Poster: Oxidative Stress

Abs # 136: Protein Oxidation in Peroxisomes

Presenter:	Donaldson, Robert P, robdon@gwu.edu
Authors	Donaldson, Robert P (A)   Yanik, Tulin O (A)   Kwak, Yoon  (A)   Balakrishnan, Sudheer  (A)
Affiliations:	(A): George Washington University

Oxidative metabolism in peroxisomes generates H₂O₂ and free radicals. These reactive oxygen species (ROS) may not all be captured by the protective enzymes and have the potential to cause oxidative damage to proteins. We investigated the oxidation, specifically carbonylation, of proteins in glyoxysomes isolated from the endosperm of germinating castor beans (Ricinus communis). Carbonylation of glyoxysomal protein was detected by dinitrophenylhydrazine (DNP) derivatization and detection with DNP antibody in immunoblots. Several of the glyoxysomal proteins became oxidized during a 2 h incubation, especially when ascorbate and Fe or Cu was present. The carbonylated proteins included the glyoxylate cycle enzymes, isocitrate lyase (ICL), malate synthase, and malate dehydrogenase, as well as catalase (CAT). A few of the proteins were found to be oxidized in freshly isolated glyoxysomes, suggesting that some protein oxidation occurs in vivo. However, oxidation had no significant effect on the measured enzymatic activities of any of these proteins, that is there was no correlation of enzymatic activity for each of these proteins with the intensity of carbonylation of the respective protein. Thus, in addition to being protected by the presence of enzymes that degrade ROS, such as superoxide dismutases, CAT and ascorbate peroxidase, the glyoxysomal proteins are apparently resistant to the effects of carbonylation and have inherent antioxidant capacity. ICL and its product glyoxylate were degraded by H₂O₂ when CAT was inactive. ICL may be subject to sulfhydryl oxidation rather that carbonylation. ICL may be protected by a physical association with CAT which was detected by gel filtration and chemical cross-linking.