake in neuronal and non-neuronal cells. Using immunofluorescence and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, the expression of glucose and ascorbic acid transporters in non-neuronal cells was studied. Like neurons, HEK293 cells expressed GLUT1, GLUT3, and SVCT2. With radioisotope-based methods, only intracellular ascorbic acid, but not extracellular, inhibits 2-deoxyglucose transport in HEK293 cells. As monocarboxylates such as pyruvate and lactate, are important metabolic sources, we analyzed the ascorbic acid effect on lactate transport in cultured neurons and HEK293 cells. Intracellular ascorbic acid was able to stimulate lactate transport in both cell types. Extracellular ascorbic acid did not affect this transport. Our data show that ascorbic acid inhibits glucose transport and stimulates lactate transport in neuronal and non-neuronal cells. Mammalian cells frequently present functional glucose and monocarboxylate transporters, and we describe here a general effect in which ascorbic acid functions like a glucose/monocarboxylate uptake switch in tissues expressing ascorbic acid transporters.
... To assess perturbations in glucose phosphorylation , measuring the hexokinase enzyme activity will be performed in kidney, liver and brain tissues of Dahl rats. This will be done as previously described [106,107]. ...
Despite the marked advances in research on insulin resistance (IR) in humans and animal models of insulin resistance, the mechanisms underlying high salt-induced insulin resistance remain unclear. Insulin resistance is a multifactorial disease with both genetic and environmental factors (such as high salt) involved in its pathogenesis. High salt triggers insulin resistance in genetically susceptible patients and animal models of insulin resistance. One of the mechanisms by which high salt might precipitate insulin resistance is through its ability to enhance an oxidative stress-induced inflammatory response that disrupts the insulin signaling pathway. The aim of this hypothesis is to discuss two complementary approaches to find out how high salt might interact with genetic defects along the insulin signaling and inflammatory pathways to predispose to insulin resistance in a genetically susceptible model of insulin resistance. The first approach will consist of examining variations in genes involved in the insulin signaling pathway in the Dahl S rat (an animal model of insulin resistance and salt-sensitivity) and the Dahl R rat (an animal model of insulin sensitivity and salt-resistance), and the putative cellular mechanisms responsible for the development of insulin resistance. The second approach will consist of studying the over-expressed genes along the inflammatory pathway whose respective activation might be predictive of high salt-induced insulin resistance in Dahl S rats.
Variations in genes encoding the insulin receptor substrates -1 and/or -2 (IRS-1, -2) and/or genes encoding the glucose transporter (GLUTs) proteins have been found in patients with insulin resistance. To better understand the combined contribution of excessive salt and genetic defects to the etiology of the disease, it is essential to investigate the following question:
Question 1: Do variations in genes encoding the IRS -1 and -2 and/or genes encoding the GLUTs proteins predict high salt-induced insulin resistance in Dahl S rats?
A significant amount of evidence suggested that salt-induced oxidative stress might predict an inflammatory response that upregulates mediators of inflammation such as the nuclear factor- kappa B (NF-kappa B), the tumor necrosis factor-alpha (TNF-α) and the c-Jun Terminal Kinase (JNK). These inflammatory mediators disrupt the insulin signaling pathway and predispose to insulin resistance. Therefore, the following question will be thoroughly investigated:
Question 2: Do variations in genes encoding the NF-kappa B, the TNF-α and the JNK, independently or in synergy, predict an enhanced inflammatory response and subsequent insulin resistance in Dahl S rats in excessive salt environment?
Finally, to better understand the combined role of these variations on glucose metabolism, the following question will be addressed:
Question 3: What are the functional consequences of gene variations on the rate of glucose delivery, the rate of glucose transport and the rate of glucose phosphorylation in Dahl S rats?
The general hypothesis is that "high-salt diet in combination with defects in candidate genes along the insulin signaling and inflammatory pathways predicts susceptibility to high salt-induced insulin resistance in Dahl S rats".
The interplay between genetic alterations and metabolic dysregulation is increasingly recognized as a pivotal axis in cancer pathogenesis. Both elements are mutually reinforcing, thereby expediting the ontogeny and progression of malignant neoplasms. Intriguingly, recent findings have highlighted the translocation of metabolites and metabolic enzymes from the cytoplasm into the nuclear compartment, where they appear to be intimately associated with tumor cell proliferation. Despite these advancements, significant gaps persist in our understanding of their specific roles within the nuclear milieu, their modulatory effects on gene transcription and cellular proliferation, and the intricacies of their coordination with the genomic landscape. In this comprehensive review, we endeavor to elucidate the regulatory landscape of metabolic signaling within the nuclear domain, namely nuclear metabolic signaling involving metabolites and metabolic enzymes. We explore the roles and molecular mechanisms through which metabolic flux and enzymatic activity impact critical nuclear processes, including epigenetic modulation, DNA damage repair, and gene expression regulation. In conclusion, we underscore the paramount significance of nuclear metabolic signaling in cancer biology and enumerate potential therapeutic targets, associated pharmacological interventions, and implications for clinical applications. Importantly, these emergent findings not only augment our conceptual understanding of tumoral metabolism but also herald the potential for innovative therapeutic paradigms targeting the metabolism–genome transcriptional axis.
Cancer treatment is hampered by resistance to conventional therapeutic strategies, including chemotherapy, immunotherapy, and targeted therapy. Redox homeostasis manipulation is one of the most effective innovative treatment techniques for overcoming drug resistance. Reactive oxygen species (ROS), previously considered intracellular byproducts of aerobic metabolism, are now known to regulate multiple signaling pathways as second messengers. Cancer cells cope with elevated amounts of ROS during therapy by upregulating the antioxidant system, enabling tumor therapeutic resistance via a variety of mechanisms. In this review, we aim to shed light on redox modification and signaling pathways that may contribute to therapeutic resistance. We summarized the molecular mechanisms by which redox signaling-regulated drug resistance, including altered drug efflux, action targets and metabolism, enhanced DNA damage repair, maintained stemness, and reshaped tumor microenvironment. A comprehensive understanding of these interrelationships should improve treatment efficacy from a fundamental and clinical research point of view.
Significance
Hexokinases are key enzymes that are responsible for the first reaction of glycolysis, but they also moonlight other cellular processes, including mitochondrial redox signaling regulation. Modulation of hexokinase activity and spatiotemporal location by reactive oxygen and nitrogen species as well as other gasotransmitters serves as the basis for a unique, underexplored method of tight and flexible regulation of these fundamental enzymes.
Recent advances
Redox modifications of thiols serve as a molecular code that enables the precise and complex regulation of hexokinases. These reactions are also used by multiple parasites to cause widespread and severe diseases, including malaria, Chagas disease, and sleeping sickness. Redox-active molecules affect each other, and the moonlighting activity of hexokinases provides another feedback loop that affects the cellular redox status and is hijacked in malignantly transformed cells.
Critical issues
Several compounds affect the redox status of hexokinases in vivo. These include the dehydroascorbic acid (oxidized form of vitamin C), contraceptive pyrrolidinium pyrrolidine-1-carbodithioate, ethanol metabolism-generated peroxynitrite, alloxan (a glucose analog), and isobenzothiazolinone ebselen. However, very limited information is available regarding which amino acid residues in hexokinases are affected by redox signaling. Except in cases of monogenic diabetes, direct evidence is absent for disease phenotypes that are associated with variations within motifs that are susceptible to redox signaling.
Future directions
Further studies should address the propensity of hexokinases and their disease-associated variants to participate in redox regulation. Robust and straightforward proteomic methods are needed to understand the context and consequences of hexokinase-mediated redox regulation in health and disease.
A U937 cell clone, in which low micromolar concentrations of ascorbic acid (AA) and dehydroascorbic acid (DHA) are taken up at identical rates, was used to investigate possible interactions between transport systems mediating cellular uptake of the two forms of the vitamin. Results obtained with different experimental approaches showed that DHA potently and reversibly inhibits AA uptake through Na(+)-AA cotransporters. Hence, a progressive increase in extracellular DHA concentrations in the presence of a fixed amount of AA caused an initial decrease in the net amount of vitamin C accumulated, and eventually, at higher levels, it caused an accumulation of the vitamin solely based on DHA uptake through hexose transporters. DHA-dependent inhibition of AA uptake was also detected in various other cell types. Taken together, our results provide evidence of a novel biological effect mediated by concentrations of DHA compatible with those produced at inflammatory sites.
