Brain mitochondrial complex I inactivation by oxidative modification.
In vitro oxidation of the brain mitochondrial complex I by the hydroxyl radical generating system ascorbate/Fe(III)/O(2) has been carried out. Complex I inactivation, by oxidation, has been studied using a method based on the resolution of proteins by blue native polyacrylamide gel electrophoresis (BN-PAGE), followed by total protein quantification by staining with Coomassie brilliant blue, in-gel activity quantification, and quantification of oxidized proteins by labelling with DIG-hydrazide and immunodetection with an anti-DIG-AP. Quantification was carried out by densitometry procedure. Our results show that oxidation is a continuous process, increasing rapidly at the beginning, reaching a plateau after 8 h of incubation. There is practically no inactivation until a threshold value of damage is reached. Below this, the complex activity is resistant to the aggression of oxygen-reactive substances and free radicals, but once the threshold value is passed, activity is lost rapidly.
Bautista
J
J
Departamento de Bioquímica, Universidad de Sevilla, Seville, 41012, Spain. jdbaut@cica.es
Corpas
R
R
Ramos
R
R
Cremades
O
O
Gutiérrez
J F
JF
Alegre
S
S
D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism; D001921:Brain / Q000166:cytology / Q000201:enzymology*; D004076:Digoxigenin; D004579:Electron Transport; D042967:Electron Transport Complex I; D004591:Electrophoresis, Polyacrylamide Gel; D017665:Hydroxyl Radical / Q000378:metabolism; D015151:Immunoblotting; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D016877:Oxidants / Q000378:metabolism*; D051381:Rats; D013194:Staining and Labeling
D016877:Oxidants; D017665:Hydroxyl Radical; D007501:Iron; D009247:NADH, NADPH Oxidoreductases; D042967:Electron Transport Complex I; D004076:Digoxigenin; D001205:Ascorbic Acid