Abstract
The expression of the phospholipase A&sub2; activity (aiPLA&sub2;) of peroxiredoxin 6 (Prdx6) in the cell cytoplasm is physiologically relevant for the repair of peroxidized cell membranes, but aiPLA&sub2; 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&sub2; 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&sub2; 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&sub2;O&sub2; or the presence of oxidized phospholipid as substrate also significantly increased aiPLA&sub2; activity at pH 7. We conclude that the oxidation of the peroxidatically active Cys47 of Prdx6 results in an increase of aiPLA&sub2; activity at pH 7 without effect on the activity of the enzyme at pH 4.