Inactivation of myocardial dihydrolipoamide dehydrogenase by myeloperoxidase systems: effect of halides, nitrite and thiol compounds

Dihydrolipoamide dehydrogenase (LADH) lipoamide reductase activity decreased whereas enzyme diaphorase activity increased after LADH treatment with myeloperoxidase (MPO) dependent systems (MPO/H₂O₂/halide, MPO/NADH/halide and MPO/H₂O₂/nitrite systems. LADH inactivation was a function of the composition of the inactivating system and the incubation time. Chloride, iodide, bromide, and the thiocyanate anions were effective complements of the MPO/H₂O₂ system. NaOCl inactivated LADH, thus supporting hypochlorous acid (HOCl) as putative agent of the MPO/H₂O₂/NaCl system. NaOCl and the MPO/H₂O₂/NaCl system oxidized LADH thiols and NaOCl also oxidized LADH methionine and tyrosine residues. LADH inactivation by the MPO/ NADH/halide systems was prevented by catalase and enhanced by superoxide dismutase, in close agreement with H₂O₂ production by the LADH/NADH system. Similar effects were obtained with lactoperoxidase and horseradish peroxidase suplemented systems. L-cysteine, N-acetylcysteine, penicillamine, N-(2-mercaptopropionylglycine), Captopril and taurine protected LADH against MPO systems and NaOCl. The effect of the MPO/H₂O₂/NaNO₂ system was prevented by MPO inhibitors (sodium azide, isoniazid, salicylhydroxamic acid) and also by L-cysteine, L-methionine, L-tryptophan, L-tyrosine, L-histidine and reduced glutathione. The summarized observations support the hypothesis that peroxidase-generated “reactive species” oxidize essential thiol groups at LADH catalytic site.     

Inactivation of Heart Dihydrolipoamide Dehydrogenase by Copper Fenton Systems. Effect of Thiol Compounds and Metal Chelators

Copper Fenton systems (Cu(II)/H₂O₂ and Cu(II)/Asc) inactivated the lipoamide reductase and enhanced the diaphorase activity of pig-heart lipoamide dehydrogenase (LADH). Cupric ions alone were less effective. As a result of Cu(II)/H₂O₂ treatment, the number of titrated thiols in LADH decreased from 6 to 1 per subunit. NADH and ADP (not NAD⁺ or ATP) enhanced LADH inactivation by Cu(II). NADH also enhanced the effect of Cu(II)/H₂O₂. Dihydrolipoamide, dihydrolipoic acid, Captopril, acetylcysteine, EDTA, DETAPAC, histidine, bathocuproine, GSSG and trypanothione prevented LADH inactivation. 100 μM GSH, DL-dithiothreitol, N-(2-mercaptopropionylglicine) and penicillamine protected LADH against Cu(II)/Asc and Cu(II), whereas 1.0 mm GSH and DL-dithiothreitol also protected LADH against Cu(II)/H₂O₂. Allopurinol provided partial protection against Cu(II)/H₂O₂. EthanoI, mannitol, Na benzoate and superoxide dismutase failed to prevent LADH inactivation by Cu(II)/H₂O₂ or Cu(II). Catalase (native or denaturated) and bovine serum albumin protected LADH but that protection should be due to Cu binding. LADH inhibited deoxyribose oxidation and benzoate hydroxylation by Cu(II)/H₂O₂. It is concluded that site-specifically generated HO, radicals were responsible for LADH inactivation by Cu(II) Fenton systems. The latter effect is discussed in the context of ischemia-reoxygenation myocardial injury.     

Trypanosoma cruzi dihydrolipoamide dehydrogenase is inactivated by myeloperoxidase-generated “reactive species”

