Immuno-spin trapping of a post-translational carboxypeptidase B1 radical formed by a dual role of xanthine oxidase and endothelial nitric oxide synthase in acute septic mice.
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.
Chatterjee
Saurabh
S
Free Radical Metabolites Group, Laboratory of Pharmacology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA. chatterjees2@niehs.nih.gov
Ehrenshaft
Marilyn
M
Bhattacharjee
Suchandra
S
Deterding
Leesa J
LJ
Bonini
Marcelo G
MG
Corbett
Jean
J
Kadiiska
Maria B
MB
Tomer
Kenneth B
KB
Mason
Ronald P
RP
D000493:Allopurinol / Q000008:administration & dosage; D000578:Amidines / Q000008:administration & dosage; D000818:Animals; D001596:Benzylamines / Q000008:administration & dosage; D043424:Carboxypeptidase B / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000008:administration & dosage; D003497:Cyclic N-Oxides / Q000008:administration & dosage; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000032:analysis* / Q000276:immunology; D047468:Immunoprecipitation; D008070:Lipopolysaccharides / Q000008:administration & dosage; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D012772:Shock, Septic / Q000139:chemically induced / Q000201:enzymology / Q000276:immunology*; D013154:Spleen / Q000276:immunology / Q000378:metabolism; D014969:Xanthine Oxidase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000578:Amidines; D001596:Benzylamines; D003432:Cross-Linking Reagents; D003497:Cyclic N-Oxides; D005609:Free Radicals; D008070:Lipopolysaccharides; C104820:N-(3-(aminomethyl)benzyl)acetamidine; D000493:Allopurinol; C017245:5,5-dimethyl-1-pyrroline-1-oxide; D052250:Nitric Oxide Synthase Type III; D014969:Xanthine Oxidase; D043424:Carboxypeptidase B