It is well established by our group that the oxidation of proteins, either by metal- catalyzed oxidation systems or other oxidative stresses, leads to the increased formation of carbonyl groups and decreased catalytic activity. In order to continue to function, cells must be able to remove or repair such damaged proteins. This project has focused upon identifying mechanisms that might repair or eliminate damaged proteins. An extensive search has been made for carbonyl reductase activity in eye, lung, and liver extracts, using oxidized glutamine synthetase and a variety of reducing agents as substrates. No such activity was observed using isoelectric focusing, tritium incorporation, or disappearance of carbonyl groups as detection systems. In addition, a range of purified enzymes, which might be expected to possess peptide-bound carbonyl reductase activity were checked for such activity. Since it has previously been shown in vivo that oxidized proteins are more susceptible to degradation by the multicatalytic proteasome and that this represents the major cellular pathway whereby such proteins are degraded, this project is currently examining whether the activity and subunit composition of the proteasome are altered in response to an oxidative stress by hydrogen peroxide in an HeLa cell culture system.