Abstract The oxidative role(s) of p66Shc protein has been increasingly expanded over the last decade. However, its relation with the most potent antioxidant molecule, i.e. ascorbic acid has never been studied. We have previously shown that p66Shc mediates rac1 activation, reactive oxygen species (ROS) production and cell death. Here we studied the effect of ascorbic acid on the pathway involving p66Shc and rac1. Our results indicate a decrease in the expression of p66Shc in a dose- and time-dependent manner. We studied the effect of ascorbic acid on rac1 expression and its activity. Ascorbic acid has no effect on total rac1 expression; however, rac1 activation was inhibited in a dose-dependent manner. Results suggest that the decrease in rac1 activity is mediated through ascorbic acid-modulated p66Shc expression. The decrease in rac1 activity was evident in cells transfected with the p66shc mutant (proline motif mutant, at residues P47 to P50). Our studies indicate that p66Shc-mediated ROS upregulation is significantly decreased in the presence of ascorbic acid. Cell migration experiments point towards the inhibition of p66Shc-rac1-mediated migration in the presence of ascorbic acid. Finally, results are suggestive that ascorbic acid-mediated decrease in Shc expression occurs through an increased Shc ubiquitination. Overall, the study brings out the novel role of ascorbic acid in antioxidant signal transduction.
Ascorbate is a widespread and efficient antioxidant that has multiple functions in plants, traditionally associated with the reactions of photosynthesis. This review aims to look at ascorbate from an evolutionary perspective. Cyanobacteria, algae, and bryophytes contain lower concentrations of ascorbate than higher plants, where the molecule accumulates in high concentrations in both photosynthetic and non-photosynthetic organs and tissues. This increase in ascorbate concentration is paralleled by an increase in the number of isoforms of ascorbate peroxidase and the ascorbate regenerating enzymes mono- and dehydroascorbate reductase. One way of understanding the rise in ascorbate concentrations is to consider ascorbate as a molecule among others that has been subject to selection pressures during evolution, due to its cost or benefit for the cell and the organism. Ascorbate has a low cost in terms of synthesis and toxicity, and its benefits include protection of the glutathione pool and proper functioning of a range of enzymes. The hypothesis presented here is that these features would have favoured increasing roles for the molecule in the development and growth of multicellular organisms. This review then focuses on this diversity of roles for ascorbate in both photosynthetic and non-photosynthetic tissues of higher plants, including fruits and seeds, as well as further functions the molecule may possess by looking at other species. The review also highlights one of the trade-offs of domestication, which has often reduced or diluted ascorbate content in the quest for increased fruit growth and yield, with unknown consequences for the corresponding functional diversity, particularly in terms of stress resistance and adaptive responses to the environment.
The ascorbic acid (ASC) system functions dynamically in seeds, although the strategies for ASC production and utilization may vary according to seed developmental and functional stages. In orthodox seeds, ASC content and ASC peroxidase activity increase during the early stages of development, then decrease during the desiccation stage, so that, at quiescence, seeds have neither ASC nor ASC peroxidase, but retain a small amount of dehydroascorbic acid (DHA) and significant activities of ASC recycling enzymes. ASC and ASC peroxidase activity re-start after a few hours from the onset of imbibition. In contrast, the ASC system is little affected during germination of recalcitrant seeds. Although the presence of the ASC system in seeds has often been considered only within the framework of seed antioxidant defences, ASC function in seeds is also likely to be related to its action as a specific co-substrate required for the activity of dioxygenases (e.g. 1-aminocyclopropane carboxylate oxidase, gibberellic acid hydroxylases and 9-cis-epoxycarotenoid dioxygenases) involved in the synthesis of ethylene, gibberellins and abscisic acid, respectively. The possible role of ASC in coordinating the activities of these key enzymes is discussed.
In vitro embryogenesis via androgenesis and somatic embryogenesis represents an efficient propagation tool as well as a suitable
model system for investigating structural, physiological, and molecular events governing embryo development. One of the major
problems encountered by tissue culturists is the poor efficiency of embryos produced in vitro and their inability to regenerate
viable plants, which denote sub-optimal culture conditions. Judicious alterations of the glutathione (GSH) and ascorbate (AsA)
redox state have a profound effect on morphogenesis and improve the quality of the embryos by promoting a zygotic-like histodifferentiation
pattern and producing well organized meristems. This approach has been investigated successfully during the development and
germination of both Brassica napus (canola, an angiosperm) microspore-derived embryos (MDEs) and Picea glauca (white spruce, a conifer) somatic embryos. The imposition of a reduced glutathione environment during the early embryonic
phases induces cellular proliferation and increases the number of immature embryos, possibly by promoting the synthesis of
nucleotides required for energetic processes and mitotic activity. Continuation of embryo development is best conducted if
the glutathione pool is experimentally switched towards an oxidized state; a condition favoring histodifferentiation and post-embryonic
growth in both canola and spruce. Structural analyses showed that the oxidized glutathione environment favors the proper formation
of the shoot apical meristem (SAM), which acquires a “zygotic-like” appearance. The apical poles of glutathione-treated embryos
are well organized and display a proper expression and localization of meristem marker genes. These conditions are not found
in control embryos which develop abnormal SAMs characterized by the presence of intercellular spaces and differentiation of
meristematic cells. Such meristems fail to reactivate at germination resulting in embryo abortion. Physiological and molecular
studies have further demonstrated that the oxidized glutathione environment induces several responses, including changes in
ascorbate metabolism, abscisic acid and ethylene synthesis, as well as alterations in storage product deposition patterns.
If an oxidized glutathione environment favors embryo formation, a reduced ascorbate redox state is required to promote germination
and conversion, i.e. the emergence of functional shoots and roots. Specifically, a highly reduced ascorbate environment induces
cell proliferation and de-novo meristem formation within those SAMs with an abnormal architecture. Overall it appears that
precise changes in glutathione and ascorbate metabolism are required to ensure proper embryo formation and regeneration.
KeywordsAscorbate-Glutathione-Meristem-Microspore-derived and somatic embryos-Germination
High doses of synthetic antioxidative vitamins: A, E, C and β-carotene are often used on long-term basis in numerous preventive and therapeutic medical applications. Instead of expected health effects, the use of those vitamins may however lead to cases of hypervitaminosis and even to intoxication. The article points out main principles of safety which are to be observed during supplementation with antioxidative vitamins. Toxic effects resulting from erroneous administration of high doses of those substances on organs and systems of the organism are also discussed. Attention is drawn to interactions of antioxidative vitamins with concomitantly used drugs, as well as intensification of adverse effects caused by various exogenous chemical factors. Moreover, the article presents the evaluation of supplementation with these vitamins, which was performed in large studies.
Ascorbate (AA), an antioxidant substance known as vitamin C, exists in the brain at a high concentration, although transfer into the brain after systemic administration of AA itself is limited. Intraperitoneal administration of dehydroascorbate (DHA) resulted in a rapid and progressive increase in extracellular AA in rat striatum in a dose-dependent manner. DHA administration increased 2,3- and 2,5-dihydroxybenzoate (2,3- and 2,5-DHBA) formation from salicylate in parallel with the increase in extracellular AA. Intrastriatal administration of active AA oxidase (AAO), but not the inactivated enzyme, completely suppressed the increase in 2,3- and 2,5-DHBA formation after the DHA administration. These findings suggest that extracellular AA might stimulate hydroxyl radical (OH) generation in the striatum. This is supported by the observation of dose-dependent OH generation upon intrastriatal administration of AA itself. In addition, deferoxamine, an iron chelator, decreased basal 2,3- and 2,5-DHBA formation and strongly, though not completely, suppressed the DHA-induced increase of 2,3- and 2,5-DHBA formation. Therefore, increased extracellular AA might function as a prooxidant and stimulate OH generation in cooperation with iron in rat striatum.
