Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:EXPT00568 (ascorbate)
 23,072 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The interaction of recombinant ascorbate peroxidase (APX) with its physiological substrate, ascorbate, has been studied by electronic and NMR spectroscopies, and by phenylhydrazine-modification experiments. The binding interaction for the cyanide-bound derivative (APX-CN) is consistent with a 1:1 stoichiometry and is characterised by an equilibrium dissociation binding constant. Kd, of 11.6 +/- 0.4 microM (pH 7.002, mu = 0.10 M, 25.0 degrees C). Individual distances between the non-exchangeable substrate protons of APX-CN and the haem iron were determined by paramagnetic-relaxation NMR measurements, and the data indicate that the ascorbate binds 0.90-1.12 nm from the haem iron. The reaction of ferric APX with the suicide substrate phenylhydrazine yields predominantly (60%) a covalent haem adduct which is modified at the C20 carbon, indicating that substrate binding and oxidation is close to the exposed C20 position of the haem, as observed for other classical peroxidases. Molecular-modelling studies, using the NNM-derived distance restraints in conjunction with the crystal structure of the enzyme [Patterson, W. R. & Poulos, T. L. (1995) Biochemistry 34, 4331-4341], are consistent with binding of the substrate close to the C20 position and a possible functional role for alanine 134 (proline in other class-III peroxidases) is implicated.
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PMID:Chemical, spectroscopic and structural investigation of the substrate-binding site in ascorbate peroxidase. 934 87

Heat-shock protein 90 (Hsp 90) has been implicated in both protection against oxidative inactivation and inhibition of the multicatalytic proteinase (MCP, also known as 20 S proteasome). We report here that the protective and inhibitory effects of Hsp 90 depend on the activation state of the proteasome. Hsp 90 (and also alpha-crystallin) inhibits the N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activity (Cbz=benzyloxycarbonyl; MCA=7-amido-4-methylcoumarin) when the rat liver MCP is in its latent form, but no inhibitory effects are observed when the MCP is in its active form. Metal-catalysed oxidation of the active MCP inactivates the Ala-Ala-Phe-MCA-hydrolysing (chymotrypsin-like), N-Boc-Leu-Ser-Thr-Arg-MCA-hydrolysing (trypsin-like; Boc=t-butyloxycarbonyl), N-Cbz-Leu-Leu-Glu-beta-naphthylamine-hydrolysing (peptidylglutamyl-peptide hydrolase) and N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activities, whereas these activities are actually increased when the MCP is in its latent form. Hsp 90 protects against oxidative inactivation of the trypsin-like and N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activities of the MCP active form, and alpha-crystallin protects the trypsin-like activity. The specificity of the Hsp 90-mediated protection was assessed by a quantitative analysis of the two-dimensional electrophoretic pattern of MCP subunits before and after oxidation of the MCP, in the presence or absence of Hsp 90. Treatment of the FAO hepatoma cell line with iron and ascorbate was found to inactivate the MCP. Hsp 90 overexpression obtained by challenging the cells with iron was associated with a decreased susceptibility to oxidative inactivation of the MCP trypsin-like activity. Depletion of Hsp 90 by using antisense oligonucleotides resulted in an increased susceptibility to oxidative inactivation of the MCP trypsin-like activity, providing evidence for the physiological relevance of Hsp 90-mediated protection of the MCP.
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PMID:Protection from oxidative inactivation of the 20S proteasome by heat-shock protein 90. 965 82

Guinea-pigs were maintained for 5 weeks on a diet containing three different concentrations of vitamin C: a) traces (none added), b) medium (0.05% w/w) and high (0.5% w/w). Twenty-four hours before killing the animals received one i.p. dose of 3 g ethanol per kg body weight (a model of short-term acute intoxication). In a parallel experiment which lasted 5 weeks, the animals were treated every week with two i.p. doses of 1 g ethanol per kg body weight followed by the final acute intoxication (3g ethanol/kg) (a model of long-term chronic alcoholization). In both experiments, the guinea-pigs with the highest tissue concentration of vitamin C proved to have significantly decreased residual levels of ethanol and acetaldehyde in the liver and the brain, a decreased activity of alanine- and aspartate aminoacyl transferases in the serum and decreased contents of triacylglycerols and cholesterol in the serum and liver in comparison with the vitamin C-unsupplemented group. The regression curve expressing vitamin C levels versus residual ethanol and acetaldehyde concentrations in the liver confirmed the highly significant negative correlation between them. Administration of the guinea-pigs with large amounts of vitamin C appears to accelerate ethanol and acetaldehyde metabolism and reduce some of their adverse health effects.
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PMID:Influence of vitamin C status on ethanol metabolism in guinea-pigs. 970 98

