P-363 - Cytochrome c stimulates the superoxide anion production by cytochrome b5 reductase in neuronal synaptic plasma membrane vesicles

Abstract

The plasma membrane NADH oxidase activity of synaptosomes and cerebellar granule neurons releases superoxide anion (O2•-) as a collateral product. In this work, we have measured the effect of cytochrome c (Cyt c) on the NADH-dependent O2•- production by rat neuronal synaptic plasma membrane vesicles. In these membranes, the Cyt c stimulated O2•- production was inhibited by antibodies against cytochrome b5 reductase (Cb5R). O2•- production by Cb5R was confirmed using purified soluble and membrane recombinant enzymes. Addition of Cyt c to the assay induced a burst of O2•- concomitant to the reduction of Cyt c by Cb5R. The low dissociation constant measured for Cyt c/Cb5R complex (0.40 ±0.05 and 0.38 ±0.02 μM for soluble and membrane Cb5R, respectively), support that Cb5R is a main molecular partner of Cyt c upon its release from mitochondria during apoptosis. O2•- production and Cyt c reduction are consequences of the stimulated NADH consumption by Cb5R upon complex formation with Cyt c. Participation of Cb5R in the apoptosis signalling network is suggested.