The copper-based drug Casiopeina II-gly (CasII-gly) shows potent antineoplastic effect and diminishes mitochondrial metabolism on several human and rodent malignant tumors. To elucidate whether CasII-gly also affects glycolysis, (a) the flux through the complete pathway and the initial segment and (b) the activities of several glycolytic enzymes of AS-30D hepatocarcinoma cells were determined. CasII-gly (IC₅₀ = 0.74-6.7 μM) was more effective to inhibit 24-72 h growth of several human carcinomas than 3-bromopyruvate (3BrPyr) (IC₅₀ = 45-100 μM) with no apparent effect on normal human-proliferating lymphocytes and HUVECs. In short-term 60-min experiments, CasII-gly increased tumor cell lactate production and glycogen breakdown. CasII-gly was 1.3-21 times more potent than 3BrPyr and cisplatin to inhibit tumor HK. As CasII-gly inhibited the soluble and mitochondrial HK activities and the flux through the HK-TPI glycolytic segment, whereas PFK-1, GAPDH, PGK, PYK activities and HPI-TPI segment flux were not affected, the data suggested glycogenolysis activation induced by HK inhibition. Accordingly, glycogen-depleted as well as oligomycin-treated cancer cells became more sensitive to CasII-gly. The inhibition time-course of HK by CasII-gly was slower than that of OxPhos in AS-30D cells, indicating that glycolytic toxicity was secondary to mitochondria, the primary CasII-gly target. In long-term 24-h experiments with HeLa cells, 5 μM CasII-gly inhibited OxPhos (80%), glycolysis (40%), and HK (42%). The present data indicated that CasII-gly is an effective multisite anticancer drug simultaneously targeting mitochondria and glycolysis.
Toxicological and environmental issues are associated with the extensive use of agricultural pesticides, although the knowledge of their toxic effects as commercial formulations is still far from being complete. This work investigated the impact of three herbicides as commercial formulations on the oxidative status of a wild type Saccharomyces cerevisiae strain. With yeast being a well-established model of eukaryotic cells, especially as far as regards the stress response, these results may be indicative of potential damages on higher eukaryotes. It was found that herbicide-mediated toxicity towards yeast cells could be the result of an increased production of hydroperoxides (like in the case of the herbicides Pointer and Silglif) or advanced oxidation protein products and lipid peroxidation (especially in the case of the herbicide Proper Energy). Through a redox-proteomic approach it was found also that, besides a common signature, each herbicide showed a specific pattern for protein thiols oxidation.
Vitamin C (ascorbic acid (AA)) is very popular for its antioxidant properties. Consequently, many other important aspects of this multifaceted molecule are often underestimated or even ignored. In the present paper, we have tried to bring to the foreground some of these aspects, including the peculiarities of the AA biosynthetic pathway in different organisms, the remarkable function of AA as a co-substrate of many important dioxygenases, the role of AA-regenerating enzymes and the known pathways of AA catabolism, as well as the intriguing function of AA in gene expression.
In the current view of most biochemists and physiologists, the role of L-ascorbic acid (AA) in cell metabolism would be more or less confined to the scavenging of reactive oxygen species. Nevertheless, many data have been collected in our and other laboratories concerning the involvement of AA in many different aspects of cell metabolism. At the present time the molecular sites of action of AA have not been completely elucidated, but recent findings on the specific requirement of AA for the activity of several 2-oxoacid-dependent dioxygenases involved in cell signalling and the activation of transcription factors open new fascinating perspectives for further research.
Reactive oxygen species (ROS) are key intermediates in cellular signal transduction pathways whose function may be counterbalanced
by antioxidants. Acting as an antioxidant, ascorbic acid (AA) donates two electrons and becomes oxidized to dehydroascorbic
acid (DHA). We discovered that DHA directly inhibits IκBα kinase β (IKKβ) and IKKα enzymatic activity in vitro, whereas AA
did not have this effect. When cells were loaded with AA and induced to generate DHA by oxidative stress in cells expressing
a constitutive active IKKβ, NF-κB activation was inhibited. Our results identify a dual molecular action of vitamin C in signal
transduction and provide a direct linkage between the redox state of vitamin C and NF-κB signaling events. AA quenches ROS
intermediates involved in the activation of NF-κB and is oxidized to DHA, which directly inhibits IKKβ and IKKα enzymatic
activity. These findings define a function for vitamin C in signal transduction other than as an antioxidant and mechanistically
illuminate how vitamin C down-modulates NF-κB signaling.
A detailed computer model of human erythrocyte metabolism was shown to predict three steady states, two stable and one unstable. The most extreme steady state is characterized by almost zero concentrations of all the phosphorylated intermediates. The "normal" steady state is remarkably robust in the face of large changes in the activity of most of the enzymes of glycolysis and the pentose phosphate pathway: this steady state can be viewed as an attractor towards which the system returns following a metabolic perturbation. Focus is given to three responses of the system: (1) the 'energy charge' that pertains to the concentration of ATP relative to all purine nucleotides; (2) redox power expressed as the ratio of reduced-to-total glutathione and (3) the concentration of 2,3-bisphosphoglycerate, that directly affects the oxygen affinity of haemoglobin thus affecting the main physiological function of the cell. The collapse of the normal steady state in what can be viewed topologically as a catastrophe is posited as one key element of erythrocyte senescence and it is particularly important for erythrocyte destruction in patients with an inborn enzyme deficiency.
The interaction between nickel and yeast hexokinase was studied. The binding of nickel showed a positive cooperativity, and saturation was not reached. The nickel binding induced modifications in the secondary structure of the protein; thus, a lost of alpha helix and beta turns, as well as an increase of the random structure and beta sheet was observed. The monomer/dimmer equilibrium of the protein was modified in the presence of nickel, and the monomer state was mainly obtained at the highest nickel concentrations studied. These changes on the protein structure caused a decrease in the enzyme activity. According to kinetic studies, nickel caused a non-competitive inhibition when glucose was the variable substrate and a linear competitive inhibition when ATP was the variable substrate.
The aim of this study was to label ascorbic acid with (99m)Tc and to investigate its radiopharmaceutical potential in rats. Ascorbic acid was labeled with (99m)Tc using the stannous chloride method. The radiochemical purity of [(99m)Tc]ascorbic acid ((99m)Tc-AA) was determined by RTLC, paper electrophoresis, and RHPLC methods. The labeling yield was found to be 93+/-5.0%. The maximum labeling yield of (99m)Tc-AA was determined at pH 5 and 25 degrees C. The biodistribution studies related to (99m)Tc-AA were done in male albino Wistar rats. (99m)Tc-AA, which has a specific activity of 13.02 GBq/mmol, was administered into the tail vein of the rats. The rats were sacrificed at 15, 30, 60, and 120 min after the injection by heart puncture under ether anaesthesia. The organs were weighed after removal. Their activities were counted using a Cd(Te) detector equipped with a RAD 501 count system. The %ID/g (% of injected dose per gram of tissue weight) in each organ and in blood was calculated. Maximum uptake of (99m)Tc-AA was observed in prostate and kidneys at the 60th min. (99m)Tc-AA may be a promising radiopharmaceutical for the imaging of prostate and kidneys.
Ascorbate and dehydroascorbate transport was investigated in rat liver microsomal vesicles using radiolabeled compounds and
a rapid filtration method. The uptake of both compounds was time- and temperature-dependent, and saturable. Ascorbate uptake
did not reach complete equilibrium, it had low affinity and high capacity. Ascorbate influx could not be inhibited by glucose,
dehydroascorbate, or glucose transport inhibitors (phloretin, cytochalasin B) but it was reduced by the anion transport inhibitor
4,4′-diisothiocyanostilbene-2,2′-disulfonic acid and by the alkylating agent N-ethylmaleimide. Ascorbate uptake could be stimulated by ferric iron and could be diminished by reducing agents (dithiothreitol,
reduced glutathione). In contrast, dehydroascorbate uptake exceeded the level of passive equilibrium, it had high affinity
and low capacity. Glucose cis inhibited andtrans stimulated the uptake. Glucose transport inhibitors were also effective. The presence of intravesicular reducing compounds
increased, while extravesicular reducing environment decreased dehydroascorbate influx. Our results suggest that dehydroascorbate
transport is preferred in hepatic endoplasmic reticulum and it is mediated by a GLUT-type transporter. The intravesicular
reduction of dehydroascorbate leads to the accumulation of ascorbate and contributes to the low intraluminal reduced/oxidized
glutathione ratio.
Reactive oxygen species have been implicated in oxidative modifications of proteins, in many cases represented as carbonyls, which can lead to a variety of diseases and the age-associated decline of physiological functions. Considerable progress, as well as controversy, about oxidatively modified proteins and aging has unfolded in the last few years. In this article we critically evaluate changes in protein carbonyl content as a marker of the oxidative stress associated with age and other relevant issues on the degradation of oxidatively modified proteins.
