Premium
Role of Se‐glutathione peroxidase‐1(GPX1) in catalyzing nitrotyrosine formation in murine hepatic proteins
Author(s) -
Yang Fang,
Lei Xin Gen
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.156.8
Subject(s) - gpx1 , chemistry , nitrotyrosine , peroxynitrite , microbiology and biotechnology , biochemistry , peroxidase , enzyme , in vitro , glutathione , glutathione peroxidase , biology , nitric oxide synthase , superoxide
We reported that primary hepatocytes of GPX1 null mice (GPX1−/−) were more resistant to peroxynitrate (PN)‐induced cell death than those of the wild‐type mice (WT). The present study was conducted to determine if and how GPX1 catalyzed nitrotyrosine formation in murine hepatic proteins in vitro. In Exp. 1, liver samples of GPX1−/− and WT (9 to12 wk old) were homogenized in 100 mM Tris HCl, 0.25M sucrose buffer, pH 7.2, and centrifuged (105,000 g) for 1 h at 4°C. The supernatant (500 μg protein) was treated with 0.18 μmol peroxynitrite (Calbiochem, San Diego, CA) at 37°C for 5 min. Samples from the WT, but not the GPX1−/−, showed strong bands of nitrotyrosine by Western analysis using mouse anti‐nitrotyrosine antibody (Upstate Biotechnology, Lake Placid, NY). In Exp. 2, bovine GPX1 enzyme (Sigma‐Aldrich, St. Louis, MO) was added to liver homogenates of both genotypes before the PN treatment. There was a GPX1 activity‐dependent increase in nitrotyrosine formation in hepatic proteins of both genotypes. In Exp. 3, nitrated proteins of liver homogenates were immuno‐precipitated by anti‐nitrotyrosine agarose beads and subject to nanoLC‐MS/MS after the PN treatment. Presence of intrinsic or extrinsic GPX1 activity in the reaction mixture resulted in five unique nitrated proteins. In conclusion, GPX1 enzyme was able to catalyze or promote the PN‐mediated mouse hepatic protein nitration in vitro [NIH DK 53018 to XGL].