Dihydrolipoamide dehydrogenase (LADH) from Trypanosoma cruzi was inactivated by treatment with myeloperoxidase (MPO)-dependent systems. With MPO/H₂O₂/NaCl, LADH lipoamide reductase and diaphorase activities significantly decreased as a function of incubation time. Iodide, bromide, thiocyanide and chloride effectively supplemented the MPO/H₂O₂ system, KI and NaCl being the most and the least effective supplements, respectively. LADH inactivation by MPO/H₂O₂/NaCl and by NaOCl was similarly prevented by thiol compounds such as GSH, L-cysteine, N-acetylcysteine, penicillamine and N-(2-mercaptopropionyl-glycine) in agreement with the role of HOCl in LADH inactivation by MPO/H₂O₂/NaCl. LADH was also inactivated by MPO/NADH/halide, MPO/H₂O₂/NaNO₂ and MPO/NADH/NaNO₂ systems. Catalase prevented the action of the NADH-dependent systems, thus supporting H₂O₂ production by NADH-supplemented LADH. MPO inhibitors (4-aminobenzoic acid hydrazide, and isoniazid), GSH, L-cysteine, L-methionine and L-tryptophan prevented LADH inactivation by MPO/H₂O₂/NaNO₂. Other MPO systems inactivating LADH were (a) MPO/H₂O₂/chlorpromazine; (b) MPO/H₂O₂/monophenolic systems, including L-tyrosine, serotonin and acetaminophen and (c) MPO/H₂O₂/di- and polyphenolic systems, including norepinephrine, catechol, nordihydroguaiaretic acid, caffeic acid, quercetin and catechin. Comparison of the above effects and those previously reported with pig myocardial LADH indicates that both enzymes were similarly affected by the MPO-dependent systems, allowance being made for T. cruzi LADH diaphorase inactivation and the greater sensitivity of its LADH lipoamide reductase activity towards the MPO/H₂O₂/NaCl system and NaOCl.     

Inactivation of Yeast Glutathione Reductase by Fenton Systems: Effect of Metal Chelators, Catecholamines and Thiol Compounds

Oxygen radical generating systems, namely, Cu(II)/ H₂O₂, Cu(II)/ascorbate, Cu(II)/NAD(P)H, Cu(II)/ H₂O₂/catecholamine and Cu(II)/H₂O₂/SH-compounds irreversibly inhibited yeast glutathione reductase (GR) but Cu(II)/H₂O₂ enhanced the enzyme diaphorase activity. The time course of GR inactivation by Cu(II)/H₂O₂ depended on Cu(II) and H₂O₂ concentrations and was relatively slow, as compared with the effect of Cu(II)/ascorbate. The fluorescence of the enzyme Tyr and Trp residues was modified as a result of oxidative damage. Copper chelators, catalase, bovine serum albumin and HO^˙ scavengers prevented GR inactivation by Cu(II)/H₂O₂ and related systems. Cysteine, N-acetylcysteine, N-(2-dimercaptopropi-onylglycine and penicillamine enhanced the effect of Cu(II)/H₂O₂ in a concentration- and time-dependent manner. GSH, Captopril, dihydrolipoic acid and dithiotreitol also enhanced the Cu(II)/H₂O₂ effect, their actions involving the simultaneous operation of pro-oxidant and antioxidant reactions. GSSG and try-panothione disulfide effectively protected GR against Cu(II)/H₂O₂ inactivation. Thiol compounds prevented GR inactivation by the radical cation ABTS*⁺. GR inactivation by the systems assayed correlated with their capability for HO* radical generation. The role of amino acid residues at GR active site as targets for oxygen radicals is discussed.     

Trypanosoma cruzi trypanothione reductase is inactivated by peroxidase-generated phenothiazine cationic radicals

Trypanosoma cruzi trypanothione reductase (TR) was irreversibly inhibited by peroxidase/H₂O2/phenothiazine (PTZ) systems. TR inactivation depended on (a) time of incubation with the phenothiazine system; (b) the peroxidase nature and (c) the PTZ structure and concentration. With the most effective systems, TR inactivation kinetics were biphasic, with a relatively fast initial phase during which about 75% of the enzyme activity was lost, followed by a slower phase leading to total enzyme inactivation. GSH prevented TR inactivation by the peroxidase/H₂O₂/PTZ^+· systems. Production of PTZ^+· cation radicals by PTZ peroxidation was essential for TR inactivation. Horseradish peroxidase, leukocyte myeloperoxidase (MPO) and the pseudo-peroxidase myoglobin (Mb) were effective catalysts of PTZ^+· production. Promazine, thioridazine, chlorpromazine, propionylpromazine prochlorperazine, perphenazine and trimeprazine were effective constituents of the HRP/H₂O₂/PTZ system. The presence of substituents at the PTZ nucleus position 2 exerted significant influence on PTZ activity, as shown by the different effects of 2-trifluoromethyl and 2-H or 2-chlorophenothiazines. The PTZ^+· cation radicals disproportionation regenerated the non-radical PTZ molecule and produced the PTZ sulfoxide that was inactive on TR. Thiol compounds including GSH interacted with PTZ^+· cation radicals transferring an electron from the sulfide anion to the PTZ^+·, thus nullifying the PTZ^+· biological and chemical activities.     