A definitive conclusion on the age-related increase of protein carbonyls is currently viewed as having to await further confirmation using detailed analysis with new methodologies. Controversial methodological measurements and characterizations of protein carbonyls are discussed, emphasizing the merits of immunoblot analysis using two-dimensional gel electrophoresis. The degradation of oxidatively modified proteins has not yet been studied in depth in relation to their possible accumulation in old tissues. Recent efforts to establish a causal relation between the effect of oxidative stress on proteins and physiological declines with age are discussed briefly.
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.
A number of metal-catalyzed oxidation (MCO) systems mediate the oxidative inactivation of enzymes. This oxidation is accompanied by conversion of the side chains of some amino acid residues to carbonyl derivatives (for review, see Stadtman, E. R. (1986) Trends Biochem. Sci. 11, 11-12). To identify the amino acid residues which are sensitive to MCO oxidation, several enzymes/proteins and amino acid homopolymers were exposed to various MCO systems. The carbonyl groups which were formed were converted to their corresponding 3H-labeled hydroxy derivatives. After acid hydrolysis, the labeled free amino acids were separated by ion exchange chromatography. Each protein or polymer gave rise to several different labeled amino acids. The elution profiles of the labeled amino acids obtained from preparations of Escherichia coli glutamine synthetase which had been oxidized by MCO systems comprised of either Fe(II)/O2 or ascorbate/Fe(II)/O2 both in the presence and absence of EDTA were qualitatively the same. From a comparison of the elution profiles of labeled amino acids from various proteins with those obtained from homopolymers, it is evident that the side chains of histidine, arginine, lysine, and proline are particularly sensitive to oxidation by the MCO systems. This conclusion is supported also by direct amino acid analysis of acid hydrolysates which shows that the oxidation of glutamine synthetase, enolase, and phosphoglycerate kinase is associated with the loss of at least 1 histidine residue per subunit. From the results of studies with homopolymers, it is apparent that glutamic semialdehyde is a major product of both proline and arginine residues. In addition, hydroxyproline and unlabeled glutamic acid were identified among the hydrolysis products of oxidized poly-L-proline, and unlabeled aspartic acid was identified as a product of poly-L-histidine oxidation.
We have previously described the oxidative inactivation of several key metabolic enzymes by a variety of mixed function oxidation systems. Because many of the enzymes which are inactivated have been shown by others to accumulate as inactive or less active forms during cellular aging, we have examined the levels of oxidatively modified proteins in two model systems used for studies on aging. The results show that levels of oxidatively modified proteins increase with age in circulating erythrocytes, and this change is correlated with the loss of marker enzyme activity. Our studies also show that in cultured fibroblasts from normal donors the levels of oxidatively modified proteins increase only after the age of 60. However, the levels of oxidatively modified proteins in fibroblasts from individuals with progeria or Werner's syndrome are significantly higher than age-matched controls. Moreover, treatment of glucose-6-phosphate dehydrogenase with a mixed function oxidation system leads to oxidative modification and increased heat lability of the enzyme. Taken together these results suggest that loss of functional enzyme activity and increased heat lability of enzymes during aging may be due in part to oxidative modification by mixed function oxidation systems.
Exposure of red blood cells to oxygen radicals can induce hemoglobin damage and stimulate protein degradation, lipid peroxidation, and hemolysis. To determine if these events are linked, rabbit erythrocytes were incubated at 37 degrees C with various oxygen radical-generating systems and antioxidants. Protein degradation, measured by the production of free alanine, increased more than 11-fold in response to xanthine (X) + xanthine oxidase (XO). A similar increase in proteolysis occurred when the cells were incubated with acetaldehyde plus XO, with ascorbic acid plus iron (Asc + Fe), or with hydrogen peroxide (H2O2) alone. Upon addition of XO, increased proteolysis was evident within 5 min and was linear for up to 5 h. In contrast, lipid peroxidation, as shown by the production of malonyldialdehyde, conjugated dienes, or lipid hydroperoxides was observed only after 2 h of incubation with X + XO, acetaldehyde + XO, or H2O2. Ascorbate plus Fe2+ induced both protein degradation and lipid peroxidation; however, the addition of various antioxidants (urate, xanthine, glucose, or butylated hydroxytoluene) decreased lipid peroxidation without affecting proteolysis. Thus, these processes seem to occur by distinct mechanisms. Furthermore, at low concentrations of XO, protein degradation was clearly increased in the absence of detectable lipid peroxidation products. Hemolysis occurred only in a small number of cells (9%) and followed the appearance of lipid peroxidation products. Thus, an important response of red cells to oxygen radicals is rapid degradation of damaged cell proteins. Increased proteolysis seems to occur independently of membrane damage and to be a more sensitive indicator of cell exposure to oxygen radicals than is lipid peroxidation.
The first step in the proteolytic degradation of bacterial glutamine synthetase is a mixed function oxidation of one of the 16 histidine residues in the glutamine synthetase subunit (Levine, R.L. (1983) J. Biol. Chem. 258, 11823-11827). A model system, consisting of oxygen, a metal ion, and ascorbic acid, mimics the bacterial system in mediating the oxidative modification of glutamine synthetase. This model system was studied to gain an understanding of the mechanism of oxidation and of factors which control the susceptibility of the enzyme to oxidation. Availability of substrates and the extent of covalent modification of the enzyme (adenylylation) interact to modulate susceptibility of the enzyme to oxidation. This interaction provides the biochemical basis for physiologic regulation of intracellular proteolysis of glutamine synthetase. The oxidative modification requires hydrogen peroxide. While the reaction may involve Fenton chemistry, the participation of free radicals, superoxide anion, and singlet oxygen could not be demonstrated.
Intracellular proteolytic degradation of glutamine synthetase occurs in two distinct steps in Escherichia coli (Levine, R. L., Oliver, C. N., Fulks, R. M., and Stadtman, E. R. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2120-2124). In the first step, a mixed function oxidation modifies the glutamine synthetase. The modified enzyme, which is catalytically inactive, becomes susceptible to proteolytic attack. In the second step, a protease specific for the modified enzyme catalyzes the actual proteolytic degradation. The oxidatively modified glutamine synthetase was studied to determine the chemical differences between it and the native enzyme. Only a single alteration was found; one of sixteen histidine residues/subunit was altered by the oxidative modification. The modification introduced a carbonyl group into the protein, permitting isolation of a stable dinitrophenylhydrazone. No other differences were detected between the native and modified proteins. Specifically, the cysteine, methionine, phenylalanine, tyrosine, and tryptophan contents were not altered. A number of other prokaryotic and eukaryotic enzymes are also susceptible to oxidative modification. This covalent modification may be important in intracellular proteolysis, in mammalian host defense systems, in prevention of autolysis, in aging processes, and in oxygen toxicity.
Rabbit hexokinase (EC 2.7.1.1) has been shown to exist in the soluble fraction of reticulocytes as two distinct molecular forms, designated hexokinase Ia and hexokinase Ib, which are separable by ion exchange chromatography and polyacrylamide gel electrophoresis. Hexokinase Ia was found to be similar to the brain enzyme, while hexokinase Ib differs from every other previously reported hexokinase isozyme. Reticulocyte hexokinase Ia and Ib have been purified 55,000-and 50,000-fold, respectively, by a combination of ion exchange chromatography, affinity chromatography, and preparative polyacrylamide gel electrophoresis, as proteins homogeneous by sodium dodecyl sulfate-gel electrophoresis. The native proteins have the same molecular weight of 105,000 by gel filtration and sedimentation velocity on sucrose density gradients. Sodium dodecyl sulfate-polyacrylamide gels have a molecular weight of 104,000, indicating that the two forms are monomers. Hexokinase Ia had a pI of 6.2 to 6.3 pH units while hexokinase Ib had a pI of 5.7 to 5.8 pH units by isoelectric focusing. The two enzymes were specific for Mg.ATP and Mg.ITP as the nucleotide substrates. Several hexoses could be phosphorylated by hexokinase Ia and Ib with different affinities.
We performed a detailed kinetic analysis of the uptake of dehydroascorbic acid by HL-60 cells under experimental conditions
that enabled the differentiation of dehydroascorbic acid transport from the intracellular reduction/accumulation of ascorbic
acid. Immunoblotting and immunolocalization experiments identified GLUT1 as the main glucose transporter expressed in the
HL-60 cells. Kinetic analysis allowed the identification of a single functional activity involved in the transport of dehydroascorbic
acid in the HL-60 cells. Transport was inhibited in a competitive manner by both 3-O-methyl-D-glucose and 2-deoxy-D-glucose. In turn, dehydroascorbic acid competitively inhibited the transport of both sugars.
