Cytochrome b5 reductase is the component from neuronal synaptic plasma membrane vesicles that generates superoxide anion upon stimulation by cytochrome c

Abstract

In this work, we measured the effect of cytochrome c on the NADH-dependent superoxide anion production by synaptic plasma membrane vesicles from rat brain. In these membranes, the cytochrome c stimulated NADH-dependent superoxide anion production was inhibited by antibodies against cytochrome b5 reductase linking the production to this enzyme. Measurement of the superoxide anion radical generated by purified recombinant soluble and membrane cytochrome b5 reductase corroborates the production of the radical by different enzyme isoforms. In the presence of cytochrome c, a burst of superoxide anion as well as the reduction of cytochrome c by cytochrome b5 reductase was measured. Complex formation between both proteins suggests that cytochrome b5 reductase is one of the major partners of cytochrome c upon its release from mitochondria to the cytosol during apoptosis. Superoxide anion production and cytochrome c reduction are the consequences of the stimulated NADH consumption by cytochrome b5 reductase upon complex formation with cytochrome c and suggest a major role of this enzyme as an anti-apoptotic protein during cell death.

Keywords

• Cb5R, Cytochrome b5 reductase
• DTPA, Diethylenetriaminepentaacetic acid
• DHE, Dihydroethidium
• E+, Ethidium
• FAD, Flavin adenine dinucleotide
• NADH, Reduced nicotinamide adenine dinucleotide
• NBT, Nitroblue tetrazolium nitroblue tetrazolium
• SPMV, Synaptic plasma membrane vesicles
• TB, Terrific Broth terrific Broth
• SOD, Superoxide dismutase
• XA, Xanthine xanthine
• XO, Xanthine oxidase