Inactivation of Lipoamide Dehydrogenase by Cobalt(II) and Iron(II) Fenton Systems: Effect of Metal Chelators, Thiol Compounds and Adenine Nucleotides

Fe(II)-and Co(II)-Fenton systems (FS) inactivated the lipoamide reductase activity but not the diaphorase activity of pig-heart lipoamide dehydrogenase (LADH). The Co(II) system was the more effective as LADH inhibitor. Phosphate ions enhanced the Fe(II)-FS activity. EDTA, DETAPAC, DL-histidine, DL-cysteine, glutathione, DL-dithiothreitol, DL-lipoamide, DL-thioctic acid, bathophenthroline, trypanothione and ATP, but not ADP or AMP, prevented LADH inactivation. Reduced disulfide compounds were more effective protectors than the parent compounds. Mg ions counteracted ATP protective action. Glutathione and DL-dithiothreitol partially restored the lipoamide dehydrogenase activity of the Fe(II)-FS-inhibited LADH. DL-histidine exerted a similar action on the Co(II)-FS-inhibited enzyme. Ethanol, mannitol and benzoate did not prevent LADH inactivation by the assayed Fenton systems and, accordingly, it is postulated that site-specific generated HO'radicals were responsible for LADH inactivation. With the Co(II)-FS, oxygen reactive species other than HO, might contribute to LADH inactivation.     

Phenothiazine radicals inactivate Trypanosoma cruzi dihydrolipoamide dehydrogenase: enzyme protection by radical scavengers

Phenothiazine cation radicals (PTZ + •) irreversibly inactivated Trypanosoma cruzi dihydrolipoamide dehydrogenase (LADH). These radicals were obtained by phenothiazine (PTZ) peroxidation with myeloperoxidase (MPO) or horseradish peroxidase (HRP/H 2 O 2 ) systems. LADH inactivation depended on PTZ structure and incubation time. After 10 min incubation of LADH with the MPO-dependent systems, promazine, trimeprazine and thioridazine were the most effective; after 30 min incubation, chlorpromazine, prochlorperazine and promethazine were similarly effective. HRP-dependent systems were equally or more effective than the corresponding MPO-dependent ones. Chloro, trifluoro, propionyl and nitrile groups at position 2 of the PTZ ring significantly decreased molecular activity, specially with the MPO/H 2 O 2 systems. Comparison of inactivation values for LADH and T. cruzi trypanothione reductase demonstrated a greater sensitivity of LADH to chlorpromazine and perphenazine and a 10-fold lower sensitivity to promazine, thioridazine and trimeprazine. Alkyl-amino, alkyl-piperidinyl or alkyl-piperazinyl groups at position 10 modulated PTZ activity to a limited degree. Production of PTZ + • radicals was demonstrated by optical and ESR spectroscopy methods. PTZ + • radicals stability depended on their structure as demonstrated by promazine and thioridazine radicals. Thiol compounds such as GSH and N -acetylcysteine, l -tyrosine, l -tryptophan, the corresponding peptides, ascorbate and Trolox, prevented LADH inactivation by the MPO/H 2 O 2 /thioridazine system, in close agreement with their action as PTZ + • scavengers. NADH (not NAD + ) produced transient protection of LADH against thioridazine and promazine radicals, the protection kinetics being affected by the relatively fast rate of NADH oxidation by these radicals. The role of the observed effects of PTZ radicals for PTZ cytotoxicity is discussed.     

Caspases are reversibly inactivated by hydrogen peroxide

Hydrogen peroxide (H₂O₂) is known to both induce and inhibit apoptosis, however the mechanisms are unclear. We found that H₂O₂ inhibited the activity of recombinant caspase-3 and caspase-8, half-inhibition occurring at about 17 μM H₂O₂. This inhibition was both prevented and reversed by dithiothreitol while glutathione had little protective effect. 100–200 μM H₂O₂ added to macrophages after induction of caspase activation by nitric oxide or serum withdrawal substantially inhibited caspase activity. Activation of H₂O₂-producing NADPH oxidase in macrophages also caused catalase-sensitive inactivation of cellular caspases. The data suggest that the activity of caspases in cells can be directly but reversibly inhibited by H₂O₂.     