A second functional component identified in experiments measuring the accumulation of ascorbic acid appears to be associated
with the intracellular reduction of dehydroascorbic acid to ascorbic acid and is not directly involved in the transport of
dehydroascorbic acid via GLUT1. Transport of dehydroascorbic acid by HL-60 cells was independent of the presence of external
Na, whereas the intracellular accumulation of ascorbic acid was found to be a Na-sensitive process. Thus, the transport of dehydroascorbic acid via glucose transporters is a Na-independent process which is kinetically and biologically separable from the reduction of dehydroascorbic acid to ascorbic
acid and its subsequent intracellular accumulation.
The truncated gene of hexokinase, mini-hexokinase, starting with methionine 455 and ending at the C terminus was expressed
in Escherichia coli. Mini-hexokinase lost its ability to ameliorate inhibition of glucose-6-P-inhibited mini-hexokinase in the presence of phosphate
(Pi). We suggest that the Pi site either resides in the N-terminal half of hexokinase I or requires the N-terminal portion of the enzyme. Site-directed
mutagenesis was performed to obtain two mutants of mini-hexokinase: C606S and C628S. Both are thought to be associated with
the active site of hexokinase I. These mutants exhibited a 3-fold increase in Kfor glucose but no change in either the Kfor ATP or the k. The circular dichroism (CD) spectra showed no differences among the wild-type or mutant enzymes. These results suggest that
Cys and Cys are not involved in glucose binding directly. The putative ATP-binding site of full-length human brain hexokinase may involve
Arg and Gly, and these residues were mutated to Ile. For the mutant R539I, the k value decreased 114-fold relative to wild-type hexokinase, whereas the Kvalues for ATP and glucose changed only slightly. No change was observed in the Kvalue for 1,5-anhydroglucitol 6-phosphate. CD spectra showed only a slight change in secondary structure. For the mutant G679I,
overexpressed hexokinase is insoluble. We suggest that Arg is important for catalysis because it stablizes the transition state product ADP-hexokinase. Gly is probably important for proper folding of the protein.
It is unknown whether ascorbate alone (vitamin C), its oxidized metabolite dehydroascorbic acid alone, or both species are transported into human cells. This problem was addressed using specific assays for each compound, freshly synthesized pure dehydroascorbic acid, the specially synthesized analog 6-chloroascorbate, and a new assay for 6-chloroascorbate. Ascorbate and dehydroascorbic acid were transported and accumulated distinctly; neither competed with the other. Ascorbate was accumulated as ascorbate by sodium-dependent carrier-mediated active transport. Dehydroascorbic acid transport and accumulation as ascorbate was at least 10-fold faster than ascorbate transport and was sodium-independent. Once transported, dehydroascorbic acid was immediately reduced intracellularly to ascorbate. The analog 6-chloroascorbate had no effect on dehydroascorbic acid transport but was a competitive inhibitor of ascorbate transport. The Ki for 6-chloroascorbate (2.9-4.4 microM) was similar to the Km for ascorbate transport (9.8-12.6 microM). 6-Chloroascorbate was itself transported and accumulated in fibroblasts by a sodium-dependent transporter. These data provide new information that ascorbate and dehydroascorbic acid are transported into human neutrophils and fibroblasts by two distinct mechanisms and that the compound available for intracellular utilization is ascorbate.
Ascorbic acid (vitamin C) accumulation in activated human neutrophils is increased as much as 10-fold above the mM concentrations present in normal neutrophils. Internal concentrations as high as 14 mM are achieved when external vitamin is at physiologic concentration. The mechanism is by oxidation of external vitamin to dehydroascorbic acid, preferential transmembrane translocation of dehydroascorbic acid, and intracellular reduction to ascorbic acid within minutes. These data indicate that vitamin C accumulation is enhanced in activated human neutrophils and that human neutrophils utilize and recycle oxidized external vitamin C under physiologic conditions.
Human hexokinase type I, one of the four isozymes consisting of a single polypeptide chain of about 100 kDa, has been cloned in the pET expression plasmid in a truncated form lacking a segment of 11 apolar amino acids at the N-terminus. The protein has been overexpressed in E.coli and purified to homogeneity. Truncated hexokinase I has been crystallized in the presence of glucose 6-phosphate and of the ATP analogue AMP-PNP. The crystals belong to the monoclinic space group P21, with unit cell constants: a = 84.2 Å, b = 177.7 Å, c = 88.2 Å, β = 90.8° and diffract to 3.0 A resolution.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
The properties of human and rabbit erythrocyte glucose‐6‐phosphate dehydrogenase have been investigated during aging in vivo . The erythrocytes of different age have been prepared by 2 procedures: (a) by inducing a reticulocytosis by phenylhydrazine administration to rabbits and (b) by separating the young and old red cells of man and rabbit according to the different resistence to osmotic lysis.
During aging, human and rabbit glucose‐6‐phosphate dehydrogenase shows a decrease of the catalytic activity and some marked modifications of its properties. Thermolability and NADPase‐dependent inactivation increase; the protective effect of NADP and the affinity for both glucose‐6‐phosphate and NADP substrates are decreased; no modifications have been demonstrated as regards pH optimum and electrophoretic patterns in starch gel. It is possible that these modifications begin after the expulsion of ribosome material.
According to the literature concerning the behaviour of other enzymes in the physiological state and of glucose‐6‐phosphate dehydrogenase of some pathological conditions, it is possible that the modified properties of the enzyme during aging in vivo can be related to some structural modifications of the protein.
N e -(Carboxymethyl)lysine (CML) has been identified as a product of oxidation of glucose adducts to protein in vitro and has been detected in human tissue proteins and urine [Ahmed, M.U., Thorpe, S.R., & Baynes, J.W. (1986) J. Biol. Chem. 261, 4889-4894; Dunn, J.A., Patrick, J.S., Thorpe, S.R. & Baynes, J.W. (1989) Biochemistry 28, 9464-9468]. In the present study we show that CML is also formed in reactions between ascorbate and lysine residues in model compounds and protein in vitro. The formation of CML from ascorbate and lysine proceeds spontaneously at physiological pH and temperature under air. Kinetic studies indicate that oxidation of ascorbic acid to dehydroascorbate is required. Threose and N e -threuloselysine, the Amadori adduct of threose to lysine, were identified in the ascorbate reaction mixtures, suggesting that CML was formed by oxidative cleavage of N e -threuloselysine. Support for this mechanism was obtained by identifying CML as a product of reaction between threose and lysine and by analysis of the relative rates of formation of threuloselysine and CML in reactions of ascorbate or threose with lysine. The detection of CML as a product of reaction of ascorbate and threose with lysine suggests that other sugars, in addition to glucose, may be sources of CML in proteins in vivo. The proposed mechanism for formation of CML from ascorbate is an example of autoxidative glycosylation of protein and suggests that CML may also be an indicator of autoxidative glycosylation of proteins in vivo
Ascorbic acid as well as dehydroascorbic acid were shown to penetrate the platelet membrane in vitro. The percentage uptake by platelets of control guinea pigs (ascorbic acid 0.22 ± 0.014; dehydroascorbic acid 0.54 ± 0.033; p = 0.001) as well as of vitamin C-deficient animals (ascorbic acid 0.40 ± 0.026; dehydroascorbic acid 0.88 ± 0.060; p = 0.001 SEM given) were found to be significantly different. The uptake of ascorbic acid as well as of dehydroascorbic acid were significantly enhanced compared to control animals (p = 0.001). However, the ratios of uptake of dehydroascorbic acid to ascorbic acid were unaffected (control animals 2.30 ± 0.24; vitamin C-deficient animals (2.30 ± 0.17).The uptake of ascorbic acid was found to be fairly dependent on the concentration and was not influenced by ouabain. On the contrary, the uptake of dehydroascorbic acid did not show a linear dependency on the concentration and was inhibited by 10−3 M ouabain. These findings suggest an active transport mechanism for the uptake of dehydroascorbic acid whereas in case of ascorbic acid the facilitated diffusion mechanism is discussed.After incubation of platelets of control and vitamin C-deficient animals with [1-14C]ascorbic acid as well as with [1-14C]dehydroascorbic acid only [1-14C] ascorbic acid was detectable in the platelets and only [1-14C]ascorbic acid was released by the platelets.
