Metal-catalyzed oxidation induces carbonylation of peroxisomal proteins and loss of enzymatic activities.
Peroxisomes are involved in oxidative metabolic reactions and have the capacity to generate large amounts of reactive oxygen species that could damage biomolecules including their own resident proteins. The purpose of this study was to determine whether peroxisomal proteins are susceptible to oxidation and whether oxidative damage affects their enzymatic activity. Peroxisomal proteins were subjected to metal-catalyzed oxidation (MCO) with CuCl(2)/ascorbate and derivatized with 2,4-dinitrophenylhydrazine which allowed for spectrophotometric quantification of carbonylation. Immunochemical detection of carbonylated peroxisomal proteins, resolved by gel electrophoresis and detected with anti-DNP antibodies, revealed five oxidatively modified proteins with the following molecular weights: 80, 66, 62, 55, and 50 kDa. The proteins at 66, 62, and 55 kDa were identified as malate synthase (MS), isocitrate lyase, and catalase (CAT), respectively. MS and CAT were estimated to contain 2-3 mol of carbonyl/mol of protein as a result of MCO. Enzymatic assays revealed varying degrees of activity loss. Isocitrate lyase and malate synthase showed significant loss of activity while catalase and malate dehydrogenase were less inhibited by carbonylation. Our findings show that peroxisomal proteins are vulnerable to MCO damage and thus may also be affected by in vivo exposure to reactive oxygen species.
Nguyen
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AT
Department of Biological Sciences, The George Washington University, Washington, DC 20052, USA.
Donaldson
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RP
D001205:Ascorbic Acid / Q000737:chemistry / Q000494:pharmacology; D002367:Ricinus communis / Q000201:enzymology*; D002384:Catalysis; D003300:Copper / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D004798:Enzymes / Q000032:analysis* / Q000378:metabolism; D016166:Free Radical Scavengers / Q000494:pharmacology; D010084:Oxidation-Reduction / Q000187:drug effects; D020675:Peroxisomes / Q000201:enzymology*; D010940:Plant Proteins / Q000032:analysis* / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects / Q000502:physiology; D017382:Reactive Oxygen Species / Q000378:metabolism; D012639:Seeds / Q000201:enzymology*
D004798:Enzymes; D016166:Free Radical Scavengers; D010940:Plant Proteins; D017382:Reactive Oxygen Species; D003300:Copper; D001205:Ascorbic Acid