Role of thiocyanate, bromide and hypobromous acid in hydrogen peroxide-induced apoptosis

We have previously reported that H₂O₂-induced apoptosis in HL-60 human leukemia cells takes place in the presence of chloride, requires myeloperoxidase (MPO), and occurs through oxidative reactions involving hypochlorous acid and chloramines. We now report that when chloride is replaced by the pseudohalide thiocyanate, there is little or no H₂O₂-induced apoptosis. Furthermore, thiocyanate inhibits H₂O₂-induced apoptosis when chloride is present at physiological concentrations, and this occurs at thiocyanate concentrations that are present in human serum and saliva. In contrast, bromide can substitute for chloride in H₂O₂-induced apoptosis, but results in a lower percent of the cells induced into apoptosis. Hypobromous acid is likely a short-lived intermediate in this H₂O₂/MPO/bromide apoptosis, and reagent hypobromous acid and bromamines induce apoptosis in HL-60 cells. We conclude that the physiologic concentrations of thiocyanate found in human plasma could modulate the cytototoxicity of H₂O₂ and its resulting highly toxic MPO-generated hypochlorous acid by competing with chloride for MPO. Furthermore, the oxidative products of the reaction of thiocyanate with MPO are relatively innocuous for human leukemic cells in culture. In contrast, bromide can support H₂O₂/MPO/halide apoptosis, but is less potent than chloride and it has no effect in the presence of physiological levels of chloride.     

Oxidative aggregation of ceruloplasmin induced by hydrogen peroxide is prevented by pyruvate

Ceruloplasmin (CP) is a blue copper glycoprotein with multiple physiological functions including ferroxidase and oxidase activities. CP is also an important serum oxygen free radical (OFR) scavenger and antioxidant, exerting cardioprotective and antifibrillatory actions. Although it has been reported that CP activities can be inhibited by OFR, the intimate mechanism of this inactivation is still not clear. Exposure of bovine CP to H₂O₂ induced inactivation of the protein as well as structural alterations as indicated by loss of protein bands by SDS-PAGE. Both phenomena were H₂O₂ concentration and time dependent. HPLC gel filtration and capillary electrophoresis analysis of CP treated with H₂O₂ revealed an aggregation of the protein. Quantification of dityrosine formation by fluorescence indicated the involvement of dityrosine bridging, which could be responsible for aggregation of CP under oxidative attack. Oxidative damage to CP under H₂O₂ treatment was completely prevented by pyruvate, suggesting that the association of CP with antioxidants could extend the range of the protective action of this protein.     

Hydrogen sulphide: a novel physiological inhibitor of LDL atherogenic modification by HOCl

Hypochlorite (HOCl), the product of the activated myeloperoxidase/H₂O₂/chloride (MPO/H₂O₂/Cl^- ) system is favored as a trigger of LDL modifications, which may play a pivotal role in early atherogenesis. As HOCl has been shown to react with thiol-containing compounds like glutathione and N-acetylcysteine protecting LDL from HOCl modification, we have tested the ability of hydrogen sulfide (H₂S)—which has recently been identified as an endogenous vasorelaxant—to counteract the action of HOCl on LDL. The results show that H₂S could inhibit the atherogenic modification of LDL induced by HOCl, as measured by apolipoprotein alterations. Beside its HOCl scavenging potential, H₂S was found to inhibit MPO (one may speculate that this occurs via H₂S/heme interaction) and destroy H₂O₂. Thus, H₂S may interfere with the reactants and reaction products of the activated MPO/H₂O₂/Cl^-  system. Our data add to the evidence of an anti-atherosclerotic action of this gasotransmitter taking the role of HOCl in the atherogenic modification of LDL into account.     

Non-oxygen-forming pathways of hydrogen peroxide degradation by bovine liver catalase at low hydrogen peroxide fluxes