Reversed-phase high-performance liquid chromatographic methods for the complete separation of all 4-dimethylaminoazobenzene-4′-sulphonyl chloride and 4-N,N-dimethylaminoazobenzene-4′-thiohydantoin amino acids on the same Supelcosil LC-18 column at room temperature are described. The procedures are simple and reproducible, and the systems are easily interconvertible. The use of a fixed-wavelength detector at 436 nm permits amino acid analysis at levels lower than 1 pmol with a stable baseline.
The leucocyte membrane was found to be permeable to both ascorbic acid and dehydroascorbic acid in either direction. Mean ratios of uptake of dehydroascorbic acid to ascorbic acid by leucocytes of normally fed guinea pigs (1.41 ± 0.16) and of vitamin C-deficient animals (1.91 ± 0.22) are given. No difference in uptake behaviour was observed using leucocytes of animals depleted of vitamin C for up to 16 days. However, ratios for uptake decreased rapidly in leucocytes of such animals being depleted for longer than 20 days, but this was most probably due to inanition and resulting metabolic disorders. After incubation with ascorbic acid, in both groups, only ascorbic acid was found in leucocytes and only ascorbic acid was released during re-incubation. After incubation with dehydroascorbic acid only ascorbic acid was found to be present in leucocyctes of the control group, whereas in the deficient group both, ascorbic acid and dehydroascorbic acid, were found and both compounds were released. After incubation with dehydroascorbic acid in the control group, mainly ascorbic acid was released.
Iron--EDTA was shown to catalyse OH. production from H2O2 and ascorbate by a mechanism largely independent of superoxide. When ascorbate and superoxide were both present, the ascorbate mechanism was more important than superoxide as a source of OH., and would appear to be more significantly biologically.
A modified method has been described for the estimation of ascorbic acid (AA) and dehydroascorbic acid (DHA) in blood and plasma. DHA is practically absent in the blood of normal human beings. On the other hand, diabetic patients have a persistently high blood DHA level. The DHA from diabetic blood has been isolated as the 2,4-dinitrophenylhydrazone derivative and identified by thin-layer chromatography and spectrophotometry.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
A study was made of the interaction of plasma ascorbate and ascorbate free radical (AFR) with exogenously added iron. The quantitative determination of AFR has the advantage that transient increases in ascorbate oxidation can be directly monitored by e.p.r. spectroscopy. An AFR signal was found in the plasma of all donors and was unaffected by superoxide dismutase, catalase and the strong iron chelator deferoxamine. These findings and the rapid decrease in AFR under a nitrogen atmosphere suggest that plasma AFR is probably a result of air auto-oxidation. Iron loading of plasma did not affect the intensity of the AFR signal until the iron concentration approached or exceeded the plasma latent iron-binding capacity. In iron-overloaded plasma, the intensity of the AFR signal increased to about 10 times the normal level before decreasing rapidly to undetectable levels after 15-20 min. Determination of plasma ascorbate showed that the disappearance of AFR was due to a complete loss of the vitamin. When 50 microM-ascorbate was loaded with iron in iso-osmotic phosphate buffer there was an increase in the AFR signal, independent of the iron concentration, which was stable at least for 15 min. Thus the rate of ascorbate loss in the iso-osmotic phosphate buffer was considerably lower than in iron-overloaded plasma. The addition of different iron chelators produced comparable effects on the intensity of the AFR signal in both iron-overloaded plasma and ascorbate solution. These results suggest that the characteristic behaviour of plasma AFR after iron loading is due to its specific iron-binding capacity and to plasma ferroxidase activity. The ferroxidase activity of plasma is important to promote the transfer of Fe2+ into transferrin without a transient ascorbate oxidation. Spin-trapping studies with 5,5-dimethyl-1-pyrroline N-oxide and N-t-butyl-alpha-phenylnitrone revealed that iron-overloaded plasma was unable to produce spin-trap adducts even in the presence of 50-300 microM-hydrogen peroxide or 100 microM-azide. Evidence of OH. radical formation was obtained only after the addition of EDTA. Therefore, iron-overloaded plasma itself does not produce a Fenton reaction and, if ascorbate does indeed have a free-radical-mediated pro-oxidant role, it is not detectable in plasma by spin-trapping experiments.
A number of systems that generate oxygen free radicals catalyze the oxidative modification of proteins. Such modifications
mark enzymes for degradation by cytosolic neutral alkaline proteases. Protein oxidation contributes to the pool of damaged
enzymes, which increases in size during aging and in various pathological states. The age-related increase in amounts of oxidized
protein may reflect the age-dependent accumulation of unrepaired DNA damage that, in a random manner, affects the concentrations
or activities of numerous factors that govern the rates of protein oxidation and the degradation of oxidized protein.
Experiments were performed to evaluate the nonenzymatic reaction between glutathione (GSH) and dehydroascorbic acid (DHA). Though both ascorbic acid and glutathione disulfide (GSSG) are formed from this reaction, previous work has focused almost exclusively on measurements of ascorbic acid. In contrast, there is very little information about the formation of GSSG under the same conditions as those used to produce ascorbic acid. The emphasis on ascorbic acid stems from the fact that a spectrophotometric technique is available for its measurement, whereas 1H-NMR or an amino acid analyzer has been used to measure GSSG. The present experiments use a simple, rapid method for accurately and precisely measuring the concentrations of GSSG in a solution. The spectrophotometric (340 nm) procedure uses NADPH and glutathione reductase; analysis time is very short, many replicate samples can be tested and as little as 0.05-0.1 mM GSSG can be detected. Using this method, it is shown that there is an equimolar production of GSSG and ascorbic acid from GSH and DHA and that the decrease in GSH is stoichiometrically related to the increase in the concentration of GSSG. The present findings provide additional insight into the interaction between the GSH/GSSG redox couple and the ascorbic acid/DHA redox couple.
Hexokinase-degrading activity (HKDA) is associated with hexokinase throughout purification. It is not ubiquitin-dependent. With glucose present, HKDA requires MgATP. It is triggered by removal (or ATP-dependent consumption) of glucose, and masked when glucose is restored. PCMB, TLCK and TPCK suppress hexokinase and activate HKDA. Glucose removal and active-site blockage appear to induce HKDA.
Rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) contains 21 cysteine residues. On the basis of the amino acid sequence of the enzyme, these are predicted to be distributed among 14 peptides produced by tryptic digestion. Ten of these peptides, containing cysteine residues derivatized by reaction with the specific sulfhydryl reagent 2-bromoacetamido-4-nitrophenol have been identified in HPLC peptide maps; the four missing peptides are predicted to be relatively large and hydrophobic in character, properties that may have prevented their detection under the present conditions. The sequences encompassed by the 10 identified peptides include 12 of the 21 cysteine residues in the enzyme. The relative reactivity of these sulfhydryl groups with 2-bromoacetamido-4-nitrophenol has been assessed, and is in general accord with what might be predicted on the basis of their accessibility in the previously proposed structure for this enzyme. The effect of various ligands on reactivity of identified sulfhydryl groups has been determined; unique patterns of altered reactivity, resulting from ligand-induced conformational changes, have been observed. Biphasic effects were observed with increasing concentrations of either glucose 6-phosphate (Glc-6-P) or Pi. In both cases, decreased reactivity of sulfhydryls in the N-terminal half of the molecule was observed at low concentrations of the ligand, while further increase in ligand concentration resulted in decreased reactivity of sulfhydryl groups in the C-terminal half. In contrast, sulfhydryls in both N- and C-terminal halves were protected concomitantly by increasing concentrations of Glc. These results are consistent with previous studies that indicated (a) the existence of two sites for binding of Glc-6-P or Pi, a high affinity site in the N-terminal half and a site with lower affinity in the C-terminal half of the brain hexokinase molecule, and (b) binding of Glc to a single site located in the C-terminal half but evoking conformational effects throughout the molecule; the glucose analog, N-acetylglucosamine, previously shown to have more limited effects on conformation, affected reactivity of sulfhydryl groups only in the C-terminal half of the molecule. As reflected by effects on reactivity of sulfhydryl groups, conformational changes induced by binding of nucleotides depends markedly on the specific nature of the purine or pyrimidine base as well as the length and chelation status of the polyphosphate side chain. These results focus attention on specific regions of the molecule (the immediate environment of the sulfhydryl groups) that are affected by the binding of these ligands.
