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Characterization of plant sulfiredoxin and role of sulphinic form of 2-Cys peroxiredoxin.

Iglesias-Baena I, Barranco-Medina S, Lázaro-Payo A, López-Jaramillo FJ, Sevilla F, Lázaro JJ - J. Exp. Bot. (2010)

Bottom Line: The antioxidant function of 2-Cys peroxiredoxin (Prx) involves the oxidation of its conserved peroxidatic cysteine to sulphenic acid that is recycled by a reductor agent.An enzyme recently discovered, named sulfiredoxin (Srx), reduces the sulphinic 2-Cys Prx (Prx-SO(2)H).This work presents the first systematic biochemical characterization of the Srx/Prx system from plants and contributes to a better understanding of its physiological function.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Cellular and Molecular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, C/Profesor Albareda 1, E-18008 Granada, Spain.

ABSTRACT
The antioxidant function of 2-Cys peroxiredoxin (Prx) involves the oxidation of its conserved peroxidatic cysteine to sulphenic acid that is recycled by a reductor agent. In conditions of oxidative stress, the peroxidatic cysteine can be overoxidized to sulphinic acid inactivating the Prx. An enzyme recently discovered, named sulfiredoxin (Srx), reduces the sulphinic 2-Cys Prx (Prx-SO(2)H). To explore the physiological functions of Srx in plants we have cloned, expressed and purified to homogeneity a Srx from Arabidopsis thaliana (AtSrx), as well as five variants by site-directed mutagenesis on amino acids involved in its activity. The activity of sulfiredoxin, determined by a new method, is dependent on the concentration of the sulphinic form of Prx and the conserved Srx is capable of regenerating the functionality of both pea and Arabidopsis Prx-SO(2)H. Molecular modelling of AtSrx and the facts that the R28Q variant shows a partial inactivation, that the activity of the E76A variant is equivalent to that of the native enzyme and that the double mutation R28Q/E76A abolishes the enzymatic activity suggests that the pair His100-Glu76 may be involved in the activation of C72 in the absence of R28. The knock-out mutant plants without Srx or 2-Cys Prx exhibited phenotypical differences under growth conditions of 16 h light, probably due to the signalling role of the sulphinic form of Prx. These mutants showed more susceptibility to oxidative stress than wild-type plants. This work presents the first systematic biochemical characterization of the Srx/Prx system from plants and contributes to a better understanding of its physiological function.

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Correlation between Srx activity measured by the determination of released phosphate and that using specific antibodies against hyperoxidized Prx. Reaction mixture containing 50 mM TRIS-HCl (pH 7.5), 1 mM MgCl2, 250 μM ATP, 10 mM GSH, 15 μM Prx-SO2H, and 5 μM Srx was incubated at 30 °C. (A) At the indicated times, aliquots were withdrawn and subjected to Western blot with specific antibodies against Prx-SO2H and 2-Cys Prx (control). (B) The concentration of Prx-SO2H reduced was determined from the corresponding Western blot band intensity (by analysed imaging) and plotted against the concentration of phosphate determined in the same samples.
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fig4: Correlation between Srx activity measured by the determination of released phosphate and that using specific antibodies against hyperoxidized Prx. Reaction mixture containing 50 mM TRIS-HCl (pH 7.5), 1 mM MgCl2, 250 μM ATP, 10 mM GSH, 15 μM Prx-SO2H, and 5 μM Srx was incubated at 30 °C. (A) At the indicated times, aliquots were withdrawn and subjected to Western blot with specific antibodies against Prx-SO2H and 2-Cys Prx (control). (B) The concentration of Prx-SO2H reduced was determined from the corresponding Western blot band intensity (by analysed imaging) and plotted against the concentration of phosphate determined in the same samples.

Mentions: The simplicity of colorimetric assay is especially attractive and further evidences of its feasibility were obtained by comparison with the method based on the detection and quantification of the redox state of the Prx by Western blotting (Chang et al., 2004). To this end, the Srx activity of aliquots withdrawn at different times from a sample containing 5 μM AtSrx, 15 μM Prx-SO2H, 10 mM GSH, and 250 μM ATP was determined by quantification of both the sulphinic Prx with specific antibodies and the phosphate by the colorimetric assay. As shown in Fig. 4A, the intensity of the band detected by antibody against the hyperoxidized Prx decreased as the incubation time with AtSrx is longer whereas the intensity of the band detected with antibodies against 2-Cys Prx remained unchanged. The plot of the amount Prx-SO2H reduced by AtSrx estimated from the Western blot, versus the concentration of phosphate (Pi) resulting from the hydrolysis of ATP, reveals a linear relationship with R coefficient of 0.94 and an estimated slope of 0.98 (Fig. 4B) that validates the colorimetric assay for the determination of the sulfiredoxin activity.