Heme catalases are considered to degrade two molecules of H₂O₂ to two molecules of H₂O and one molecule of O₂ employing the catalatic cycle. We here studied the catalytic behaviour of bovine liver catalase at low fluxes of H₂O₂ (relative to catalase concentration), adjusted by H₂O₂-generating systems. At a ratio of a H₂O₂ flux (given in μM/min^- 1) to catalase concentration (given in μM) of 10 min^- 1 and above, H₂O₂ degradation occurred via the catalatic cycle. At lower ratios, however, H₂O₂ degradation proceeded with increasingly diminished production of O₂. At a ratio of 1 min^- 1, O₂ formation could no longer be observed, although the enzyme still degraded H₂O₂. These results strongly suggest that at low physiological H₂O₂ fluxes H₂O₂ is preferentially metabolised reductively to H₂O, without release of O₂. The pathways involved in the reductive metabolism of H₂O₂ are presumably those previously reported as inactivation and reactivation pathways. They start from compound I and are operative at low and high H₂O₂ fluxes but kinetically outcompete the reaction of compound I with H₂O₂ at low H₂O₂ production rates. In the absence of NADPH, the reducing equivalents for the reductive metabolism of H₂O₂ are most likely provided by the protein moiety of the enzyme. In the presence of NADPH, they are at least in part provided by the coenzyme.     

Nitrite reduces the effects of HOCl on unsaturated phosphatidylcholines--a MALDI-TOF MS study

The concentration of nitrite (NO₂⁻) increases under inflammatory conditions. However, the physiological role of nitrite is so far controversial discussed: it was reported that effects of HOCl (an important inflammation mediator) on phospholipids (PL) may be enhanced but also reduced in the presence of nitrite.

In this paper a simple model system was used: unsaturated phosphatidylcholine (PC) vesicles were treated with HOCl in the presence of varying NaNO₂ concentrations and the yield of reaction products was determined by MALDI-TOF MS: the extent of chlorohydrin generation was significantly reduced in the presence of NaNO₂ because HOCl is consumed by the oxidation of NO₂⁻ to NO₃⁻.

Similar results were obtained when HOCl was generated by the myeloperoxidase (MPO)/H₂O₂/Cl⁻ system or the experiments were carried out in the presence of a simple peptide. It is concluded that the transient products of the reaction between HOCl and NO₂⁻ do not have a sufficient reactivity to modify PL.     

Singlet Oxygen-Trapping Reaction as a Method of 1O2 Detection: Role of Some Reducing Agents

The production of singlet oxygen by H₂O₂ disproportionation and via the oxidation of H₂O₂ by NaOCl in a neutral medium was monitored by spin trapping with 2,2,6,6 tetramethyl-4-piperidone (TMPone). The singlet oxygen formed in both reactions oxidized 2,2,6,6 tetramethyl-4-piperidone to give nitroxide radicals. However the production of nitroxide radicals was relatively small considering the concentrations of H₂O₂ and NaOCl used in the reaction systems. Addition of electron donating agents: ascorbate, Fe²⁺ and desferrioxamine leads to an increase in the production of nitroxide radicals. We assumed that a very slow step of the reaction sequence, the homolytic breaking of the O-O bond of N-hydroperoxide (formed as an intermediate product during the reaction of ¹O₂ with TMPone) could be responsible for the relatively small production of nitroxide radicals. Electron donating agents added to the reaction system probably raise the rate of the hydroperoxide decomposition by allowing a more rapid heterolytic cleavage of the O-O bond leading to a greater production of nitroxide radicals. The largest effect was observed in the presence of desferrioxamine. Its participation in this process is proved by the concomitant appearance of desferrioxamine nitroxide radicals. The results obtained demonstrate that the method proposed by several authors and tested in this study to detect singlet oxygen is not convenient for precise quantitative studies. The reactivity of TMPone towards O₂^-7HO₂' and 'OH has been also investigated. It has been found that both O₂^-7HO₂' and 'OH radicals formed in a phosphate buffer solution (pH 7.4, 37°C), respectively by a xanthine-oxidase/hypoxanthine system and via H₂O₂ UV irradiation, do not oxidize 2,2,6,6 tetramethyl-4-piperidone to nitroxide radicals.     

Lethality of hydrogen peroxide in wild type and superoxide dismutase mutants of Escherichia coli. (A hypothesis on the mechanism of H2O2-induced inactivation of Escherichia coli)

