scholarly journals Nitric oxide-dependent NAD linkage to glyceraldehyde-3-phosphate dehydrogenase: possible involvement of a cysteine thiyl radical intermediate

1996 ◽  
Vol 319 (2) ◽  
pp. 369-375 ◽  
Author(s):  
Maurizio MINETTI ◽  
Donatella PIETRAFORTE ◽  
A. M. Michela DI STASI ◽  
Cinzia MALLOZZI
Keyword(s):  
Nitric Oxide ◽  
Free Radical ◽  
Active Site ◽  
Superoxide Radical ◽  
Spin Trapping ◽  
Cysteine Residue ◽  
Thiyl Radical ◽  
Radical Intermediate ◽  
No Donors ◽  
High Enzyme

Previous studies have demonstrated that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) undergoes NAD(H) linkage to an active site thiol when it comes into contact with •NO-related oxidants. We found that a free-radical generator 2,2´-azobis-(2-amidinopropane) hydrochloride (AAPH), which does not release either •NO or •NO-related species, was indeed able to induce the NAD(H) linkage to GAPDH. We performed spin-trapping studies with purified apo-GAPDH to identify a putative thiol intermediate produced by AAPH as well as by •NO-related oxidants. As •NO sources we used •NO gas and two •NO-donors, S-nitroso-N-acetyl-D,L-penicillamine and 3-morpholinosydnonimine hydrochloride (SIN-1). Because SIN-1 produces •NO and a superoxide radical simultaneously, we also tested the effects of peroxynitrite. All the •NO-related oxidants were able to induce the linkage of NAD(H) to GAPDH and the formation of a protein free-radical identified as a thiyl radical (inhibited by N-ethylmaleimide). •NO gas and the •NO-donors required molecular oxygen to induce the formation of the GAPDH thiyl radical, suggesting the possible involvement of higher nitrogen oxides. Thiyl radical formation was decreased by the reconstitution of GAPDH with NAD+. Apo-GAPDH was a strong scavenger of AAPH radicals, but its scavenging ability was decreased when its cysteine residues were alkylated or when it was reconstituted with NAD+. In addition, after treatment with AAPH, a thiyl radical of GAPDH was trapped at high enzyme concentrations. We suggest that the NAD(H) linkage to GAPDH is mediated by a thiyl radical intermediate not specific to •NO or •NO-related oxidants. The cysteine residue located at the active site of GAPDH (Cys-149) is oxidized by free radicals to a thiyl radical, which reacts with the neighbouring coenzyme to form Cys-NAD(H) linkages. Studies with the NAD+ molecule radiolabelled in the nicotinamide or adenine portion revealed that both portions of the NAD+ molecule are linked to GAPDH.

scholarly journals Nitric Oxide Inhibits Caspase Activation and Apoptotic Morphology but Does Not Rescue Neuronal Death

2005 ◽  
Vol 25 (3) ◽  
pp. 348-357 ◽  
Author(s):  
Ping Zhou ◽  
Liping Qian ◽  
Costantino Iadecola
Keyword(s):  
Nitric Oxide ◽  
Cell Death ◽  
Cell Viability ◽  
Cell Survival ◽  
Caspase 3 ◽  
Apoptotic Cell ◽  
Cysteine Residue ◽  
Caspase Activity ◽  
No Donors ◽  
Apoptotic Morphology

Nitric oxide (NO) has been shown to inhibit apoptotic cell death by S-nitrosylation of the catalytic-site cysteine residue of caspases. However, it is not clear whether in neurons NO-mediated caspase inactivation leads to improved cell survival. To address this issue, we studied the effect of NO donors on caspase activity and cell survival in cortical neuronal culture treated with the apoptosis inducer staurosporine (STS) and camptothecin. In parallel, cell viability was assessed by the MTS assay and MAP2 staining. We found that NO donors ((±)- S-nitroso- N-acetylpenicillamine, S-nitrosoglutathione, and NONOates) dose-dependently inhibited caspase-3 and -9 activity induced by STS and camptothecin. The reduction in caspase-3 activity was, in large part, because of the blockage of the proteolytic conversion of pro-caspase-3 to active caspase-3. NO donors also inhibited the appearance of the classical apoptotic nuclear morphology. However, inhibition of both caspase activity and apoptotic morphology was not associated with enhancement of cell viability. Thus, inhibition of caspase and apoptotic morphology by NO donors does not improve neuronal survival. The data suggest that inhibition of caspase by NO unmasks a caspase-independent form of cell death. A better understanding of this form of cell death may provide new strategies for neuroprotection in neuropathologies, such as ischemic brain injury, associated with apoptosis.