Mg2(+)-chelates of several nucleoside triphosphates were shown to increase the inactivation of rat brain hexokinase (ATP:D-hexose-6-phosphotransferase, EC 2.7.1.1) by 0.6 M guanidine hydrochloride, with ATP-Mg2+ having the greatest effect; unchelated forms did not significantly affect inactivation. Since catalytic activity has been associated with the C-terminal half of the molecule, these results were interpreted as indicating a destabilization of this C-terminal region by binding of these chelates to the substrate nucleotide sites, with the particular effectiveness of ATP-Mg2+ reflecting the specificity for this species as a phosphoryl donor. These compounds were also shown to bind to the N-terminal half of the enzyme, as judged by their ability to protect against denaturation by guanidine hydrochloride and subsequent digestion with trypsin. Both free and Mg2(+)-chelated forms afforded protection, with the unchelated nucleotides being most effective; a preference for ATP was seen only with the chelated forms. Thus, it was concluded that the N-terminal half of hexokinase contains a relatively nonspecific nucleotide binding site, distinct from the substrate nucleotide site previously shown to reside in the C-terminal half. On the basis of this same ability to protect the N-terminal half against denaturation and proteolysis, several other polyanionic ligands were shown to bind to this region of the molecule. These included inorganic phosphate, its analogs, sulfate and arsenate, and its homologs, pyrophosphate and tripolyphosphate. All of these anionic ligands were also shown to antagonize inhibition by the glucose 6-phosphate (Glc-6-P) analog, 1,5-anhydroglucitol 6-phosphate. The allosteric site for binding of Glc-6-P has previously been shown to reside in the N-terminal half of the molecule, and it is suggested that the antagonism of inhibition by Glc-6-P (or its analog) by these anionic ligands results from interaction with an anion binding site for which the 6-phosphate group of inhibitory hexose 6-phosphates must compete. A model depicting possible relationships between ligand binding sites on brain hexokinase, and how their interactions might lead to observed regulatory properties, is developed based on these and previous studies of ligand binding as well as evidence that mammalian hexokinases (Mr 100,000) have evolved by duplication and fusion of a gene coding for an ancestral hexokinase with Mr 50,000 and which, like the mammalian enzyme, was sensitive to inhibition by Glc-6-P.
A simple and fast reversed-phase high-performance liquid chromatographic method has been developed for the complete separation of 35 dimethylaminoazobenzene sulfonyl (DABS)-amino acids and by-products. This method allows simultaneous determination of primary and secondary amino acids which can be present in protein and peptide hydrolysates and also detects the presence of cysteic acid, S-sulfocysteine, hydroxyproline, taurine, norleucine, cystine, and delta-hydroxylysine. The precolumn derivatization of amino acids with dimethylaminoazobenzene sulfonyl chloride (DABS-Cl) is simple and quick (10 min at 70 degrees C) and allows the complete reaction of primary and secondary amino acids. The separation of the compounds under investigation is achieved in 25 min using a reversed-phase 3-microns Supelcosil LC-18 column at room temperature. The versatility of the proposed method is documented by amino acid determination on protein samples obtained using different hydrolysis techniques (HCl, methane-sulfonic acid, and NaOH), with attention given to the detection of tryptophan in protein samples with high sugar concentration. Furthermore, we have reported the experimental conditions necessary to apply this method to the amino acid analysis of very low amount of proteins (1 to 5 micrograms) electroeluted from a stained band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The stability of DABS-derivatives, the short time of analysis, the high reproducibility and sensitivity of the system, and the complete resolution of all compounds of interest make this method suitable for routine analysis. Furthermore, we have also developed a fast reversed-phase high-performance liquid chromatographic method for the complete separation of dimethylaminoazobenzene thiohydantoin (DABTH)-amino acids. The separation of the compounds under investigation is obtained, at room temperature, in less than 18 min using a reversed-phase Supelcosil LC-18 DB column, 3-micron particles, and also allows the complete separation of DABTH-Ile, DABTH-Leu, and DABTH-Norleu. The short time of analysis, together with the high reproducibility of the system and its sensitivity at picomole levels, make this method very suitable for the identification of DABTH-amino acids released during microsequencing studies of proteins and peptides with the dimethylaminoazobenzene isothiocyanate reagent. In addition, we have shown that it is possible to obtain complete separation of DABTH-amino acids also under isocratic conditions.(ABSTRACT TRUNCATED AT 400 WORDS)
In recent years it has become clear that various free radicals and related oxidants can cause serious damage to intracellular enzymes and other proteins. Several investigators have shown that in extreme cases this can result in an accumulation of oxidatively damaged proteins as useless cellular debris. In other instances, proteins may undergo scission reactions with certain radicals/oxidants, resulting in the direct formation of potentially toxic peptide fragments. Data has also been gathered (recently) demonstrating that various intracellular proteolytic enzymes or systems can recognize, and preferentially degrade, oxidatively damaged proteins (to amino acids). In this hypothesis paper I present evidence to suggest that proteolytic systems (of proteinases, proteases, and peptidases) may function to prevent the formation or accumulation of oxidatively damaged protein aggregates. Proteolytic systems can also preferentially degrade peptide fragments and may thus prevent a wide variety of potentially toxic consequences. I propose that many proteolytic enzymes may be important components of overall antioxidant defenses because they can act to ameliorate the consequences of oxidative damage. A modified terminology is suggested in which the primary antioxidants are such agents as vitamin E, beta-carotene, and uric acid and such enzymes as superoxide dismutase, glutathione peroxidase, and DT-diaphorase. In this classification scheme, proteolytic systems, DNA repair systems, and certain lipolytic enzymes would be considered as secondary antioxidant defenses. As secondary antioxidant defenses, proteolytic systems may be particularly important in times of high oxidative stress, during periods of (primary) antioxidant insufficiency, or with advancing age.
A glucose analog, N-(bromoacetyl)-D-glucosamine (GlcNBrAc), previously used to label the glucose binding sites of rat muscle Type II and bovine brain Type I hexokinases, also inactivates rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) with pseudo-first-order kinetics. Inactivation occurs predominantly via a "specific" pathway involving formation of a complex between hexokinase and GlcNBrAc, but significant nonspecific (i.e., without prior complex formation) inactivation also occurs, and equations to describe this behavior are derived. Inactivation is dependent on deprotonation of a residue with an alkaline pKa, consistent with the modified residue being a sulfhydryl group as reported to be the case with the hexokinase of bovine brain. The affinity label modifies three residues (per molecule of enzyme) at indistinguishable rates, but only one of these residues appears to be critical for activity. Amino acid analysis of the modified enzyme indicates derivatization of three cysteine residues; there was no indication of modification of other residues potentially reactive with haloacetyl derivatives. Kinetic analysis and effects of protective ligands were consistent with location of the critical sulfhydryl at the glucose binding site. Peptide mapping techniques permitted localization of the critical residue, and thus the glucose binding site, in a 40-kDa domain at the C-terminus of the enzyme. This is the same domain recently shown to include the ATP binding site. Thus, catalytic function is assigned to the C-terminal domain of rat brain hexokinase.
Exposure of cellular membranes to dehydroascorbic acid can result in a loss of membrane integrity. Renal brush border or basolateral membrane vesicles pre-incubated with dehydroascorbic acid demonstrate a decrease in initial transport rates of D-glucose and a loss of intravesicular volume. The activity of brush border membrane specific leucine aminopeptidase is increased in vesiculated membrane preparations following exposure of the vesicles to either dehydroascorbic acid or Triton X-100. Erythrocytes in isotonic buffer with dehydroascorbic acid lose membrane integrity as demonstrated by a release of hemoglobin.
Using a novel high performance liquid chromatography (HPLC) determination of ascorbic acid and dehydroascorbic acid, we have measured the relative amounts of ascorbate and dehydroascorbate in 20 normal controls and in paired sera and synovial fluid from 13 patients with rheumatoid arthritis (RA). In complete contrast to previous published findings we were able to detect dehydroascorbate in normal human sera (12.0 +/- 3.7 mumol/l), while the mean and range of ascorbate measured in normals was 69.6 +/- 20.6 mumol/l. These findings were completely reversed in rheumatoid sera (21.8 +/- 8.6 mumol/l and 5.1 +/- 5.0 mumol/l for dehydroascorbate and ascorbic acid respectively). In several rheumatoid sera no ascorbate could be detected. In paired synovial fluid and serum samples, there was always more dehydroascorbate detected in synovial fluids than in the corresponding sera (p less than 0.01). The data suggests that the reduced level of ascorbate and increased level of dehydroascorbate may be a reflection of the increased antioxidant and free-radical scavenging activity of the vitamin in RA, especially within the inflamed joint.
