Limits...
Differential inhibition of Arabidopsis superoxide dismutases by peroxynitrite-mediated tyrosine nitration.

Holzmeister C, Gaupels F, Geerlof A, Sarioglu H, Sattler M, Durner J, Lindermayr C - J. Exp. Bot. (2014)

Bottom Line: Here, we investigated the in vitro effects of nitric oxide derivatives on the seven SOD isoforms of Arabidopsis thaliana.S-nitrosoglutathione, which causes S-nitrosylation of cysteine residues, did not influence SOD activities.The corresponding Tyr34 of human manganese SOD is also nitrated, suggesting that this might be an evolutionarily conserved mechanism for regulation of manganese SODs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemical Plant Pathology, Helmholtz Zentrum München-German Research Center for Environmental Health, 85764 München/Neuherberg, Germany.

No MeSH data available.


Effect of GSNO on enzyme activity of cysteine containing SODs. Recombinant MnSOD, FeSOD3, Cu/ZnSOD1, Cu/ZnSOD2, and Cu/ZnSOD3 were treated with 250 µM (light grey) and 500 µM (white) GSNO for 20min (RT, in dark). Control treatment was done with 500 µM GSNO in presence of 5mM DTT (dark grey). Afterwards the activity was determined. Treatment with light-inactivated GSNO was used as control. These activities were set to 100%. Values represent means±SD of three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4321555&req=5

Figure 3: Effect of GSNO on enzyme activity of cysteine containing SODs. Recombinant MnSOD, FeSOD3, Cu/ZnSOD1, Cu/ZnSOD2, and Cu/ZnSOD3 were treated with 250 µM (light grey) and 500 µM (white) GSNO for 20min (RT, in dark). Control treatment was done with 500 µM GSNO in presence of 5mM DTT (dark grey). Afterwards the activity was determined. Treatment with light-inactivated GSNO was used as control. These activities were set to 100%. Values represent means±SD of three independent experiments.

Mentions: The total SOD activity in atgsnor plants is lower than in WT plants (Supplementary Fig. S5), which is probably related to the higher levels of NO-derivatives in the mutant (Feechan et al., 2005). As the decreased SOD activity in atgsnor cannot be explained by transcriptional regulation (Supplementary Fig. S6), we hypothesized that it is regulated on the protein level. The two most important NO-dependent post-translational modifications are S-nitrosylation of Cys residues and nitration of Tyr residues. Assuming that SOD activity might be inhibited by S-nitrosylation of critical Cys residues, MSD1, FSD3, and all three CSDs, were treated with the S-nitrosylating agent GSNO, as these isoform have at least one cysteine residue. However, none of these SODs was inhibited by GSNO (Fig. 3). Next, we tested the effect of ONOO– on SOD activity. To this end, all SODs, which have at least one Tyr residue (MSD1, all three FSDs, and CSD3) were treated with different concentrations of ONOO–. A concentration-dependent inhibition of MSD1, FSD3, and CSD3 could be observed, whereas the activity of the other two tested FSD isoforms was not affected by this treatment (Fig. 4). Especially MSD1 seems to be very sensitive to this treatment. Its activity decreased to about 10% with 500 µM ONOO–, whereas the activity of FSD3 and CSD3 was reduced to 65%. However, it has to be mentioned that the observed differences in the efficiency of ONOO–-dependent inhibition of the different SODs could be caused by different ratio of applied protein and ONOO–. For a better comparison we calculated the ratio of applied protein per nmol ONOO– for the highest ONOO– concentration used (500 µM) (Fig. 4).


Differential inhibition of Arabidopsis superoxide dismutases by peroxynitrite-mediated tyrosine nitration.

Holzmeister C, Gaupels F, Geerlof A, Sarioglu H, Sattler M, Durner J, Lindermayr C - J. Exp. Bot. (2014)

Effect of GSNO on enzyme activity of cysteine containing SODs. Recombinant MnSOD, FeSOD3, Cu/ZnSOD1, Cu/ZnSOD2, and Cu/ZnSOD3 were treated with 250 µM (light grey) and 500 µM (white) GSNO for 20min (RT, in dark). Control treatment was done with 500 µM GSNO in presence of 5mM DTT (dark grey). Afterwards the activity was determined. Treatment with light-inactivated GSNO was used as control. These activities were set to 100%. Values represent means±SD of three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4321555&req=5

Figure 3: Effect of GSNO on enzyme activity of cysteine containing SODs. Recombinant MnSOD, FeSOD3, Cu/ZnSOD1, Cu/ZnSOD2, and Cu/ZnSOD3 were treated with 250 µM (light grey) and 500 µM (white) GSNO for 20min (RT, in dark). Control treatment was done with 500 µM GSNO in presence of 5mM DTT (dark grey). Afterwards the activity was determined. Treatment with light-inactivated GSNO was used as control. These activities were set to 100%. Values represent means±SD of three independent experiments.
Mentions: The total SOD activity in atgsnor plants is lower than in WT plants (Supplementary Fig. S5), which is probably related to the higher levels of NO-derivatives in the mutant (Feechan et al., 2005). As the decreased SOD activity in atgsnor cannot be explained by transcriptional regulation (Supplementary Fig. S6), we hypothesized that it is regulated on the protein level. The two most important NO-dependent post-translational modifications are S-nitrosylation of Cys residues and nitration of Tyr residues. Assuming that SOD activity might be inhibited by S-nitrosylation of critical Cys residues, MSD1, FSD3, and all three CSDs, were treated with the S-nitrosylating agent GSNO, as these isoform have at least one cysteine residue. However, none of these SODs was inhibited by GSNO (Fig. 3). Next, we tested the effect of ONOO– on SOD activity. To this end, all SODs, which have at least one Tyr residue (MSD1, all three FSDs, and CSD3) were treated with different concentrations of ONOO–. A concentration-dependent inhibition of MSD1, FSD3, and CSD3 could be observed, whereas the activity of the other two tested FSD isoforms was not affected by this treatment (Fig. 4). Especially MSD1 seems to be very sensitive to this treatment. Its activity decreased to about 10% with 500 µM ONOO–, whereas the activity of FSD3 and CSD3 was reduced to 65%. However, it has to be mentioned that the observed differences in the efficiency of ONOO–-dependent inhibition of the different SODs could be caused by different ratio of applied protein and ONOO–. For a better comparison we calculated the ratio of applied protein per nmol ONOO– for the highest ONOO– concentration used (500 µM) (Fig. 4).

Bottom Line: Here, we investigated the in vitro effects of nitric oxide derivatives on the seven SOD isoforms of Arabidopsis thaliana.S-nitrosoglutathione, which causes S-nitrosylation of cysteine residues, did not influence SOD activities.The corresponding Tyr34 of human manganese SOD is also nitrated, suggesting that this might be an evolutionarily conserved mechanism for regulation of manganese SODs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemical Plant Pathology, Helmholtz Zentrum München-German Research Center for Environmental Health, 85764 München/Neuherberg, Germany.

No MeSH data available.