F1F0-ATPASE, EARLY TARGET OF THE RADICAL INITIATOR 2,2'-AZOBIS-(2-AMIDINOPROPANE) DIHYDROCHLORIDE IN RAT-LIVER MITOCHONDRIA IN-VITRO

This study was designed to determine which enzyme activities were firs t impaired in mitochondria exposed to 2,2'-azobis-(2-amidinopropane) d ihydrochloride (AAPH), a known radical initiator. EPR spin-trapping re vealed generation of reactive oxygen species although malondialdehyde formation remained very low. With increasing AAPH concentrations, Stat e-3 respiration was progressively depressed with unaltered ADP/O ratio s. A top-down approach demonstrated that alterations were located at t he phosphorylation level. As shown by inhibitor titrations, ATP/ADP tr anslocase 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 F-0, proton channel. A comparison of ATP hydrolase activity in uncoupled and broken mitochondria reinforced this finding. In spite o f its pro-oxidant properties, AAPH was shown to act as a dose-dependen t inhibitor of cyclosporin-sensitive permeability transition initiated by Ca2+, probably as a consequence of its effect on F1F0-ATPase. Resv eratrol, a potent antiperoxidant, completely failed to prevent the dec rease in State-3 respiration caused by AAPH. The data suggest that AAP H, when used under mild conditions, acted as a radical initiator and w as capable of damaging F1F0-ATPase, thereby slowing respiratory chain activity and reducing mitochondrial antioxidant defences.