Thiourea protects against copper-induced oxidative damage by formation of a redox-inactive thiourea-copper complex

Although thiourea has been used widely to study the role of hydroxyl radicals in metal-mediated biological damage, it is not a specific hydroxyl radical scavenger and may also exert antioxidant effects unrelated to hydroxyl radical scavenging. Thus, we investigated the effects of thiourea on copper-induced oxidative damage to bovine serum albumin (1 mg/ml) in three different copper-containing systems: Cu(II)/ascorbate, Cu(II)/H₂O₂, and Cu(II)/H₂O₂/ascorbate [Cu(II), 0.1 mM; ascorbate, 1 mM; H₂O₂, 1 mM]. Oxidative damage to albumin was measured as protein carbonyl formation. Thiourea (0.1–10 mM) provided marked and dose-dependent protection against protein oxidation in all three copper-containing systems. In contrast, only minor protection was observed with dimethyl sulfoxide and mannitol, even at concentrations as high as 100 mM. Strong protection was also observed with dimethylthiourea, but not with urea or dimethylurea. Thiourea also significantly inhibited copper-catalyzed oxidation of ascorbate, and competed effectively with histidine and 1,10-phenanthroline for binding of cuprous, but not cupric, copper, as demonstrated by both UV-visible and low temperature electron spin resonance measurements. We conclude that the protection by thiourea against copper-mediated protein oxidation is not through scavenging of hydroxyl radicals, but rather through the chelation of cuprous copper and the formation of a redox-inactive thiourea-copper complex.     

Leukotriene B4, C4, D4 and E4 inactivation by hydroxyl radicals

Leukotriene B4 chemotactic activity and leukotriene C4, D4 and E4 slow reacting substance activity were rapidly decreased by hydroxyl radicals generated by two different iron-supplemented acetaldehyde-xanthine oxidase systems. At low Fe2+, leukotriene inactivation was inhibited by catalase, superoxide dismutase, mannitol and ethanol, suggesting involvement of hydroxyl radicals generated by the iron-catalyzed interaction of superoxide and H2O2 (Haber-Weiss reaction). Leukotriene inactivation increased at high Fe2+ concentrations, but was no longer inhibitable by superoxide dismutase, suggesting that inactivation resulted from a direct interaction between H2O2 and Fe2+ to form hydroxyl radicals (Fenton reaction). The inactivation of leukotrienes by hydroxyl radicals suggests that oxygen metabolites generated by phagocytes may play a role in modulating leukotriene activity.     

Radical scavenging activity and oxidative modification of citrus dehydrin.

Dehydrins are ubiquitous proteins produced by plants in response to water stress. Their functions, however, are not fully understood. The overexpression of Citrus unshiu Marcov. dehydrin (CuCOR19) enhanced cold tolerance in transgenic plants by reducing lipid peroxidation promoted by cold stress, suggesting that the CuCOR19 protein directly scavenges radicals. In this paper, we report the radical scavenging activity and oxidative modification of CuCOR19. The hydroxyl radical generated by the Fe2+/H2O2 system and peroxyl radical generated from 2, 2'-azobis (2-amidinopropane) (AAPH) were scavenged by CuCOR19, but hydrogen peroxide and superoxide were not. The scavenging activity for the hydroxyl radical and peroxyl radical of CuCOR19 was more potent than that of mannitol, and approximately equal to that of serum albumin, which is known as an antioxidative protein in mammals. CuCOR19 was degraded by the hydroxyl radical and peroxyl radical in a time- and dose-dependent manner. Mannitol and thiourea inhibited the degradation. Analysis of the amino acid composition of CuCOR19 indicated that glycine, histidine, and lysine, which are major residues in many dehydrins, were targeted by the hydroxyl radical. These results suggest that CuCOR19 is a radical scavenging protein, and may reduce oxidative damage induced by water stress in plants.     

Increased resistance to oxidative stress in transgenic plants by targeting mannitol biosynthesis to chloroplasts.

