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Metabolism ofReactive Oxygen Species
in Peroxisomes:
Rob P. DonaldsonDepartment of Biological Sciences
George Washington [email protected]
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Reactive Oxygen Metabolism in Peroxisomes
Peroxisomal oxidases produce Hydrogen Peroxide in Liver, Yeast, Leaves, Germinating oil seeds.
Peroxisomal proteins are subject to oxidation.
Prevention of protein oxidation in peroxisomes: Catalase, Ascorbate Peroxidase, Ascorbate Free Radical Reductase NADH oxidation cycle within peroxisomes
Yeast Liver Leaf
Malate Synthase
Malate Dehydrogenase
Citrate Synthase Aconitase
Fe
Isocitrate lyase
Catalase
Multifunctional hydratase dehydrogenase
Acyl CoAOxidase
H2O2
H2O2
H2O2
H2O2
H2O2
Thiolase
Acetyl-CoA
Fatty Acid
Glyoxylate
Oxidation
Glyoxylate Cycle
Succinate,Glucose
O2
Proteins that are targeted to peroxisomes:
Peroxisomal Oxidases bypass Mitochondrial ATP generation-hydroxyacid OxidaseGlycolate Oxidase (Tolbert)D-amino acid oxidaseXanthine OxidaseUrate Oxidase (de Duve)NADH OxidaseAcyl-CoA Oxidase (Beevers)
The peroxisomal oxidases generate Reactive Oxygen SpeciesAcyl-CoA + O2 ----> Enoyl-CoA + H2O2
Xanthine + O2 ----> Urate + O2•
Catalase scavenges Hydrogen Peroxide (de Duve) 2H2O2 ----> H2O + O2
Ascorbate Peroxidase, also, in membrane of plant peroxisomes
Glyxoysomal proteins, including Catalase CAT and Isocitrate Lyase ICL, are oxidized by H2O2 when CAT is inhibited by amino triazole (AT).
Immunoblot detects oxidized proteins dervitized with Dinitrophenyl Hydrazine (DNP).
Yanik, 2005
Isocitrate Lyase (ICL) is associated with Catalase (CAT) within peroxisomes.
ICL and CAT proteins co-purify,
The two proteins can be chemically cross-linked
ICL co-immunoprecipitates with CAT
Catalase Far-western blot indicates affinity for ICL
ICL CAT
Yanik, 2005
Peroxisomal proteins are oxidized by incubation with Cu (10 uM) and Ascorbate (4 mM) which bypasses Catalase protection.
Carbonylated proteins were detected as DNP derivitives with anti-DNP antibody
(B) The same nitrocellulose membrane was stripped with 3M guanidine thiocyanate. Probed with antibodies against Malate Synthase MS Isocitrate Lyase ICLCatalase CAT.
These superimposed on proteins seen with the DNP.
Spectophotometric determination of total DNP content indicates ~30 nmol DNP per mg protein at 90 min, ~2 mol carbonlyl per mol protein.
METAL CATALYZED PROTEIN OXIDATIONNguyen, 2005
Malate Synthase
Malate Dehydrogenase
Citrate Synthase Aconitase
Isocitrate lyase
Catalase
Multifunctional hydratase dehydrogenase
Acyl CoAOxidase
H2O2
H2O2H2O2
Ascorbate
H2O2
Thiolase
Acetyl-CoA
Fatty Acid
Glyoxylate
APx
AFRNADH
H2O2
H2O2H2O2H2O2
