Automated assays for superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase activity

Wheeler, Conrad R.; Salzman, Jhaine A.; Elsayed, Nadin; Omaye, Stanley T.; Korte, Don W.

doi:10.1016/0003-2697(90)90668-y

Cited by 492 publications

(273 citation statements)

References 18 publications

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“…The assay for the assessment of CAT activity is based on the reaction of the enzyme with methanol in the presence of an optimal concentration of H 2 O 2 . The formaldehyde produced was measured spectrophotometrically with 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (Purpald † ) as the chromogen (Wheeler et al 1990). Purpald † specifically forms a bicyclic heterocycle with aldehydes; this bicyclic heterocycle changes from colorless to purple upon oxidation.…”

Section: Cu/zn-sod Cat and Gpx Activity Analysismentioning

confidence: 99%

Effect of hypoosmotic and thermal stress on gene expression and the activity of antioxidant enzymes in the cinnamon clownfish,Amphiprion melanopus

Park

Shin

Choi

et al. 2011

Animal Cells and Systems

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We studied oxidative stress in cinnamon clownfish exposed to hypoosmotic (35 psu 0 17.5 psu and 17.5 psu with prolactin (PRL)) and low temperature (288C 0 248C and 208C) conditions by measuring the expression and activity of Cu/Zn-superoxide dismutase (Cu/Zn-SOD), catalase (CAT), and glutathione peroxidase (GPX). The expression and activity of the antioxidant enzymes were significantly higher after the fish were exposed to 248C, 208C, and 17.5 psu, and expression was repressed by PRL treatment. Furthermore, we measured H 2 O 2 and lipid peroxidation levels and found that they were significantly higher after exposure to the hypoosmotic and low-temperature environments. Additionally, we investigated changes in plasma AST and ALT levels after exposure to low temperature and hypoosmotic stress. These levels increased upon exposure of the clownfish to 248C, 208C, and 17.5 psu, but the levels of these parameters decreased in the 17.5 psu with PRL treatment during a salinity change. The results indicate that hypoosmotic and low-temperature conditions induce oxidative stress in cinnamon clownfish and that the parameters tested in this study may be indices of oxidative stress in the cinnamon clownfish.

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Section: Cu/zn-sod Cat and Gpx Activity Analysismentioning

confidence: 99%

Effect of hypoosmotic and thermal stress on gene expression and the activity of antioxidant enzymes in the cinnamon clownfish,Amphiprion melanopus

Park

Shin

Choi

et al. 2011

Animal Cells and Systems

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“…The method described before (Wheeler et al, 1990) was used to measure the activity of cellular GR. Briefly, to an assay cuvette containing 0.465 ml of 50 mM potassium phosphate buffer (pH 7.0) and 1 mM EDTA, 15 μl of sample and 60 μl of 20 mM GSSG were added.…”

Section: Assay For Gr Activitymentioning

confidence: 99%

Potent induction of total cellular GSH and NQO1 as well as mitochondrial GSH by 3H-1,2-dithiole-3-thione in SH-SY5Y neuroblastoma cells and primary human neurons: Protection against neurocytotoxicity elicited by dopamine, 6-hydroxydopamine, 4-hydroxy-2-nonenal, or hydrogen peroxide

et al. 2008

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Evidence suggests oxidative and electrophilic stress as a major factor contributing to the neuronal cell death in neurodegenerative disorders, especially Parkinson's disease. Consistent with this concept, administration of exogenous antioxidants has been shown to be protective against oxidative/ electrophilic neurodegeneration. However, whether induction of endogenous antioxidants and phase 2 enzymes by the unique chemoprotectant, 3H-1,2-dithiole-3-thione (D3T) in neuronal cells also affords protection against oxidative and electrophilic neurocytotoxicity has not been carefully investigated. In this study, we showed that incubation of SH-SY5Y neuroblastoma cells or primary human neurons with micromolar concentrations (10-100 μM) of D3T for 24 h resulted in significant increases in the levels of reduced glutathione (GSH) and NAD(P)H:quinone oxidoreductase 1 (NQO1), two crucial cellular defenses against oxidative and electrophilic stress. D3T treatment also caused increases in mRNA expression of γ-glutamylcysteine ligase catalytic subunit and NQO1 in SH-SY5Y cells. In addition, D3T treatment of the neuronal cells also resulted in a marked elevation of GSH content in the mitochondrial compartment. To determine the protective effects of the D3T-induced cellular defenses on neurotoxicant-elicited cell injury, SH-SY5Y cells were pretreated with D3T for 24 h and then exposed to dopamine, 6-hydroxydopamine (6-OHDA), 4-hydroxy-2-nonenal (HNE), or H 2 O 2 , agents that are known to be involved in neuron degeneration. We observed that D3T-pretreatment of SH-SY5Y cells led to significant protection against the cytotoxicity elicited by the above neurotoxicants. Similar neurocytoprotective effects of D3T-pretreatment were also observed in primary human neurons exposed to 6-OHDA or HNE. Taken together, this study demonstrates that D3T potently induces neuronal cellular GSH and NQO1 as well as mitochondrial GSH, and that such upregulated endogenous defenses are accompanied by increased resistance to oxidative and electrophilic neurocytotoxicity.