To investigate the potential role of a polyol, mannitol, in oxidative stress protection, a bacterial mannitol-1-phosphate dehydrogenase gene was targeted to chloroplasts by the addition of an amino-terminal transit peptide. Transgenic tobacco (Nicotiana tabacum) lines accumulate mannitol at concentrations ranging from 2.5 to 7 mumol/g fresh weight. Line BS1-31 accumulated approximately 100 mM mannitol in chloroplasts and was identical to the wild type in phenotype and photosynthetic performance. The presence of mannitol in chloroplasts resulted in an increased resistance to methyl viologen (MV)-induced oxidative stress, documented by the increased retention of chlorophyll in transgenic leaf tissue following MV treatment. In the presence of MV, isolated mesophyll cells of BS1-31 exhibited higher CO2 fixation than the wild type. When the hydroxyl radical probe dimethyl sulfoxide was introduced into cells, the initial formation rate of methane sulfinic acid was significantly lower in cells containing mannitol in the chloroplast compartment than in wild-type cells, indicating an increased hydroxyl radical-scavenging capacity in BS1-31 tobacco. We suggest that the chloroplast location of mannitol can supplement endogenous radical-scavenging mechanisms and reduce oxidative damage of cells by hydroxyl radicals.     

The analogous mechanisms of enzymatic inactivation induced by ascorbate and superoxide in the presence of copper

The mechanism of enzymatic inactivation of purified and membrane-bound acetylcholine esterase by ascorbate and copper was investigated. While the exposure of the enzyme to either ascorbate or copper did not cause enzymatic inactivation, the incubation of the enzyme with a combination of both ascorbate and copper resulted in a loss in acetylcholine esterase activity, which was time dependent. The enzymatic inactivation required either molecular oxygen or hydrogen peroxide under anaerobic conditions. Scavengers of hydroxyl radicals at concentrations of up to 100 mM did not provide protection to acetylcholine esterase. Only mannitol at very high concentrations (above 1 M) efficiently prevented the inactivation of the enzyme. The kinetics of the aerobic oxidation of reduced ascorbate in the presence of acetylcholine esterase and copper closely followed the rate of enzyme inactivation. Addition of the chelating agents EDTA and diethylenetriaminepentaacetic acid prevented both the oxidation of ascorbate and the inactivation of the enzyme. In the presence of low concentrations of histidine (0.5-2.0 mM), which forms high affinity complexes with copper, the rate of ascorbate oxidation was similar to that recorded in its absence. On the other hand, no enzyme inactivation was indicated in the presence of histidine. Low temperature EPR measurements have demonstrated the binding of copper to the enzyme, and have shown the reduction of the cupric enzyme to the corresponding cuprous complex. In view of these results, a general "site-specific" mechanism for biological damage can be offered, in which copper(II) ions are bound to enzymes or other biological macromolecules. Ascorbate plays a dual role: it reduces the cupric complex to the corresponding cuprous state and serves as a source for H2O2, which, in turn, reacts with the reduced copper complex, in a Fenton reaction. In this reaction, secondary hydroxyl radicals are site specifically formed, and react preferentially with the protein, at the site of their formation, causing its inactivation. This mechanism is analogous to that previously proposed (Samuni, A., Chevion, M., and Czapski, G. (1981) J. Biol. Chem. 256, 12632-12635) for the enhancement of the biological damage caused by superoxide in the presence of copper.     

Hydroxyl radical production during oxidative deposition of iron in ferritin

The chemistry of oxidative deposition of iron(III) in ferritin and apoferritin is poorly understood. This study was undertaken to look for radicals formed as the hydrous ferric oxide core is developed from Fe(II) and O2. Radicals were observed indirectly by using the spin-trapping reagent N-tert-butyl-alpha-phenylnitrone (PBN) at room temperature and directly by measuring ESR spectra of frozen solutions at 77 K. In both instances, radical production was inhibited by the hydroxyl radical scavenging agents dimethyl sulfoxide, thiourea, and mannitol and enhanced by the addition of hydrogen peroxide. These findings strongly suggest that hydroxyl radical, produced from the iron-catalyzed Haber-Weiss reaction, is a by-product of core formation in ferritin and is a precursor to the observed radicals. The yield of ESR-observable and spin-trapped radicals is quite low, being at the micromolar level when millimolar concentrations of ferrous ion are employed. Furthermore, radical production appears to be confined to the interior of the ferritin molecule, where cellular components would be protected from the oxygen-derived toxic effects of iron. It is postulated that hydroxyl radical-medicated oxidative damage to the protein, a process that may contribute to the formation of hemosiderin from ferritin, leads to the observed radicals. By serving as a sink for hydroxyl radical, the protein shell may therefore efficiently minimize damage to other biomolecules in the cell.     

