carboxypeptidase-b and 5-5-dimethyl-1-pyrroline-1-oxide

Post-translational modification of proteins due to exposure to radicals and other reactive species are markers of metabolic and inflammatory oxidative stress such as sepsis. This study uses the nitrone spin-trap DMPO and a combination of immuno-spin trapping and mass spectrometry to identify in vivo products of radical reactions in mice. We report the detection of dose-dependent production of DMPO-carboxypeptidase B1 (CPB1) adducts in the spleens of mice treated with lipopolysaccharide (LPS). Additionally, we report significant detection of DMPO-CPB1 adducts in mice experiencing normal physiological conditions. Treatments with inhibitors and experiments with knock-out mice indicate that xanthine oxidase and endothelial nitric oxide synthase are important sources of the reactive species that lead to CPB1 adduct formation. We also report a significant loss of CPB1 activity following LPS challenge in conjunction with an increase in CPB1 protein accumulation. This suggests the presence of a possible mechanism for CPB1 activity loss with compensatory protein production.

Topics: Allopurinol; Amidines; Animals; Benzylamines; Carboxypeptidase B; Cross-Linking Reagents; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Free Radicals; Immunoprecipitation; Lipopolysaccharides; Male; Mass Spectrometry; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitric Oxide Synthase Type III; Protein Processing, Post-Translational; Shock, Septic; Spleen; Xanthine Oxidase