Oxidation-reduction properties of methylglyoxal-modified protein in relation to free radical generation were investigated. Glycation of bovine serum albumin by methylglyoxal generated the protein-bound free radical, probably the cation radical of the cross-linked Schiff base, as observed in the reaction of methylglyoxal with L-alanine (Yim, H.-S., Kang, S.-O., Hah, Y. C., Chock, P. B., and Yim, M. B. (1995) J. Biol. Chem. 270, 28228-28233) or with Nalpha-acetyl-L-lysine. The glycated bovine serum albumin showed increased electrophoretic mobility suggesting that the basic residues, such as lysine, were modified by methylglyoxal. The glycated protein reduced ferricytochrome c to ferrocytochrome c in the absence of oxygen or added metal ions. This reduction of cytochrome c was accompanied by a large increase in the amplitude of the electron paramagnetic resonance signal originated from the protein-bound free radical. In addition, the glycated protein catalyzed the oxidation of ascorbate in the presence of oxygen, whereas the protein free radical signal disappeared. These results indicate that glycation of protein generates active centers for catalyzing one-electron oxidation-reduction reactions. This active center, which exhibits enzyme-like characteristic, was suggested to be the cross-linked Schiff base/the cross-linked Schiff base radical cation of the protein. It mimics the characteristics of the metal-catalyzed oxidation system. The glycated bovine serum albumin cross-linked further to the cytochrome c in the absence of methylglyoxal. The cross-linked cytochrome c maintains its oxidation-reduction properties. These results together indicate that glycated proteins accumulated in vivo provide stable active sites for catalyzing the formation of free radicals.
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PMID:Oxidation-reduction properties of methylglyoxal-modified protein in relation to free radical generation. 973 92

The amino acid utilization between human promyelocytic leukemia (HL-60), human oral squamous carcinoma (HSC-2, HSC-4, NA), human salivary gland tumor (HSG) and rat neuron cells (PC-12) were compared, using amino acid analyzer. All these cells consumed four essential amino acids (valine, methionine, isoleucine, leucine), glutamine and arginine, whereas they produced glycine, alanine and ammonia, without significantly affecting threonine, tyrosine, phenylalanine, histidine or lysine concentration. Serine and glutamine utilization varied considerably from cell to cell. HL-60 cells consumed serine and arginine at much higher rates than other cells. Serine depletion accumulated the G1 arrested cells, and produced increasing numbers of the apoptotic cells. Supplementation of serine significantly extended the period of logarithmic cell growth. During apoptosis induction of HL-60 cells by dopamine, sodium ascorbate or sodium 5,6-benzylidene-L-ascorbate, the oxidation of methionine to methionine sulfoxide was enhanced, but the consumption of serine, glutamine and arginine was reduced. In the presence of HL-60 cells, the methionine oxidation was significantly inhibited, suggesting the antioxidant action of the cells. These present study suggests the importance of re-evaluation of culture condition for each cell lines.
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PMID:Amino acid utilization during cell growth and apoptosis induction. 989 82

Human promyelocytic leukemia (HL-60), human squamous carcinoma (HSC-2, HSC-4, NA) and rat pheochromocytoma (PC-12) cell lines consumed nonpolar (Leu, Ile, Val, Cys, Met, Phe), neutral polar (Gln, Ser, Thr, Tyr) and basic polar amino acids (Arg, Lys, His), whereas they produced nonpolar (Gly, Pro, Ala) and acidic polar amino acids (Glu). The consumption rate of Ser and Arg by HL-60 cells was significantly higher than that of other cell lines. During apoptosis of HL-60 cells induced by either sodium ascorbate, sodium 5,6-benzylidene-L-ascorbate (SBA) or dopamine, the consumption of nonpolar and polar amino acids (neutral or acidic) generally declined, except for Cys, Met and Arg, whereas the production of Gly and Glu was slightly increased. Since the intracellular concentration of cGMP was not significantly changed before and after ascorbate treatment, nitric oxide might not be involved in the ascorbate-induced apoptosis. The present data demonstrates that consumption rate of nonpolar and polar amino acid, whether neutral, acidic or basic, was reduced almost evenly during the apoptosis induction. This suggests that apoptosis-associated changes in the amino acid utilization might not be significantly affected by ATP depletion, which might be caused by mitochondrial dysfunction.
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PMID:Amino acid utilization during apoptosis in HL-60 cells. 1022 62

The final step of ethylene biosynthesis in plants is catalyzed by the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACCO). In addition to ACC, Fe(II), O2, CO2, and ascorbate are required for in vitro enzyme activity. Direct evidence for the role of the Fe(II) center in the recombinant avocado ACCO has now been obtained through formation of enzyme.(substrate or cofactor).NO complexes. These NO adducts convert the normally EPR-silent ACCO complexes into EPR-active species with structural properties similar to those of the corresponding O2 complexes. It is shown here that the ternary Fe(II)ACCO.ACC.NO complex is readily formed, but no Fe(II)ACCO.ascorbate.NO complex could be observed, suggesting that ascorbate and NO are mutually exclusive in the active site. The binding modes of ACC and the structural analog alanine specifically labeled with 15N or 17O were examined by using Q-band electron nuclear double resonance (ENDOR). The data indicate that these molecules bind directly to the iron through both the alpha-amino and alpha-carboxylate groups. These observations are inconsistent with the currently favored mechanism for ACCO, in which it is proposed that both ascorbate and O2 bind to the iron as a step in O2 activation. We propose a different mechanism in which the iron serves instead to simultaneously bind ACC and O2, thereby fixing their relative orientations and promoting electron transfer between them to initiate catalysis.
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PMID:Role of the nonheme Fe(II) center in the biosynthesis of the plant hormone ethylene. 1039 20