Inactivation of mitochondrial succinate dehydrogenase by adriamycin activated by horseradish peroxidase and hydrogen peroxide

Although human cancers are widely treated with anthracycline drugs, these drugs have limited use because they are cardiotoxic. To clarify the cardiotoxic action of the anthracycline drug adriamycin (ADM), the inhibitory effect on succinate dehydrogenase (SDH) by ADM and other anthracyclines was examined by using pig heart submitochondrial particles. ADM rapidly inactivated mitochondrial SDH during its interaction with horseradish peroxidase (HRP) in the presence of H(2)O(2) (HRP-H(2)O(2)). Butylated hydroxytoluene, iron-chelators, superoxide dismutase, mannitol and dimethylsulfoxide did not block the inactivation of SDH, indicating that lipid-derived radicals, iron-oxygen complexes, superoxide and hydroxyl radicals do not participate in SDH inactivation. Reduced glutathione was extremely efficient in blocking the enzyme inactivation, suggesting that the SH group in enzyme is very sensible to ADM activated by HRP-H(2)O(2). Under anaerobic conditions, ADM with HRP-H(2)O(2) caused inactivation of SDH, indicating that oxidized ADM directly attack the enzyme, which loses its activity. Other mitochondrial enzymes, including NADH dehydrogenase, NADH oxidase and cytochrome c oxidase, were little sensitive to ADM with HRP-H(2)O(2). SDH was also sensitive to other anthracycline drugs except for aclarubicin. Mitochondrial creatine kinase (CK), which is attached to the outer face of the inner membrane of muscle mitochondria, was more sensitive to anthracyclines than SDH. SDH and CK were inactivated with loss of red color of anthracycline, indicating that oxidative activation of the B ring of anthracycline has a crucial role in inactivation of enzymes. Presumably, oxidative semiquinone or quinone produced from anthracyclines participates in the enzyme inactivation.     

Plant defenses against oxidative stress

Many reactive oxygen species such as ozone, singlet oxygen, hydroxyl radical, and organic oxyradicals have been implicated in damage to plant organs and biopolymers such as chloroplasts, cell membranes, proteins, and DNA. The principal defenses against these reactive molecules and free radicals in plants include detoxifying enzymes (catalase, superoxide dismutase, etc.) and also lower molecular weight secondary products with antioxidant activity. These latter compounds include a great variety of phenolic compounds, carotenoids, nitrogenous, and sulfur-containing materials. Some of the more important mechanisms of action of the secondary compounds will be discussed, with emphasis on the use of structural and kinetic data to identify the most effective antioxidants against peroxy radical-induced damage, which is perhaps the most important of the oxidative stresses present in the usual environment of plants. © 1995 Wiley-Liss, Inc.     

Ethanol induces peroxynitrite-mediated toxicity through inactivation of NADP+-dependent isocitrate dehydrogenase and superoxide dismutase

It has been reported that chronic alcohol administration increases peroxynitrite hepatotoxicity by enhancing concomitant production of nitric oxide and superoxide. Several studies have shown the importance of superoxide dismutase (SOD) in protecting cells against ethanol-induced oxidative stress. Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of NADP(+)-dependent isocitrate dehydrogenase (ICDH) through to supply NADPH for antioxidant systems. In this report, we demonstrate that ethanol induces the peroxynitrite-mediated cytotoxicity in HepG2 cells through inactivation of antioxidant enzymes such as ICDH and SOD. Upon exposure to 100mM ethanol for 3days to HepG2 cells, a significant decrease in the viability and activities of ICDH and SOD was observed. The ethanol-induced inactivation of antioxidant enzymes resulted in the cellular oxidative damage and modulation of redox status as well as mitochondrial dysfunction in HepG2 cells. The cytoxicity of ethanol and inactivation of antioxidant enzymes were effectively protected by manganeses(III) tetrakis(N-methyl-2-pyridyl) porphyrin, a manganese SOD mimetic, and N'-monomethyl-l-arginine, a nitric oxide synthase inhibitor. These results indicate that ethanol toxicity is mediated by peroxynitrite and the peroxynitrite-mediated damage to ICDH and SOD may be resulted in the perturbation of the cellular antioxidant defense systems and subsequently lead to a pro-oxidant condition.     

