Knockout of SOD1 decreased GPX1 activity but not GPX1 protein in murine liver

Abstract

We previously reported a 40% decrease of liver glutathione peroxidase 1 (GPX1) activity in copper,zinc superoxide dismutase knockout (SOD1‐/‐) mice. This study determined respective changes in hepatic GPX1 and SOD1 protein expression in the SOD1‐/‐ and GPX1 knockout (GPX1‐/‐) mice. Two experiments were conducted with 6 SOD1‐/‐ and GPX1‐/‐ male mice (8 wk), along with their respective wild‐type (WT) littermates. Liver SOD1 activities were similar between the GPX1‐/‐ and the WT mice (1,073 ± 72 vs. 1,128 ± 81 U/mg protein). In contrast, liver GPX1 activity was lower (P < 0.05) than WT mice (487 ± 56 vs. 822 ± 45 U/mg protein). Western blot analysis of liver homogenates showed no alteration of either enzyme protein amount from the WT by deleting the other enzyme. To explore physical interactions of SOD1 and GPX1 proteins, we performed co‐immunoprecipation (Co‐IP) with liver homogenates and HepG2 cells lysates. Samples were prepared in NP40 buffer, and Co‐IP was conducted with 100 µg protein of homogenates, 1 µg of anti‐SOD1 (Cell Signal, Boston, MA) or anti‐GPX1 (Abfrontier, Seoul, S. Korea) antibody, and 20 µl of A/G‐coupled agarose beads (Abcam, Cambridge, MA). The Western analysis showed no Co‐IP of either enzyme protein by the antibody against the other enzyme. In conclusion, the decreased liver GPX1 activity in the SOD1‐/‐ was likely mediated by factors other than changes of the enzyme protein abundance (NIH DK 53018).