The binding of glucose and glucose-6-P by pure rat brain hexokinase has been studied by using an ultrafiltration procedure [H. Paulus (1969) Anal. Biochem. 32, 91–100]. Each mole of enzyme (molecular weight 98,000) binds 1 mole of glucose or 1 mole of glucose-6-P. The dissociation constant for the enzyme-glucose complex (0.04 mm) is in excellent agreement with the kinetically determined Km for this substrate. The dissociation constant for the enzyme-glucose-6-P complex was estimated to be 1.3 μm, substantially lower than values of 7–8 μm obtained by alternative methods. This discrepancy appears to be due to retardation of the passage of the charged glucose-6-P through the ultrafiltration membrane, resulting in an effective increase in the ligand concentration at the membrane surface and thereby a decrease in the apparent dissociation constant. No appreciable retardation of the passage of the uncharged glucose molecule was observed.The binding of glucose-6-P (but not glucose) is prevented in the presence of Pi. This is in accord with a previously suggested model in which binding of Pi is considered to stabilize the enzyme in a conformation having little, if any, affinity for glucose-6-P.Serine was found as a C-terminal amino acid. The method used would not have detected C-terminal proline or tryptophan residues, and thus these cannot be excluded by the present experiments. However, in view of other results indicating that rat brain hexokinase consists of a single polypeptide chain, it seems probable that serine is indeed the only C-terminal amino acid in the molecule.
Rat brain hexokinase (ATP: d-hexose-6-phosphotransferase, EC 2.7.1.1) is rapidly inactivated by reaction with 5,5′-dithiobis-(2-nitrobenzoate). The inactivation follows monophasic first-order kinetics in either the absence of ligands (k = 0.641 min−1 at 25 °C) or in the presence of saturating levels of ATP (free or complexed with Mg2+) or P1; the inactivation rate is slightly increased ( min −1) in the presence of ATP or P1. In contrast, glucose and glucose-6-P markedly decrease the inactivation rate; inactivation in the presence of these ligands is biphasic, with two first-order rates ( min−1 and 0.01 min−1) being distinguishable.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
In contrast to normal subjects diabetic patients and very low plasma ascorbic acid and significantly high (p less than 0.001) dehydroascorbic acid irrespective of age, sex, duration of the disease, type of treatment, and glycemic control. However, there was no significant difference between the mean leukocyte ascorbate concentrations of the two populations. The in vitro rates of dehydroascorbate reduction in the hemolysate and the erythrocyte reduced glutathione levels and the glucose-6-phosphate dehydrogenase activities, which regulate the dehydroascorbate reduction, were similar in normal and diabetic subjects. The turnover of ascorbic acid was higher in the diabetics than that in the normal volunteers. Experiments with diabetic rats indicated that the increased turnover of ascorbic acid was probably due to increased oxidation of ascorbate to dehydroascorbate in tissue mitochondria. Ascorbic acid supplementation at a dose of 500 mg per day for a brief period of 15 days resulted in an increase in the plasma ascorbate level temporarily, but it did not lower the blood glucose level of the diabetic patients.
An analogue of the substrate glucose, N-(bromoacetyl)-D-glucosamine (GlcNBrAc) inactivates bovine brain mitochondrial hexokinase completely and irreversibly in a pseudo-first-order fashion at pH 8.5 and 22 degrees C. The rate of inactivation of hexokinase by this reagent does not increase linearly with increasing reagent concentration but exhibits an apparent saturation effect, suggesting the formation of a reversible complex between the enzyme and the reagent prior to the inactivation step. The pH dependence of the rate of inactivation suggests that a group on the enzyme with pKa = 9.1 is being modified by this reagent. At pH 8.0 the rate of inactivation by this reagent is very slow, and it can be shown to be a competitive inhibitor of the hexokinase reaction with respect to the substrate glucose. The substrates glucose and ATP strongly protected the enzyme against the inactivation reaction. The inactivation of the enzyme was found to be accompanied by the alkylation of two sulfhydryl residues as shown by the formation of approximately 2 mol of S-(carboxymethyl)-cysteine/mol of inactivated enzyme. Treatment of the enzyme with 14C-labeled reagent results in the incorporation of approximately 2 mol of reagent/mol of inactivated enzyme. However, the enzyme protected by glucose still shows the incorporation of approximately 1 mol of the labeled reagent/mol of the enzyme. From a tryptic digest of the enzyme inactivated by this reagent, two labeled peptides were obtained, one of which was absent if the labeling reaction was carried out in presence of glucose. These results indicate that the affinity reagent reacts with two thiols, only one of which is crucial for the activity of the enzyme and is located in the region of its active site.
Exposure of rabbit reticulocytes to Fe(II)/ascorbate induced a pronounced decay in hexokinase activity. In reticulocytes, this enzyme is present in at least three different molecular forms, Ia, Ia* and Ib, sub-types of hexokinase type I, which show different intracellular distribution. Hexokinase Ia and Ib are soluble, whereas hexokinase Ia* is almost entirely bound to the mitochondria. Anion exchange chromatography of hexokinase from intact reticulocytes exposed to Fe(II)/ascorbate revealed a selective inactivation of forms Ia and Ib, whereas the form Ia* did not show any decay. Binding to the mitochondrial membrane seems to be responsible for the observed resistance of the form Ia* to the inactivation elicited by Fe(II)/ascorbate. Indeed, by using a cell-free system in which hexokinase Ia* was solubilized using Triton X-100, the decay in hexokinase activity induced by iron/ascorbate involved all three enzymatic forms.
Dehydroascorbic acid is generated in plants and animal cells by oxidation of ascorbic acid. The reaction is believed to occur by the one-electron oxidation of ascorbic acid to semidehydroascorbate radical followed by disproportionation to dehydroascorbic acid and ascorbic acid. Semidehydroascorbic acid may recycle to ascorbic acid catalyzed by membrane-bound NADH-semidehydroscorbate reductase. However, disproportionation of the free radical occurs at a rapid rate, 10(5) M-1 s-1, accounting for measurable cellular levels of dehydroascorbate. Dehydroascorbate reductase, studied earlier and more extensively in plants, is now recognized as the intrinsic activity of thioltransferases (glutaredoxins) and protein disulfide isomerase in animal cells. These enzymes catalyze the glutathione-dependent two-electron regeneration of ascorbic acid. The importance of the latter route of ascorbic acid renewal was seen in studies of GSH-deficient rodents (Meister, A. (1992) Biochem. Pharmacol. 44, 1905-1915). GSH deficiency in newborn animals resulted in decreased tissue ascorbic acid and increased dehydroascorbate-to-ascorbate ratios. Administration of ascorbic acid daily to GSH-deficient animals decreased animal mortality and cell damage from oxygen stress. A cellular role is proposed for dehydroascorbate in the oxidation of nascent protein dithiols to disulfides catalyzed in the endoplasmic reticulum compartment by protein disulfide isomerase.
Rabbit red blood cell hexokinase (EC 2.7.1.1) has been shown to be inactivated in vitro by incubating intact erythrocytes in the presence of an oxygen-radical-generating system represented by ascorbate and iron. It was interesting to note that among the glycolytic enzymes, only hexokinase was found to be susceptible to the action of oxygen radicals, suggesting that the loss of activity of this enzyme may be one of the first signals of cellular damage in rabbit red blood cells. This statement is supported by the fact that, under the experimental conditions used, we did not observe any significant plasma membrane lipid peroxidation nor intracellular proteolysis. Furthermore, mature erythrocytes are unable to synthesize hexokinase as well as other proteins de novo; therefore, the inactivation of this enzyme, which is both the first and one of the rate-limiting enzymes of the glycolytic pathway, could play an important role in determining metabolic impairment of red blood cells, with possible physiological implications. We also investigated the effect of various radical scavengers and antioxidants (glucose, vitamin E, dithiothreitol, flavonoids) which are able to influence the inactivation of hexokinase activity to different extents. Finally, under the experimental conditions used (90 min of incubation at 37 degrees C), we did not observe any difference in the hemolysis of rabbit red blood cells incubated in the presence or absence of ascorbate and iron (hemolysis was about 1% after 90 min of incubation), suggesting that the system used was able to furnish information about the cellular damage produced by oxygen radicals without provoking cell lysis.