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“…GPx (EC 1.11.1.9) activity was measured in a 96-well plate reader at 37 °C by a coupled assay system [55]. The reaction mixture consisted of 0.2 mM H 2 O 2 , 1.0 mM GSH, 0.14 U of GR, 1.5 mM NADPH, 1.0 mM sodium azide and 0.1M phosphate buffer (pH 7.4) and 10 μl PMS in a total volume of 200 μl.…”

Section: Estimation Of Glutathione Peroxidase Activitymentioning

confidence: 99%

In vivo administration of D609 leads to protection of subsequently isolated gerbil brain mitochondria subjected to in vitro oxidative stress induced by amyloid beta-peptide and other oxidative stressors: Relevance to Alzheimer’s disease and other oxidative stress-related neurodegenerative disorders

Ansari

Joshi

Huang

et al. 2006

Free Radical Biology and Medicine

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Tricyclodecan-9-yl-xanthogenate (D609) has in vivo and in vitro antioxidant properties. D609 mimics glutathione (GSH), has a free thiol group, which upon oxidation forms a disulfide. The resulting dixanthate is a substrate for glutathione reductase, regenerating D609. Recent studies have also shown that D609 protects brain in vivo and neuronal cultures in vitro against the potential Alzheimer's disease (AD) causative factor, Aβ (1-42)-induced oxidative stress and cytotoxicity. Mitochondria are important organelles with both pro-and anti-apoptotic factor proteins. The present study was undertaken to test the hypothesis that i.p. injection of D609 would provide neuroprotection against free radical-induced, mitochondria-mediated apoptosis in vitro. Brain mitochondria were isolated from gerbils 1 h post injection intraperitonially (i.p.) with D609 and subsequently treated in vitro with the oxidants Fe 2+ /H 2 O 2 (hydroxyl free radicals), 2,2-azobis-(2-amidinopropane) dihydrochloride (AAPH, alkoxyl and peroxyl free radicals) and AD-relevant amyloid β-peptide 1-42 [Aβ (1-42)]. Brain mitochondria isolated from the gerbils previously injected i.p. with D609 and subjected to these oxidative stress inducers, in vitro, showed significant reduction in levels of protein carbonyls, protein-bound hydroxynonenal (HNE) [a lipid peroxidation product], 3-nitrotyrosine (3-NT), and cytochrome-c release compared to oxidant-treated brain mitochondria isolated from salineinjected gerbils. D609 treatment significantly maintains the GSH/GSSG ratio in oxidant-treated mitochondria. Increased activity of glutathione-S-transferase (GST), glutathione peroxidase (GPx) and glutathione reductase (GR) in brain isolated from D609-injected gerbils is consistent with the notion that D609 acts like GSH. These anti-apoptotic findings are discussed with reference to the potential use of this brain-accessible glutathione mimetic in the treatment of oxidative stress-related neurodegenerative disorders, including AD.

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Automated assays for superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase activity

Cited by 492 publications

References 18 publications

Effect of hypoosmotic and thermal stress on gene expression and the activity of antioxidant enzymes in the cinnamon clownfish,Amphiprion melanopus

Effect of hypoosmotic and thermal stress on gene expression and the activity of antioxidant enzymes in the cinnamon clownfish,Amphiprion melanopus

Potent induction of total cellular GSH and NQO1 as well as mitochondrial GSH by 3H-1,2-dithiole-3-thione in SH-SY5Y neuroblastoma cells and primary human neurons: Protection against neurocytotoxicity elicited by dopamine, 6-hydroxydopamine, 4-hydroxy-2-nonenal, or hydrogen peroxide

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