Oxidative modification of citrate synthase by peroxyl radicals and protection with novel antioxidants.
In mammals, aging is linked to a decline in the activity of citrate synthase (CS; E.C. 2.3.3.1), the first enzyme of the citric acid cycle. We used 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), a water-soluble generator of peroxyl and alkoxyl radicals, to investigate the susceptibility of CS to oxidative damage. Treatment of isolated mitochondria with AAPH for 8-24 h led to CS inactivation; however, the activity of aconitase, a mitochondrial enzyme routinely used as an oxidative stress marker, was unaffected. In addition to enzyme inactivation, AAPH treatment of purified CS resulted in dityrosine formation, increased protein surface hydrophobicity, and loss of tryptophan fluorescence. Propyl gallate, 1,8-naphthalenediol, 2,3-naphthalenediol, ascorbic acid, glutathione, and oxaloacetate protected CS from AAPH-mediated inactivation, with IC(50) values of 9, 14, 34, 37, 150, and 160 muM, respectively. Surprisingly, the antioxidant epigallocatechin gallate offered no protection against AAPH, but instead caused CS inactivation. Our results suggest that the current practice of using the enzymatic activity of CS as an index of mitochondrial abundance and the use of aconitase activity as an oxidative stress marker may be inappropriate, especially in oxidative stress-related studies, during which alkyl peroxyl and alkoxyl radicals can be generated.
Chepelev
Nikolai L
NL
Institute of Biochemistry, Carleton University, Ottawa, Ontario, Canada.
Bennitz
Joshua D
JD
Wright
James S
JS
Smith
Jeffrey C
JC
Willmore
William G
WG
D000154:Aconitate Hydratase / Q000037:antagonists & inhibitors / Q000378:metabolism; D000578:Amidines / Q000494:pharmacology / Q000633:toxicity; D000975:Antioxidants / Q000494:pharmacology*; D001205:Ascorbic Acid / Q000494:pharmacology; D002392:Catechin / Q000031:analogs & derivatives / Q000494:pharmacology / Q000633:toxicity; D002950:Citrate (si)-Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004789:Enzyme Activation; D005978:Glutathione / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D008928:Mitochondria / Q000201:enzymology; D009284:Naphthols / Q000494:pharmacology; D062907:Oxaloacetic Acid / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology* / Q000633:toxicity; D010084:Oxidation-Reduction / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects* / Q000502:physiology; D010545:Peroxides / Q000494:pharmacology* / Q000633:toxicity; D011435:Propyl Gallate / Q000494:pharmacology
D000578:Amidines; D000975:Antioxidants; D009284:Naphthols; D016877:Oxidants; D010545:Peroxides; D062907:Oxaloacetic Acid; C049375:perhydroxyl radical; C046728:2,2'-azobis(2-amidinopropane); D011435:Propyl Gallate; D002392:Catechin; C045651:epigallocatechin gallate; D002950:Citrate (si)-Synthase; D000154:Aconitate Hydratase; D005978:Glutathione; D001205:Ascorbic Acid