Citrulline, a novel compatible solute in drought-tolerant wild watermelon leaves, is an efficient hydroxyl radical scavenger.

Drought-tolerant wild watermelon accumulates high levels of citrulline in the leaves in response to drought conditions. In this work, the hydroxyl radical-scavenging activity of citrulline was investigated in vitro. The second-order rate constant for the reaction between citrulline and hydroxyl radicals was found to be 3.9x10(9) M(-1) s(-1), demonstrating that citrulline is one of the most efficient scavengers among compatible solutes examined so far. Moreover, citrulline effectively protected DNA and an enzyme from oxidative injuries. Liquid chromatography-mass spectrometry analysis revealed that at least four major products were formed by the reaction between citrulline and hydroxyl radicals. Activities of metabolic enzymes were not inhibited by up to 600 mM citrulline, indicating that citrulline does not interfere with cellular metabolism. We reasoned, from these results, that citrulline contributes to oxidative stress tolerance under drought conditions as a novel hydroxyl radical scavenger.     

Molecular characterization and redox regulation of phosphoribulokinase from the cyanobacterium Synechococcus sp. PCC 7942

We isolated and characterized a gene encoding phosphoribulokinase (PRK) from Synechococcus sp. PCC 7942. The isolated sequence consisted of a 999 bp open reading frame encoding 333 amino acid residues of PRK. The PRK contained a pair of cysteinyl residues corresponding to Cys16 and Cys55 of spinach PRK regulated by a ferredoxin-thioredoxin system. However, there were seventeen amino acid residues lacking between the two cysteinyl residues compared with those of the chloroplastic enzyme in higher plants. The recombinant PRK of Synechococcus sp. PCC 7942 accounted for about 6-13% of the total soluble protein in the Escherichia coli. The specific activity of the enzyme was 230 micro mol min(-1) (mg protein)(-1). The enzyme activity was completely inactivated by treatment with 5,5'-dithiobis (2-nitrobenzoic acid) (cysteinyl residue-specific oxidant) or was decreased by treatment with H(2)O(2), but was more tolerant to oxidation than that of chloroplast. The oxidized PRK was fully activated by treatment with excessive dithiothreitol. Furthermore, incubation with 3 mM ATP protected the oxidation of the enzyme by either 5,5'-dithiobis (2-nitrobenzoic acid) or H(2)O(2). These results suggest Synechococcus sp. PCC 7942 PRK can be regulated by reversible oxidation/reduction in vitro, but might be resistant to oxidative inactivation in vivo.     

Mechanism of lysozyme inactivation and degradation by iron.

The site-specific lysozyme damage by iron and by iron-catalysed oxygen radicals was investigated. A solution of purified lysozyme was inactivated by Fe(II) at pH 7.4 in phosphate buffer, as tested on cleavage of Micrococcus lysodeikticus cells; this inactivation was time- and iron concentration-dependent and was associated with a loss of tryptophan fluorescence. In addition, it was reversible at pH 4, as demonstrated by lysozyme reactivation and by the intensity of the 14.4-kD-band on SDS-PAGE. Desferal (1 mM) and Detapac (1 mM) added before iron, prevented lysozyme inactivation, while catalase (100 micrograms/ml), superoxide dismutase (100 micrograms/ml) and bovine serum albumin (100 micrograms/ml) gave about 30 to 40% protection by competing with lysozyme for iron binding. The denaturing effect of iron on lysozyme was studied in the presence of H2O2 (1 mM) and ascorbate (1 mM); under these conditions the enzyme underwent partly irreversible inactivation and degradation different to that produced by gamma radiolysis-generated .OH. Catalase almost fully protected lysozyme; in contrast, mannitol (10 mM), benzoate (10 mM), and formate (10 mM) provided no protection because of their inability to access the site at which damaging species are generated. In this system, radical species were formed in a site-specific manner, and they reacted essentially with lysozyme at the site of their formation, causing inactivation and degradation differently than the hydroxyl radical.     

