Identification of an NADH plus iron dependent, Ca2+ activated hydrogen peroxide production in synaptosomes.
The addition of microM Ca2+ to synaptosomes incubated in the presence of EGTA and NADH activates a slow production of H2O2, which is promptly inhibited by excess EGTA and reactivated by Ca2+. The H2O2 output is inhibited by the Fe chelator deferoxamine. Higher Ca(2+)-dependent H2O2 productions are induced in the presence of added FeCl3 in the absence of lipid peroxidation. The apparent Km for Fe is 28 microM. NADH is oxidized parallel to the production of H2O2 (NADH/H2O2 = 0.82 +/- 0.1). If NADH is omitted, some H2O2 is still generated, with electrons from ferrocytochrome c; in these conditions the inhibition by excess EGTA develops slowly being completed only after some minutes. The semimaximal activation of the H2O2 production is obtained at 1-1.2 microM free Ca2+. Millimolar Ca2+ is inhibitory. After treatment with digitonin, the H2O2 production increases by 40-50%. This 'internal' H2O2 probably corresponds to the previously described Ca(2+)-ionophore-induced H2O2 generation which is observable after glutathione depletion. In no case could a production of O2- be monitored. All the synaptosomal Ca(2+)-Fe oxidase activity is recovered in the plasma membrane fractions. NADH provides most of the reducing equivalents in the heavier fraction, which is the richest in postsynaptic components. A significant proportion of the H2O2 production utilizes electrons from cytochrome c in the lighter plasma membrane fractions.
Department of Biological Chemistry, University of Padova, Italy.
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F
Zoccarato
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Valente
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A
Alexandre
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002540:Cerebral Cortex / Q000187:drug effects* / Q000378:metabolism; D003574:Cytochrome c Group / Q000378:metabolism; D003676:Deferoxamine / Q000494:pharmacology; D006168:Guinea Pigs; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007501:Iron / Q000494:pharmacology*; D009243:NAD / Q000494:pharmacology*; D013574:Synaptosomes / Q000187:drug effects* / Q000378:metabolism
D003574:Cytochrome c Group; D009243:NAD; D006861:Hydrogen Peroxide; D007501:Iron; D003676:Deferoxamine; D002118:Calcium