The reaction of superoxide radical anion with dithiothreitol: a chain process

Zhang, Nan; Schuchmann, Heinz Peter; Sonntag, Clemens von

doi:10.1021/j100165a024

Cited by 57 publications

(41 citation statements)

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“…Others have made similar observations with cysteine (20,21). The explanation given, that superoxide somehow protects cysteine against oxidation (13,21), is not consistent with current knowledge of how superoxide interacts with thiols (11,12). After further investigation, we report here that the enzyme does not act in these systems simply by dismutating superoxide.…”

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confidence: 74%

Thiol Oxidase Activity of Copper,Zinc Superoxide Dismutase

Winterbourn

Peskin

Parsons-Mair

2002

Journal of Biological Chemistry

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The ability of copper,zinc superoxide dismutase (Cu,Zn-SOD) to catalyze autoxidation of cysteine and other thiols was investigated by measuring thiol loss and oxygen consumption. The reaction occurred equally well with the bovine and human enzymes and produced hydrogen peroxide and the corresponding disulfide. It did not occur with manganese SOD and is not, therefore, due to the dismutase activity of the enzyme. Cysteine and cysteamine were highly reactive: the K m for cysteine was 1.4 mM and V max (with 40 g/ml SOD) 35 M/ min; the equivalent values for cysteamine (with 20 g/ml SOD) were 1.4 mM and 36 M/min. With 1 mM thiol and 40 g/ml SOD, rates of oxidation of other thiols (M/min) were as follows: GSH, 1.0; dithiothreitol, 2.1; dihydrolipoic acid, 1.7; homocysteine, 1.6; cys-gly, 1.4; penicillamine, 0.6; and N-acetylcysteine, 0.1. SOD-mediated oxidation of cysteine, in the absence of chelating agents, proceeded only after a variable lag phase. The lag was decreased but not eliminated with Chelex-treated reagents and is attributed to interference by submicromolar concentrations of iron and possibly other transition metal ions. SOD-catalyzed oxidation of the other thiols was variably affected by adventitious metal ions and chelating agents. Reactions were all performed in the presence of desferrioxamine to obviate these effects. SOD-catalyzed oxidation of GSH and homocysteine was enhanced by cysteine through a thiol-disulfide exchange mechanism. This study characterizes a novel pro-oxidant thiol oxidase activity of Cu,Zn-SOD. It is a potential source of reactive oxidants and may contribute to the cytotoxicity of reactive thiols such as cysteine and cysteamine.

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confidence: 74%

Thiol Oxidase Activity of Copper,Zinc Superoxide Dismutase

Winterbourn

Peskin

Parsons-Mair

2002

Journal of Biological Chemistry

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“…DTT at 1 mM prevents RyR2 oxidation (Eager et al, 1997;Eager and Dulhunty, 1999), and its addition to the cis or trans chamber can indicate the location of the reactive -SH groups, because DTT is not highly lipid soluble at pH 7.4 and does not cross the bilayer in significant amounts. Because the thiol groups of DTT have pK a values of 9.1 and 10.2 (Zhang et al, 1991) and the pH of the bilayer solutions was buffered to 7.4, DTT would be predominantly in its water soluble, ionized form (White, 1999) and would be unable to easily cross the lipid bilayer (White, 1999;Rang, 2007).…”

Section: Dtt Protection Of Free -Sh Groups and Reduction Of -S-s-mentioning

confidence: 99%

Multiple Actions of the Anthracycline Daunorubicin on Cardiac Ryanodine Receptors

Hanna¹,

Janczura²,

Cho³

et al. 2011

Molecular Pharmacology

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Our aim was to examine the molecular basis for acute effects of the anthracycline daunorubicin on cardiac ryanodine receptor (RyR2) channels and cardiac calsequestrin (CSQ2). Cardiotoxic effects of anthracyclines preclude their chemotherapeutic use in patients with pre-existing heart conditions. To address this significant problem, the mechanisms of anthracycline toxicity must be defined but at present are poorly understood. RyR2 channel activity was assessed by measuring Ca 2ϩ release from cardiac sarcoplasmic reticulum vesicles and by examining single RyR2 channels inserted into artificial lipid bilayers. We show that 0.5 to 10 M daunorubicin increases the activity of RyR2 channels after 5 to 10 min and that activity then declines to very low levels when channels are exposed to daunorubicin concentrations of Ն2.5 M for a further 10 to 20 min. Extensive dissection of these effects shows for the first time that the activation results from a redox-independent binding of daunorubicin to the RyR2 complex. Novel data include the demonstration of daunorubicin binding to RyR2. We provide compelling evidence that RyR2 channel inhibition is due to the oxidation of free SH groups. The oxidation reaction is prevented by the presence of 1 mM dithiothreitol. We also present novel data showing that CSQ2 modifies the response of RyR2 to daunorubicin, but that the response of RyR2 is not dependent on daunorubicin binding to CSQ2. We suggest that binding of daunorubicin to RyR2 and CSQ2, and oxidation of RyR2, are all likely to contribute to anthracycline-induced cardiotoxicity during chemotherapy.