Oxidative inactivation of paraoxonase1, an antioxidant protein and its effect on antioxidant action

Paraoxonase1 (PON1), one of antioxidant proteins to protect low density lipoprotein (LDL) from the oxidation, is known to lose its activity in the oxidative environment. Here, we attempted to elucidate the possible mechanisms for the oxidative inactivation of PON1, and to examine the capability of hydroxyl radicals-inactivated PON1 to prevent against LDL oxidation. Of various oxidative systems, the ascorbate/Cu₂₊ system was the most potent in inactivating the purified PON1 (PON1) as well as HDL-bound PON1 (HDL-PON1). In contrast to a limited inactivation by Fe₂₊ (2.0 μM), the inclusion of Cu₂₊ (0.1-1.0 μM) remarkably enhanced the inactivation of PON1 in the presence of ascorbate (0.5 mM). A similar result was also obtained with the inactivation of HDL-PON1. The inactivation of PON1 by ascorbate/Cu₂₊ was pevented by catalase, but not general hydroxyl radical scavengers, supporting Cu₂₊-catalyzed oxidative inactivation. In addition, Cu₂₊ alone inactivated PON1, either soluble or HDL-bound, by different mechanisms, concentration-dependent. Separately, there was a reverse relationship between the inactivation of PON1 and its preventive action against LDL oxidation during Cu₂₊-induced oxidation of LDL. Noteworthy, ascorbate/Cu₂₊-inactivated PON1, which was charaterized by the partial loss of histidine residues, expressed a lower protection against Cu₂₊-induced LDL oxidation, compared to native PON1. Based on these results, it is proposed that metal-catalyzed oxidation may be a primary factor to cause the decrease of HDL-associated PON1 activity under oxidative stress, and radicals-induced inactivation of PON1 may lead to the decrease in its antioxidant action against LDL oxidation.     

The role of cationized catalase and cationized glucose oxidase in mucosal oxidative damage induced in the rat jejunum.

The successful prevention of hydrogen peroxide-induced damage to the rat jejunal mucosa by cationized catalase is described in this study. Biological damage was induced in a closed circulating intestinal loop of the rat by hydrogen peroxide and by hydroxyl radicals induced in situ via the metal-mediated Haber-Wiess reaction. The mucosal activity of lactate dehydrogenase and the amount of potassium ions were used to quantitatively characterize the tissue damage. Catalase was cationized by reacting it with N,N'-dimethyl-1,3-propanediamine to give a soluble product or with polyhistidine to give an insoluble product. The activity of the modified enzymes was assessed, and their ability to protect the rat jejunal mucosa against oxidative stress was studied. It was found that in all cases the cationized enzymes were superior to the native catalase in their shield capability. A significant protection against Fe(II)/H2O2 and ascorbic acid/copper ion-mediated damage was obtained when the cationized enzymes were used. In the presence of glucose, native glucose oxidase failed to cause damage in the rat jejunal mucosa; however, the cationized enzyme caused profound tissue injury. These findings indicate the potential therapeutic merit of cationized enzymes for the treatment of pathological processes in the intestine, whenever oxidative stress is involved.     

An Enhancing Effect of Exogenous Mannitol on the Antioxidant Enzyme Activities in Roots of Wheat Under Salt Stress

The role of mannitol as an osmoprotectant, a radical scavenger, a stabilizer of protein and membrane structure, and protector of photosynthesis under abiotic stress has already been well described. In this article we show that mannitol applied exogenously to salt-stressed wheat, which normally cannot synthesize mannitol, improved their salt tolerance by enhancing activities of antioxidant enzymes. Wheat seedlings (3 days old) grown in 100 mM mannitol (corresponding to −0.224 MPa) for 24 h were subjected to 100 mM NaCl treatment for 5 days. The effect of exogenously applied mannitol on the salt tolerance of plants in view of growth, lipid peroxidation levels, and activities of antioxidant enzymes in the roots of salt-sensitive wheat (Triticum aestivum L. cv. Kızıltan-91) plants with or without mannitol was studied. Although root growth decreased under salt stress, this effect could be alleviated by mannitol pretreatment. Peroxidase (POX) and ascorbate peroxidase (APX) activities increased, whereas superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR) activities decreased in Kızıltan-91 under salt stress. However, activities of antioxidant enzymes such as SOD, POX, CAT, APX, and GR increased with mannitol pretreatment under salt stress. Although root tissue extracts of salt-stressed wheat plants exhibited only nine different SOD isozyme bands of which two were identified as Cu/Zn-SOD and Mn-SOD, mannitol treatment caused the appearance of 11 different SOD activity bands. On the other hand, five different POX isozyme bands were determined in all treatments. Enhanced peroxidation of lipid membranes under salt stress conditions was reduced by pretreatment with mannitol. We suggest that exogenous application of mannitol could alleviate salt-induced oxidative damage by enhancing antioxidant enzyme activities in the roots of salt-sensitive Kızıltan-91.     