The toxicity of H₂O₂ in Escherichia coli wild type and superoxide dismutase mutants was investigated under different experimental conditions. Cells were either grown aerobically, and then treated in M9 salts or K medium, or grown anoxically, and then treated in K medium. Results have demonstrated that the wild type and superoxide dismutase mutants display a markedly different sensitivity to both modes of lethality produced by H₂O₂ (i.e. mode one killing, which is produced by concentrations of H₂O₂ lower than 5 mM, and mode two killing which results from the insult generated by concentrations of H₂O₂ higher than 10 mM). Although the data obtained do not clarify the molecular basis of H₂O₂ toxicity and/or do not explain the specific function of superoxide ions in H₂O₂-induced bacterial inactivation, they certainly demonstrate that the latter species plays a key role in both modes of H₂O₂ lethality. A mechanism of H₂O₂ toxicity in E. coli is proposed, involving the action of a hypothetical enzyme which should work as an O₂^-• generating system. This enzyme should be active at low concentrations of H₂O₂ (<5 mM) and high concentrations of the oxidant (>5 mM) should inactivate the same enzyme. Superoxide ions would then be produced and result in mode one lethality. The resistance at intermediate H₂O₂ concentrations may be dependent on the inactivation of such enzyme with no superoxide ions being produced at levels of H₂O₂ in the range 5–10 mM. Mode two killing could be produced by the hydroxyl radical in concert with superoxide ions, chemically produced via the reaction of high concentrations of H₂O₂ (>10 mM) with hydroxyl radicals. The rate of hydroxyl radical production may be increased by the higher availability of Fe²⁺ since superoxide ions may also reduce trivalent iron to the divalent form.     

The Action of Hydrogen Peroxide on the Formation of Thiobarbituric Acid-Reactive Material From Microsomes, Liposomes Or From Dna Damaged By Bleomycin Or Phenanthroline. Artefacts in the Thiobarbituric Acid Test

Incubation of rat-liver microsomes, previously azide-treated to inhibit catalase, with H₂O₂ caused a loss of cytochrome P-450 but not of cytochrome b₅. This loss of P-450 was not prevented by scavengers of hydroxyl radical, chain-breaking antioxidants or metal ion-chelating agents. Application of the thiobarbituric acid (TBA) assay to the reaction mixture suggested that H₂O₂ induces lipid peroxidation, but this was found to be due largely or completely to an effect of H²O₂ on the TBA assay. By contrast, addition of ascorbic acid and Fe(III) to the microsomes led to lipid peroxidation and P-450 degradation: both processes were inhibited by chelating agents and chain-breaking antioxidants, but not by hydroxyl radical scavengers. H₂O₂ inhibited ascorbate/Fe (III)-induced microsomal lipid peroxidation, but part of this effect was due to an action of H₂O₂ in the TBA test itself. H₂O₂ also decreased the colour measured after carrying out the TBA test upon authentic malondialdehyde, tetraethoxypropane, a DNA-Cu²⁺/o-phenanthroline system in the presence of a reducing agent, ox-brain phospholipid liposomes in the presence of Fe(III) and ascorbate, or a bleomycin-iron ion/DNA/ascorbate system. Caution must be used in interpreting the results of TBA tests upon systems containing H₂O₂.     

Redox Properties of Iron in the Binding Site(s) of F1ATPase from Mammalian Mitochondria and Thermophilic Bacterium PS3: A Comparative Study

Iron ions in the two iron centers of beef heart mito-chondrial F, ATPase, which we have been recently characterized (FEBS Letters 1996,379, 231-235), exhibit different redox properties. In fact, the ATP-dependent site is able to maintain iron in the redox state of Fe(II) even in the absence of reducing agents, whereas in the nucleotide-independent site iron is oxidized to Fe(III) upon removal of the reductant. Fe(III) ions in the two sites display different reactivity towards H₂O₂, because only Fe(III) bound in the nucleotide-independent site rapidly reacts with H₂O₂ thus mediating a 30% enzyme inactivation. Thermophilic bacterium PS3 bears one Fe(III) binding site, which takes up Fe(III) either in the absence or presence of nucleotides and is unable to maintain iron in the redox state of Fe(II) in the absence of ascorbate. Fe(III) bound in thermophilic F₁ATPase in a molar ratio 1:1 rapidly reacts with H₂O₂ mediating a 30% enzyme inactivation. These results support the presence in mitochon-drial and thermophilic F₁ATPase of a conserved site involved in iron binding and in oxidative inactivation, in which iron exhibits similar redox properties. On the other hand, at variance with thermophilic F₁ATPase, the mitochondrial enzyme has the possibility of maintaining one equivalent of Fe(II) in its peculiar ATP-dependent site, besides one equivalent of Fe(III) in the conserved nucleotide-independent site. In this case mitochondrial F, ATPase undergoes a higher inactivation (75%) upon exposure to H₂O₂. Under all conditions the inactivation is significantly prevented by PBN and DMSO but not by Cu, Zn superoxide dis-mutase, thus suggesting the formation of OH radicals as mediators of the oxidative damage. No dityrosines, carbonyls or oxidized thiols are formed. In addition, in any cases no protein fragmentation or aggregation is observed upon the treatment with H₂O₂.     