Spin trapping of a free radical intermediate formed during microsomal metabolism of hydrazine

1985 ◽  
Vol 133 (3) ◽  
pp. 1086-1091 ◽  
Author(s):  
Atsuko Noda ◽  
Hiroshi Noda ◽  
Kohji Ohno ◽  
Toshiaki Sendo ◽  
Ayako Misaka ◽  
...  
Keyword(s):  
Free Radical ◽  
Spin Trapping ◽  
Radical Intermediate ◽  
Microsomal Metabolism ◽  
Free Radical Intermediate

scholarly journals Anti-Inflammatory CeO2 Nanoparticles Prevented Cytotoxicity Due to Exogenous Nitric Oxide Donors via Induction Rather Than Inhibition of Superoxide/Nitric Oxide in HUVE Cells

Molecules ◽  
2021 ◽  
Vol 26 (17) ◽  
pp. 5416
Author(s):  
Mohd Javed Akhtar ◽  
Maqusood Ahamed ◽  
Hisham Alhadlaq
Keyword(s):  
Nitric Oxide ◽  
Superoxide Radical ◽  
Underlying Mechanism ◽  
No Production ◽  
Oxygen Species ◽  
No Donors ◽  
Mitochondrial Data ◽  
Tnf Α ◽  
Exogenous Nitric Oxide ◽  
O2 Production

The mechanism behind the cytoprotective potential of cerium oxide nanoparticles (CeO2 NPs) against cytotoxic nitric oxide (NO) donors and H2O2 is still not clear. Synthesized and characterized CeO2 NPs significantly ameliorated the lipopolysaccharide (LPS)-induced cytokines IL-1β and TNF-α. The main goal of this study was to determine the capacities of NPs regarding signaling effects that could have occurred due to reactive oxygen species (ROS) and/or NO, since NP-induced ROS/NO did not lead to toxicity in HUVE cells. Concentrations that induced 50% cell death (i.e., IC50s) of two NO donors (DETA-NO; 1250 ± 110 µM and sodium nitroprusside (SNP); 950 ± 89 µM) along with the IC50 of H2O2 (120 ± 7 µM) were utilized to evaluate cytoprotective potential and its underlying mechanism. We determined total ROS (as a collective marker of hydrogen peroxide, superoxide radical (O2•−), hydroxyl radical, etc.) by DCFH-DA and used a O2•− specific probe DHE to decipher prominent ROS. The findings revealed that signaling effects mediated mainly by O2•− and/or NO are responsible for the amelioration of toxicity by CeO2 NPs at 100 µg/mL. The unaltered effect on mitochondrial membrane potential (MMP) due to NP exposure and, again, CeO2 NPs-mediated recovery in the loss of MMP due to exogenous NO donors and H2O2 suggested that NP-mediated O2•− production might be extra-mitochondrial. Data on activated glutathione reductase (GR) and unaffected glutathione peroxidase (GPx) activities partially explain the mechanism behind the NP-induced gain in GSH and persistent cytoplasmic ROS. The promoted antioxidant capacity due to non-cytotoxic ROS and/or NO production, rather than inhibition, by CeO2 NP treatment may allow cells to develop the capacity to tolerate exogenously induced toxicity.

scholarly journals Free Radical Scavenging Activity of Aerial Parts of Artemisia pallens in Mediating Hepatoprotective Activity of RIF+INH Intoxicated Rats

2019 ◽  
Vol 14 ◽  
pp. 1-15
Author(s):  
Praveen Kumar Ashok ◽  
Poonam Rishishwar ◽  
Kumud Upadhyaya
Keyword(s):  
Nitric Oxide ◽  
Free Radical ◽  
Superoxide Radical ◽  
Radical Scavenging Activity ◽  
Radical Scavenging ◽  
Hepatoprotective Activity ◽  
Free Radical Scavenging ◽  
Scavenging Activity ◽  
Dry Extract ◽  
Artemisia Pallens

To investigate the free radical scavenging & hepatoprotective activity of phenolic rich fraction of Artemisia pallens on RIF+INH induced oxidative stress in Sprague dawley rats. Free radical scavenging and hepatoprotective activity was evaluated by using DPPH, Nitric oxide, Superoxide radical and Hydroxyl radical assay models and induced RIF+INH intoxicated rats. The total phenolic content was found to be 312.60 μg gallic acid equivalents (GAE)/gm of dry extract. The total flavonoid content was found to be 322.20 μg rutin equivalents (RUE)/gm of dry extract. In the current study, free radical scavenging activity was evaluated by using 1,1-diphenyl-2-picrylhydrazyl (DPPH), nitric oxide, superoxide radical and hydroxyl radical scavenging activity were found to be (42.25 ± 0.95) and (09.16 ± 1.62) μg/ml, (101.62 ± 1.64) and (32.41 ± 1.24) μg/ml, (72.62 ± 1.86) and (10.28 ± 1.96) μg/ml, (33.82 ± 1.12) and (12.82 ± 1.86) μg/ml, respectively. There was also a dose dependent increase in reductive ability of Artemisia pallens extract with increase in concentration and were further investigated in invivo hepatoprotective activity experiment against toxicity induced by RIF+INH. The free radical scavenging and hepatoprotective activity may be attributed to the presence of phenolic compounds and histology of the liver section of the animals treated with the extracts showed the presence of normal hepatic cords, absence of necrosis and fatty infiltration, which further evidenced the hepatoprotective activity of Artemisia pallens.

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