Carbonylation and Loss-of-Function Analyses of SBPase Reveal Its Metabolic Interface Role in Oxidative Stress, Carbon Assimilation, and Multiple Aspects of Growth and Development in Arabidopsis

Sedoheptulose-1,7-bisphosphatase (SBPase) is a Calvin cycle enzyme and functions in photosynthetic carbon fixation. We found that SBPase was rapidly carbonylated in response to methyl viologen (MV) treatments in detached leaves of Arabidopsis plants. In vitro activity analysis of the purified recombinant SBPase showed that SBPase was carbonylated by hydroxyl radicals, which led to enzyme inactivation in an H(2)O(2) dose-dependent manner. To determine the conformity with carbonylation-caused loss in enzymatic activity in response to stresses, we isolated a loss-of-function mutant sbp, which is deficient in SBPase-dependent carbon assimilation and starch biosynthesis. sbp mutant exhibited a severe growth retardation phenotype, especially for the developmental defects in leaves and flowers where SBPASE is highly expressed. The mutation of SBPASE caused growth retardation mainly through inhibition of cell division and expansion, which can be partially rescued by exogenous application of sucrose. Our findings demonstrate that ROS-induced oxidative damage to SBPase affects growth, development, and chloroplast biogenesis in Arabidopsis through inhibiting carbon assimilation efficiency. The data presented here provide a case study that such inactivation of SBPase caused by carbonyl modification may be a kind of adaptation for plants to restrict the operation of the reductive pentose phosphate pathway under stress conditions.     

Inactivation of Rabbit Muscle Creatine Kinase by Hydrogen Peroxide

The effects of xanthine + xanthine oxidase-generated reactive oxygen species (ROS) on rabbit muscle creatine kinase (CK) were studied. Xanthine (0.1 mM) + xanthine oxidase (30 mU/ml) inhibited activity of rabbit muscle CK (1.2mU/ml). Catalase (100/ml), but not SOD (100 U/ml), deferoxamine (100μM) or mannitol (20 mM), protected CK from inactivation; suggesting that H₂O₂ was responsible for inactivation. These results were different from previously reported findings on bovine heart CK that superoxide radicals inactivate the enzyme. Thus, enzymes with homologous structures may have different reactivities to different ROS. H₂O₂-induced inactivation of rabbit muscle CK was accompanied by a decrease in its thiol group content, whereas no significant changes in the protein structure were detected by SDS-PAGE or carbonyl content. These results suggest that oxidation of -SH groups by H₂O₂ seems to be a major mechanism of activation of rabbit muscle CK by xanthine + xanthine oxidase. Such inactivation of CK by H₂O₂ may be important in ROS-induced pathology.     

Radiation inactivation analysis of enzymes. Effect of free radical scavengers on apparent target sizes

In most cases the apparent target size obtained by radiation inactivation analysis corresponds to the subunit size or to the size of a multimeric complex. In this report, we examined whether the larger than expected target sizes of some enzymes could be due to secondary effects of free radicals. To test this proposal we carried out radiation inactivation analysis on Escherichia coli DNA polymerase I, Torula yeast glucose-6-phosphate dehydrogenase, Chlorella vulgaris nitrate reductase, and chicken liver sulfite oxidase in the presence and absence of free radical scavengers (benzoic acid and mannitol). In the presence of free radical scavengers, inactivation curves are shifted toward higher radiation doses. Plots of scavenger concentration versus enzyme activity showed that the protective effect of benzoic acid reached a maximum at 25 mM then declined. Mannitol alone had little effect, but appeared to broaden the maximum protective range of benzoic acid relative to concentration. The apparent target size of the polymerase activity of DNA polymerase I in the presence of free radical scavengers was about 40% of that observed in the absence of these agents. This is considerably less than the minimum polypeptide size and may reflect the actual size of the polymerase functional domain. Similar effects, but of lesser magnitude, were observed for glucose-6-phosphate dehydrogenase, nitrate reductase, and sulfite oxidase. These results suggest that secondary damage due to free radicals generated in the local environment as a result of ionizing radiation can influence the apparent target size obtained by this method.     