Hydroxyl Radical Generation in the Nadh/Microsomal Reduction of vanadate

ESR spin trapping measurements demonstrate generation of hydroxyl (.OH) radical from reduction of vanadate by rat liver microsomes/NADH without exogenous H₂O₂. Catalase decreases the .OH signal while increasing a vanadium(4+) signal. Addition of superoxide dismutase (SOD) or measurements under an argon atmosphere show decreased .OH radical production. The results suggest that during the one-electron vanadate reduction process by microsomes/NADH, molecular oxygen is reduced to H₂O₂, which then reacts with vanadium (4+) to generate .OH radical via a Fenton-like mechanism.     

Inactivation of alcohol dehydrogenase by piroxicam-derived radicals

Alcohol dehydrogenase (ADH) was used as a marker molecule to clarify the mechanism of gastric mucosal damage as a side effect of using piroxicam. Piroxicam inactivated ADH during interaction of ADH with horseradish peroxidase and H₂O₂ (HRP-H₂O₂). The ADH was more easily inactivated under aerobic than anaerobic conditions, indicating participation by oxygen. Superoxide dismutase, but not hydroxyl radical scavengers, inhibited inactivation of ADH, indicating participation by superoxide. Sulfhydryl (SH) groups in ADH were lost during incubation of piroxicam with HRP-H₂O₂. Adding reduced glutathione (GSH) efficiently blocked ADH inactivation. Other SH enzymes, including creatine kinase and glyceraldehyde-3-phosphate dehydrogenase, were also inactivated by piroxicam with HRP-H₂O₂. Thus SH groups in the enzymes seem vulnerable to piroxicam activated by HRP-H₂O₂. Spectral change in piroxicam was caused by HRP-H₂O₂. ESR signals of glutathionyl radicals occurred during incubation of piroxicam with HRP-H₂O₂ in the presence of GSH. Under anaerobic conditions, glutathionyl radical formation increased. Thus piroxicam free radicals interact with GSH to produce glutathionyl radicals. Piroxicam peroxyl radicals or superoxide, or both, seem to inactivate ADH. Superoxide may be produced through interaction of peroxyl radicals with H₂O₂. Thus superoxide dismutase may inhibit inactivation of ADH through reducing piroxicam peroxyl radicals or blocking interaction of SH groups with O₂^-, or both. Other oxicam derivatives, including isoxicam, tenoxicam and meloxicam, induced ADH inactivation in the presence of HRP-H₂O₂.     

NO和H2O2在光/暗调控蚕豆气孔运动中的作用及其相互关系

借助表皮条分析和激光扫描共聚焦显微镜技术,对NO和H2O2在光/暗调控蚕豆(Vicia faba L.)气孔运动中的作用及其相互关系进行了探索.结果显示,光下外源NO供体硝普钠(SNP)和H2O2促进气孔关闭的效应明显大于暗中,暗中NO专一性清除剂2,4-羧基苯-4,4,5,5-四甲基咪唑-1-氧-3-氧化物(cPTIO)、一氧化氮合酶(NOS)抑制剂NG-氮-L-精氨酸-甲酯(L-NAME)和H2O2清除剂抗坏血酸(Vc)、过氧化氢酶(CAT)对气孔开度的效应明显大于光下,而且光下蚕豆保卫细胞NO和H2O2水平比暗中明显降低.上述结果表明,光/暗通过影响保卫细胞NO和H2O2的水平调控气孔运动.研究还发现,光下H2O2既诱导NO水平增加,也诱导气孔关闭,cPTIO和L-NAME有效地逆转H2O2的这些效应;光下SNP既诱导H2O2水平增加,也诱导气孔关闭,SNP的上述效应又被Vc和CAT有效逆转.这些结果表明,NO和H2O2在生成及效应上均存在明显的相互作用.另外,L-NAME显著逆转暗和光下H2O2处理对气孔关闭和NO生成的效应表明,蚕豆保卫细胞中可能存在NOS,暗和光下H2O2处理可能通过提高NOS的活性促进NO水平增加,进而诱导气孔关闭.