F1F0-ATPase, early target of the radical initiator 2,2′-azobis-(2-amidinopropane) dihydrochloride in rat liver mitochondria in vitro

Abstract

This study was designed to determine which enzyme activities were first impaired in mitochondria exposed to 2,2'-azobis-(2-amidinopropane) dihydrochloride (AAPH), a known radical initiator. EPR spin-trapping revealed generation of reactive oxygen species although malondialdehyde formation remained very low. With increasing AAPH concentrations, State-3 respiration was progressively depressed with unaltered ADP/O ratios. A top-down approach demonstrated that alterations were located at the phosphorylation level. As shown by inhibitor titrations, ATP/ADP translocase activity was unaffected in the range of AAPH concentrations used. In contrast, AAPH appeared to exert a deleterious effect at the level of F1F0-ATPase, comparable with dicyclohexylcarbodi-imide, which alters Fo proton channel. A comparison of ATP hydrolase activity in uncoupled and broken mitochondria reinforced this finding. In spite of its pro-oxidant properties, AAPH was shown to act as a dose-dependent inhibitor of cyclosporin-sensitive permeability transition initiated by Ca2+, probably as a consequence of its effect on F1F0-ATPase. Resveratrol, a potent antiperoxidant, completely failed to prevent the decrease in State-3 respiration caused by AAPH. The data suggest that AAPH, when used under mild conditions, acted as a radical initiator and was capable of damaging F1F0-ATPase, thereby slowing respiratory chain activity and reducing mitochondrial antioxidant defences.

Effect of endurance training on oxidative and antioxidative function in human permeabilized muscle fibres

“…However, previous studies have shown that ADP/ATP translocase and F 1 F 0 ATPase are highly sensitive to ROS [4,10,21,35,39]. A reduced V max of these processes would explain the accentuated effect of ROS at higher flux.…”

Activity in vitro of resveratrol on granulocyte and monocyte adhesion to endothelium

“…Another study (33) showed that the antioxidant activity of different commercial wines against LDL oxidation is not a property of a single phenol compound but that the activity is widely distributed among phytochemical substances, including resveratrol. The low activity of resveratrol as an antioxidant was also shown by the fact that it completely failed to prevent the decrease in rat liver state-3 mitochondrial respiration caused by the radical initiator 2,2-azobis-(2-amidinopropane) dihydrochloride (34). It was shown recently that red wine consumption in volunteers did not affect oxidizability of LDL (35), thereby suggesting that the activity of wine in reducing coronary artery disease may not be related to the antioxidant activity of wine compounds.…”

Quantification of pepsin in rennet using a monoclonal antibody-based inhibition ELISA

“…The preparation and use of permeabilized yeast spheroplasts was made according to Averet et al (14). Measurement of respiration (in the presence of 6 mM NADH) was made with an oxygen electrode; measurement of mitochondrial membrane potential was determined with a tetraphenylphosphonium (TPP ϩ ) electrode as described elsewhere (15) under conditions where the ⌬pH component was low and did not vary. To compare different spheroplast preparation, the experiments were performed using the same amount of respiratory chains as determined by the maximal respiratory rate in presence of 1 M FCCP uncoupler.…”