Formation of 3-nitrotyrosines in carbonic anhydrase III is a sensitive marker of oxidative stress in skeletal muscle

Abstract

Oxidation of skeletal muscle proteins has been reported to occur following contractions, with ageing, and with a variety of disease states, but the nature of the oxidised proteins has not been identified. A proteomics approach was utilised to identify major proteins that contain carbonyls and/or 3-nitrotyrosine (3-NT) groups in the gastrocnemius (GTN) muscles of adult (5–11 14months of age) and old (26–28 14months of age) wild type (WT) mice and adult mice lacking copper, zinc superoxide dismutase ( Sod1 −/− mice), manganese superoxide dismutase ( Sod2 +/− mice) or glutathione peroxidase 1 ( GPx1 −/− mice). In quiescent GTN muscles of adult and old WT mice, protein carbonylation and/or formation of 3-NT occurred in several proteins involved in glycolysis, as well as creatine kinase and carbonic anhydrase III. Following contractions, the 3-NT intensity was increased in specific protein bands from GTN muscles of both adult and old WT mice. In quiescent GTN muscles from adult Sod1 −/− , Sod2 +/− or GPx1 −/− mice compared with age-matched WT mice only carbonic anhydrase III showed a greater 3-NT content. We conclude that formation of 3-NT occurs readily in response to oxidative stress in carbonic anhydrase III and this may provide a sensitive measure of oxidative damage to muscle proteins.