Oxidation of Peroxiredoxin 6 in the Presence of GSH Increases its Phospholipase A2 Activity at Cytoplasmic pH
The expression of the phospholipase A2 activity (aiPLA2) of peroxiredoxin 6 (Prdx6) in the cell cytoplasm is physiologically relevant for the repair of peroxidized cell membranes, but aiPLA2 assay in vitro indicates that, unlike assay at pH 4, activity at cytosolic pH is essentially absent with non-...
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| Format: | Article |
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MDPI AG
2018-12-01
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| Series: | Antioxidants |
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| Online Access: | http://www.mdpi.com/2076-3921/8/1/4 |
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| author | Suiping Zhou Chandra Dodia Sheldon I. Feinstein Sandra Harper Henry J. Forman David W. Speicher Aron B. Fisher |
| author_facet | Suiping Zhou Chandra Dodia Sheldon I. Feinstein Sandra Harper Henry J. Forman David W. Speicher Aron B. Fisher |
| author_sort | Suiping Zhou |
| collection | DOAJ |
| description | The expression of the phospholipase A2 activity (aiPLA2) of peroxiredoxin 6 (Prdx6) in the cell cytoplasm is physiologically relevant for the repair of peroxidized cell membranes, but aiPLA2 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 aiPLA2 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 PLA2 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 H2O2 or the presence of oxidized phospholipid as substrate also significantly increased aiPLA2 activity at pH 7. We conclude that the oxidation of the peroxidatically active Cys47 of Prdx6 results in an increase of aiPLA2 activity at pH 7 without effect on the activity of the enzyme at pH 4. |
| first_indexed | 2024-03-12T06:49:33Z |
| format | Article |
| id | doaj.art-b6c364e284a04dae9398f0b0d70d725a |
| institution | Directory Open Access Journal |
| issn | 2076-3921 |
| language | English |
| last_indexed | 2024-03-12T06:49:33Z |
| publishDate | 2018-12-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Antioxidants |
| spelling | doaj.art-b6c364e284a04dae9398f0b0d70d725a2023-09-03T00:24:16ZengMDPI AGAntioxidants2076-39212018-12-0181410.3390/antiox8010004antiox8010004Oxidation of Peroxiredoxin 6 in the Presence of GSH Increases its Phospholipase A2 Activity at Cytoplasmic pHSuiping Zhou0Chandra Dodia1Sheldon I. Feinstein2Sandra Harper3Henry J. Forman4David W. Speicher5Aron B. Fisher6Institute for Environmental Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USAInstitute for Environmental Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USAInstitute for Environmental Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USACenter for Systems and Computational Biology, The Wistar Institute, Philadelphia, PA 19104, USALeonard Davis School of Gerontology, The University of Southern California, Los Angeles, CA 19104, USACenter for Systems and Computational Biology, The Wistar Institute, Philadelphia, PA 19104, USAInstitute for Environmental Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USAThe expression of the phospholipase A2 activity (aiPLA2) of peroxiredoxin 6 (Prdx6) in the cell cytoplasm is physiologically relevant for the repair of peroxidized cell membranes, but aiPLA2 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 aiPLA2 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 PLA2 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 H2O2 or the presence of oxidized phospholipid as substrate also significantly increased aiPLA2 activity at pH 7. We conclude that the oxidation of the peroxidatically active Cys47 of Prdx6 results in an increase of aiPLA2 activity at pH 7 without effect on the activity of the enzyme at pH 4.http://www.mdpi.com/2076-3921/8/1/4substrate bindingsulfinic acidPrdx6 structuremass spectroscopic analysis |
| spellingShingle | Suiping Zhou Chandra Dodia Sheldon I. Feinstein Sandra Harper Henry J. Forman David W. Speicher Aron B. Fisher Oxidation of Peroxiredoxin 6 in the Presence of GSH Increases its Phospholipase A2 Activity at Cytoplasmic pH Antioxidants substrate binding sulfinic acid Prdx6 structure mass spectroscopic analysis |
| title | Oxidation of Peroxiredoxin 6 in the Presence of GSH Increases its Phospholipase A2 Activity at Cytoplasmic pH |
| title_full | Oxidation of Peroxiredoxin 6 in the Presence of GSH Increases its Phospholipase A2 Activity at Cytoplasmic pH |
| title_fullStr | Oxidation of Peroxiredoxin 6 in the Presence of GSH Increases its Phospholipase A2 Activity at Cytoplasmic pH |
| title_full_unstemmed | Oxidation of Peroxiredoxin 6 in the Presence of GSH Increases its Phospholipase A2 Activity at Cytoplasmic pH |
| title_short | Oxidation of Peroxiredoxin 6 in the Presence of GSH Increases its Phospholipase A2 Activity at Cytoplasmic pH |
| title_sort | oxidation of peroxiredoxin 6 in the presence of gsh increases its phospholipase a2 activity at cytoplasmic ph |
| topic | substrate binding sulfinic acid Prdx6 structure mass spectroscopic analysis |
| url | http://www.mdpi.com/2076-3921/8/1/4 |
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