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Oxidation of Peroxiredoxin 6 in the Presence of GSH Increases its Phospholipase A(2) Activity at Cytoplasmic pH

The expression of the phospholipase A(2) activity (aiPLA(2)) of peroxiredoxin 6 (Prdx6) in the cell cytoplasm is physiologically relevant for the repair of peroxidized cell membranes, but aiPLA(2) assay in vitro indicates that, unlike assay at pH 4, activity at cytosolic pH is essentially absent wit...

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Autores principales: Zhou, Suiping, Dodia, Chandra, Feinstein, Sheldon I., Harper, Sandra, Forman, Henry J., Speicher, David W., Fisher, Aron B.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6357108/
https://www.ncbi.nlm.nih.gov/pubmed/30586895
http://dx.doi.org/10.3390/antiox8010004
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author Zhou, Suiping
Dodia, Chandra
Feinstein, Sheldon I.
Harper, Sandra
Forman, Henry J.
Speicher, David W.
Fisher, Aron B.
author_facet Zhou, Suiping
Dodia, Chandra
Feinstein, Sheldon I.
Harper, Sandra
Forman, Henry J.
Speicher, David W.
Fisher, Aron B.
author_sort Zhou, Suiping
collection PubMed
description The expression of the phospholipase A(2) activity (aiPLA(2)) of peroxiredoxin 6 (Prdx6) in the cell cytoplasm is physiologically relevant for the repair of peroxidized cell membranes, but aiPLA(2) assay in vitro indicates that, unlike assay at pH 4, activity at cytosolic pH is essentially absent with non-oxidized substrate. However, the addition of glutathione (GSH) to the assay medium significantly increased aiPLA(2) activity at cytosolic pH, while oxidized GSH (GSSG) and several other thiols had no effect. By mass spectroscopy (ESI MS), the addition of GSH to Prdx6 paradoxically led to oxidation of its conserved Cys47 residue to a sulfinic acid. The effect of GSH on PLA(2) activity was abolished by incubation under anaerobic conditions, confirming that auto-oxidation of the protein was the mechanism for the GSH effect. Analysis by circular dichroism (CD) and tryptophan fluorescence showed alterations of the protein structure in the presence of GSH. Independently of GSH, the oxidation of Prdx6 by exposure to H(2)O(2) or the presence of oxidized phospholipid as substrate also significantly increased aiPLA(2) activity at pH 7. We conclude that the oxidation of the peroxidatically active Cys47 of Prdx6 results in an increase of aiPLA(2) activity at pH 7 without effect on the activity of the enzyme at pH 4.
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spelling pubmed-63571082019-02-04 Oxidation of Peroxiredoxin 6 in the Presence of GSH Increases its Phospholipase A(2) Activity at Cytoplasmic pH Zhou, Suiping Dodia, Chandra Feinstein, Sheldon I. Harper, Sandra Forman, Henry J. Speicher, David W. Fisher, Aron B. Antioxidants (Basel) Article The expression of the phospholipase A(2) activity (aiPLA(2)) of peroxiredoxin 6 (Prdx6) in the cell cytoplasm is physiologically relevant for the repair of peroxidized cell membranes, but aiPLA(2) assay in vitro indicates that, unlike assay at pH 4, activity at cytosolic pH is essentially absent with non-oxidized substrate. However, the addition of glutathione (GSH) to the assay medium significantly increased aiPLA(2) activity at cytosolic pH, while oxidized GSH (GSSG) and several other thiols had no effect. By mass spectroscopy (ESI MS), the addition of GSH to Prdx6 paradoxically led to oxidation of its conserved Cys47 residue to a sulfinic acid. The effect of GSH on PLA(2) activity was abolished by incubation under anaerobic conditions, confirming that auto-oxidation of the protein was the mechanism for the GSH effect. Analysis by circular dichroism (CD) and tryptophan fluorescence showed alterations of the protein structure in the presence of GSH. Independently of GSH, the oxidation of Prdx6 by exposure to H(2)O(2) or the presence of oxidized phospholipid as substrate also significantly increased aiPLA(2) activity at pH 7. We conclude that the oxidation of the peroxidatically active Cys47 of Prdx6 results in an increase of aiPLA(2) activity at pH 7 without effect on the activity of the enzyme at pH 4. MDPI 2018-12-24 /pmc/articles/PMC6357108/ /pubmed/30586895 http://dx.doi.org/10.3390/antiox8010004 Text en © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Zhou, Suiping
Dodia, Chandra
Feinstein, Sheldon I.
Harper, Sandra
Forman, Henry J.
Speicher, David W.
Fisher, Aron B.
Oxidation of Peroxiredoxin 6 in the Presence of GSH Increases its Phospholipase A(2) Activity at Cytoplasmic pH
title Oxidation of Peroxiredoxin 6 in the Presence of GSH Increases its Phospholipase A(2) Activity at Cytoplasmic pH
title_full Oxidation of Peroxiredoxin 6 in the Presence of GSH Increases its Phospholipase A(2) Activity at Cytoplasmic pH
title_fullStr Oxidation of Peroxiredoxin 6 in the Presence of GSH Increases its Phospholipase A(2) Activity at Cytoplasmic pH
title_full_unstemmed Oxidation of Peroxiredoxin 6 in the Presence of GSH Increases its Phospholipase A(2) Activity at Cytoplasmic pH
title_short Oxidation of Peroxiredoxin 6 in the Presence of GSH Increases its Phospholipase A(2) Activity at Cytoplasmic pH
title_sort oxidation of peroxiredoxin 6 in the presence of gsh increases its phospholipase a(2) activity at cytoplasmic ph
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6357108/
https://www.ncbi.nlm.nih.gov/pubmed/30586895
http://dx.doi.org/10.3390/antiox8010004
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