Characterization of plant sulfiredoxin and role of sulphinic form of 2-Cys peroxiredoxin.

Iglesias-Baena I, Barranco-Medina S, Lázaro-Payo A, López-Jaramillo FJ, Sevilla F, Lázaro JJ - J. Exp. Bot. (2010)

Correlation between Srx activity measured by the determination of released phosphate and that using specific antibodies against hyperoxidized Prx. Reaction mixture containing 50 mM TRIS-HCl (pH 7.5), 1 mM MgCl2, 250 μM ATP, 10 mM GSH, 15 μM Prx-SO2H, and 5 μM Srx was incubated at 30 °C. (A) At the indicated times, aliquots were withdrawn and subjected to Western blot with specific antibodies against Prx-SO2H and 2-Cys Prx (control). (B) The concentration of Prx-SO2H reduced was determined from the corresponding Western blot band intensity (by analysed imaging) and plotted against the concentration of phosphate determined in the same samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2837264&req=5

fig4: Correlation between Srx activity measured by the determination of released phosphate and that using specific antibodies against hyperoxidized Prx. Reaction mixture containing 50 mM TRIS-HCl (pH 7.5), 1 mM MgCl2, 250 μM ATP, 10 mM GSH, 15 μM Prx-SO2H, and 5 μM Srx was incubated at 30 °C. (A) At the indicated times, aliquots were withdrawn and subjected to Western blot with specific antibodies against Prx-SO2H and 2-Cys Prx (control). (B) The concentration of Prx-SO2H reduced was determined from the corresponding Western blot band intensity (by analysed imaging) and plotted against the concentration of phosphate determined in the same samples.
Mentions: The simplicity of colorimetric assay is especially attractive and further evidences of its feasibility were obtained by comparison with the method based on the detection and quantification of the redox state of the Prx by Western blotting (Chang et al., 2004). To this end, the Srx activity of aliquots withdrawn at different times from a sample containing 5 μM AtSrx, 15 μM Prx-SO2H, 10 mM GSH, and 250 μM ATP was determined by quantification of both the sulphinic Prx with specific antibodies and the phosphate by the colorimetric assay. As shown in Fig. 4A, the intensity of the band detected by antibody against the hyperoxidized Prx decreased as the incubation time with AtSrx is longer whereas the intensity of the band detected with antibodies against 2-Cys Prx remained unchanged. The plot of the amount Prx-SO2H reduced by AtSrx estimated from the Western blot, versus the concentration of phosphate (Pi) resulting from the hydrolysis of ATP, reveals a linear relationship with R coefficient of 0.94 and an estimated slope of 0.98 (Fig. 4B) that validates the colorimetric assay for the determination of the sulfiredoxin activity.

Bottom Line: The antioxidant function of 2-Cys peroxiredoxin (Prx) involves the oxidation of its conserved peroxidatic cysteine to sulphenic acid that is recycled by a reductor agent.An enzyme recently discovered, named sulfiredoxin (Srx), reduces the sulphinic 2-Cys Prx (Prx-SO(2)H).This work presents the first systematic biochemical characterization of the Srx/Prx system from plants and contributes to a better understanding of its physiological function.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Cellular and Molecular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, C/Profesor Albareda 1, E-18008 Granada, Spain.

ABSTRACT
The antioxidant function of 2-Cys peroxiredoxin (Prx) involves the oxidation of its conserved peroxidatic cysteine to sulphenic acid that is recycled by a reductor agent. In conditions of oxidative stress, the peroxidatic cysteine can be overoxidized to sulphinic acid inactivating the Prx. An enzyme recently discovered, named sulfiredoxin (Srx), reduces the sulphinic 2-Cys Prx (Prx-SO(2)H). To explore the physiological functions of Srx in plants we have cloned, expressed and purified to homogeneity a Srx from Arabidopsis thaliana (AtSrx), as well as five variants by site-directed mutagenesis on amino acids involved in its activity. The activity of sulfiredoxin, determined by a new method, is dependent on the concentration of the sulphinic form of Prx and the conserved Srx is capable of regenerating the functionality of both pea and Arabidopsis Prx-SO(2)H. Molecular modelling of AtSrx and the facts that the R28Q variant shows a partial inactivation, that the activity of the E76A variant is equivalent to that of the native enzyme and that the double mutation R28Q/E76A abolishes the enzymatic activity suggests that the pair His100-Glu76 may be involved in the activation of C72 in the absence of R28. The knock-out mutant plants without Srx or 2-Cys Prx exhibited phenotypical differences under growth conditions of 16 h light, probably due to the signalling role of the sulphinic form of Prx. These mutants showed more susceptibility to oxidative stress than wild-type plants. This work presents the first systematic biochemical characterization of the Srx/Prx system from plants and contributes to a better understanding of its physiological function.

Show MeSH
Related in: MedlinePlus