Inhibition of rat brain mitochondrial electron transport chain activity by dopamine oxidation products during extended in vitro incubation: implications for Parkinson's disease

Several studies on mitochondrial functions following brief exposure (5-15 min) to dopamine (DA) in vitro have produced extremely variable results. In contrast, this study demonstrates that a prolonged exposure (up to 2 h) of disrupted or lysed mitochondria to DA (0.1-0.4 mM) causes a remarkable and dose-dependent inhibition of complex I and complex IV activities. The inhibition of complex I and complex IV activities is not prevented by the antioxidant enzyme catalase (0.05 mg/ml) or the metal-chelator diethylenetriaminepentaacetic acid (0.1 mM) or the hydroxyl radical scavengers like mannitol (20 mM) and dimethyl sulphoxide (20 mM) indicating the non-involvement of *OH radicals and Fenton's chemistry in this process. However, reduced glutathione (5 mM), a quinone scavenger, almost completely abolishes the DA effect on mitochondrial complex I and complex IV activities, while tyrosinase (250 units/ml) which catalyses the conversion of DA to quinone products dramatically enhances the former effect. The results suggest the predominant involvement of quinone products instead of reactive oxygen radicals in long-term DA-mediated inactivation of complex I and complex IV. This is further indicated from the fact that significant amount of quinones and quinoprotein adducts (covalent adducts of reactive quinones with protein thiols) are formed during incubation of mitochondria with DA. Monoamine oxidase A (MAO-A) inhibitor clorgyline also provides variable but significant protection against DA induced inactivation of complex I and complex IV activities, presumably again through inhibition of quinoprotein formation. Mitochondrial ability to reduce tetrazolium dye 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) in presence of a respiratory substrate like succinate (10 mM) is also reduced by nearly 85% following 2 h incubation with 0.4 mM DA. This effect of DA on mitochondrial function is also dose-dependent and presumably mediated by quinone products of DA oxidation. The mitochondrial dysfunction induced by dopamine during extended periods of incubation as reported here have important implications in the context of dopaminergic neuronal death in Parkinson's disease (PD).     

Oxidative Inactivation of Paraoxonase1, an Antioxidant Protein and its Effect on Antioxidant Action

Paraoxonase1 (PON1), one of antioxidant proteins to protect low density lipoprotein (LDL) from the oxidation, is known to lose its activity in the oxidative environment. Here, we attempted to elucidate the possible mechanisms for the oxidative inactivation of PON1, and to examine the capability of hydroxyl radicals-inactivated PON1 to prevent against LDL oxidation. Of various oxidative systems, the ascorbate/Cu²⁺ system was the most potent in inactivating the purified PON1 (PON1) as well as HDL-bound PON1 (HDL-PON1). In contrast to a limited inactivation by Fe²⁺ (2.0?μM), the inclusion of Cu²⁺ (0.1–1.0?μM) remarkably enhanced the inactivation of PON1 in the presence of ascorbate (0.5?mM). A similar result was also obtained with the inactivation of HDL-PON1. The inactivation of PON1 by ascorbate/Cu²⁺ was pevented by catalase, but not general hydroxyl radical scavengers, supporting Cu²⁺-catalyzed oxidative inactivation. In addition, Cu²⁺ alone inactivated PON1, either soluble or HDL-bound, by different mechanisms, concentration-dependent. Separately, there was a reverse relationship between the inactivation of PON1 and its preventive action against LDL oxidation during Cu²⁺-induced oxidation of LDL. Noteworthy, ascorbate/Cu²⁺-inactivated PON1, which was charaterized by the partial loss of histidine residues, expressed a lower protection against Cu²⁺-induced LDL oxidation, compared to native PON1. Based on these results, it is proposed that metal-catalyzed oxidation may be a primary factor to cause the decrease of HDL-associated PON1 activity under oxidative stress, and radicals-induced inactivation of PON1 may lead to the decrease in its antioxidant action against LDL oxidation.