carboxypeptidase-b and Shock--Septic

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

Endogenous opioid peptides have been implicated in the pathophysiology of shock (1-5). In anaesthetised mongrel dogs, administration of E coli endotoxin caused a rise in plasma met-enkephalin-like immunoreactivity (MLI). Biochemical characterisation of MLI by gel filtration chromatography revealed various molecular forms: 31K, 8K, 3-5K and the native pentapeptide in approximately equal amounts. After enzymatic treatment of column fractions the 31K form predominated (90.7%). This is the first demonstration of elevated MLI in endotoxin shock.

Topics: Animals; Carboxypeptidase B; Carboxypeptidases; Chromatography, Gel; Dogs; Enkephalin, Methionine; Escherichia coli; Female; Male; Molecular Weight; Shock, Septic; Trypsin