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“…To determine whether thiol oxidation played a role in the modification of GAPDH with NADH, transition metal catalysts and oxygen, which stimulate thiol oxidation (3,5,22,23,31), were removed (Fig. 4).…”

Section: Resultsmentioning

confidence: 99%

“…Thiols undergo oxidation with resultant concentration-and time-dependent generation of superoxide (3,5,7,22,23,31). Dithiols (e.g.…”

Section: Resultsmentioning

confidence: 99%

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Thiols Mediate Superoxide-dependent NADH Modification of Glyceraldehyde-3-phosphate Dehydrogenase

Rivera–Nieves¹,

Thompson²,

Levine³

et al. 1999

Journal of Biological Chemistry

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Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is covalently modified by NAD in the presence of nitric oxide (NO) and dithiothreitol. Replacement of NAD with NADH in the presence of SIN-1 (3-morpholinosydnonimine) and dithiothreitol increased modification 25-fold. We now demonstrate that in contrast to NO-mediated attachment of NAD, covalent attachment of NADH to GAPDH proceeds in the presence of low molecular weight thiols, independent of NO. Removal of oxygen and transition metal ions inhibited modification, consistent with a role for reactive oxygen species; inhibition by superoxide dismutase, stimulation by xanthine oxidase/hypoxanthine, and the lack of an effect of catalase supported the hypothesis that superoxide, generated from thiol oxidation, was involved. Electrospray mass spectrometry showed covalent linkage of the NADH molecule to GAPDH. Characterization of the product of phosphodiesterase cleavage demonstrated that linkage to GAPDH occurred through the nicotinamide of NADH. Lys-C digestion of GAPDH, followed by peptide isolation by high performance liquid chromatography, matrix-assisted laser desorption ionization time-of-flight analysis, and Edman sequencing, demonstrated that NADH attachment occurred at Cys-149, the active-site thiol. This thiol linkage was stable to HgCl 2 . Thus, linkage of GAPDH to NADH, in contrast to NAD, occurs in the presence of thiol, is independent of NO, and is mediated by superoxide.Superoxide and nitric oxide (NO) are important free radical mediators of host immunity, either through their direct actions or as precursors of other reactive species (1). Nitric oxide is generated from L-arginine by NO synthases (2), and activated phagocytic cells (i.e. macrophages, neutrophils, monocytes, and eosinophils) produce superoxide through single-electron reduction of molecular oxygen (3). The latter process, catalyzed by NADPH oxidase, is pivotal to the production of other superoxide-derived reactive oxygen species (ROS), 1 i.e. hydroxyl radical, singlet oxygen, hydrogen peroxide, and peroxynitrite. Its importance in immunity is best exemplified by chronic granulomatous disease, a condition in which defects in the NADPH oxidase complex result in predisposition to bacterial and fungal infections (4). In addition to enzymatic synthesis, superoxide may be generated from oxidation of reduced flavins, catecholamines, tetrahydrofolates (3), and low molecular weight thiols, e.g. dithiothreitol (DTT), cysteine, or glutathione (5).Thiols may either stimulate (6) or inhibit (7, 8) NO-dependent reactions. Stimulatory actions are mostly attributed to their ability to transfer NO via transnitrosation reactions (6, 9), whereas inhibitory actions may result from thiol-mediated superoxide generation (7,8). Superoxide reacts rapidly with NO to form peroxynitrite (ONOO Ϫ ) (1, 3, 10, 11), which is rapidly degraded at physiological pH (1, 11). In smooth muscle relaxation and neurotransmission, NO, serving as a signaling molecule, reacts with the heme group of guanylate cyclase and activates that ...

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The reaction of superoxide radical anion with dithiothreitol: a chain process

Cited by 57 publications

References 1 publication

Thiol Oxidase Activity of Copper,Zinc Superoxide Dismutase

Thiol Oxidase Activity of Copper,Zinc Superoxide Dismutase

Multiple Actions of the Anthracycline Daunorubicin on Cardiac Ryanodine Receptors

Thiols Mediate Superoxide-dependent NADH Modification of Glyceraldehyde-3-phosphate Dehydrogenase

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