The structure and property of cross-linked amino acids and proteins produced by a three- carbon alpha-dicarbonyl methylglyoxal in glycation reaction were investigated. Our results showed that these reactions generated yellow fluorescent products and several free radical species. From the reaction with alanine, three types of free radicals were identified by EPR spectroscopy: 1) the cross-linked radical cation, methylglyoxal diaklylimine cation radical; 2) the methylglyoxal radical anion as the counterion; 3) the superoxide radical anion produced only in the presence of oxygen. Glycation of bovine serum albumin by methylglyoxal also generated the protein-bound, cross-linked free radical, probably the cation radical of the cross-linked Schiff base as observed with alanine. The glycated protein reduced ferricytochrome c to ferrocytochrome c in the absence of oxygen or added metal ions. This reduction of cytochrome c was accompanied by a large increase in the amplitude of the electron paramagnetic resonance signal originated from the protein-bound free radical. In addition, the glycated protein catalyzed the oxidation of ascorbate in the presence of oxygen while the protein-free radical signal disappeared. These results indicate that glycation of protein generates active centers for catalyzing one-electron oxidation-reduction reactions. This active center, which exhibits enzyme-like character, was suggested to be the cross-linked Schiff base/the cross-linked Schiff base radical cation of the protein. It mimics the characteristics of metal-catalyzed oxidation system. These results together indicate that glycated proteins accumulated in vivo provide stable active-sites for catalyzing the formation of free radicals.
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PMID:Enzyme-like activity of glycated cross-linked proteins in free radical generation. 1086 38

The cytotoxic effects of ginkgetin, a natural biflavone isolated from Selaginella moellendorffii Hieron, were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in three different human cell lines: ovarian adenocarcinoma (OVCAR-3), cervical carcinoma (HeLa) and foreskin fibroblast (FS-5). The concentrations of ginkgetin required to induce 50% death (EC50) in OVCAR-3, HeLa, and FS-5 were 3.0, 5.2, and 8.3 microg/ml, respectively. Morphological changes in cells and their nuclei, DNA fragmentation with a characteristic pattern of inter-nucleosomal ladder, and double-stranded DNA breaks were detected following treatment with 3 microg/ml of this biflavone for 24 h. Incubation with 5 microg/ml ginkgetin led to increased intracellular levels of hydrogen peroxide as early as 30 min. The cytotoxicity of ginkgetin was partially inhibited by pretreating cells with vitamin C, vitamin E or catalase. Catalase not only afforded the best protective effect among three antioxidants, but also reduced both the DNA fragmentation and double-stranded DNA breakage induced by ginkgetin. Moreover, the involvement of caspase(s) in ginkgetin-induced apoptosis was demonstrated by the activation of caspase 3 after drug treatment and the suppression of cell death by a broad-spectrum caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk). However, the protective effects of z-VAD-fmk and catalase were not additive. Taken together, our results indicated that the apoptosis induced by ginkgetin (especially at 5 microg/ml) is mediated mainly through the activation of caspase(s) by the hydrogen peroxide generated possibly through autooxidation of this biflavone.
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PMID:Studies on the cytotoxic mechanisms of ginkgetin in a human ovarian adenocarcinoma cell line. 1093 37

The effects of two oxygen radical generating systems (H2O2 and ascorbate/Fe+2) on erythrocyte deformability, osmotic fragility, lipid peroxidation and protein degradation were studied. Incubation of erythrocytes with different concentrations of H202 (5-20 mM) or ascorbate/Fe+2 (10/0.1-40/0.4 mM) caused a loss of deformability and increased osmotic fragility. The loss of deformability has occurred in a dose-dependent fashion and was proportional to the extent of malonyldialdehyde (an indicator of lipid peroxidation) and alanine production (an indicator of protein degradation). Prior exposure of the erythrocytes to carbon monoxide (known to inhibit heme-protein degradation) prevented almost completely the loss in deformability caused by H2O2, indicating that the loss in deformability was due mainly to protein degradation rather than to lipid peroxidation. Erythrocytes incubated with either of the two systems have also shown morphologic changes characterized by a dose-dependent increase in echinocyte formation. The results indicate the importance of oxidatively damaged proteins in compromising the rheologic behaviour of the erythrocytes, particularly when the free radicals are involved.
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PMID:Exposure of human erythrocytes to oxygen radicals causes loss of deformability, increased osmotic fragility, lipid peroxidation and protein degradation. 1121 9


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