Reconstituted microsomal lipid peroxidation: ADP-Fe 3+-dependent peroxidation of phospholipid vesicles containing NADPH-cytochrome P450 reductase and cytochrome P450

Abstract

A reconstituted lipid peroxidation system consisting of rat liver microsomal NADPH-cytochrome P450 reductase and cytochrome P450 incorporated into phospholipid vesicles was developed and characterized. Peroxidation of the vesicles required NADPH and ADP-Fe 3+, just as in the NADPH-dependent peroxidation of microsomes. The peroxidation of the vesicles was inhibited 30–50% by superoxide dismutase, depending upon their cytochrome P450 content: those with higher cytochrome P450 contents exhibited greater rates of malondialdehyde formation which were less sensitive to inhibition by superoxide dismutase. When cytochrome P450 was incorporated into vesicles, EDTA-Fe 3+ was not required for lipid peroxidation distinguishing this system from the one previously described by Pederson and Aust [ Biochem. Biophys. Res. Commun. 48, 789; 1972]. Since at least 50% of the malondialdehyde formation in the vesicular system was not inhibited by superoxide dismutase, alternative means of iron reduction (O 2 ▪ -independent) were examined. It was found that rat liver microsomes or a reconstituted mixed function oxidase system consisting of NADPH-cytochrome P450 reductase and cytochrome P450 in dilauroylphosphatidylcholine micelles reduced ADP-Fe 3+ under anerobic conditions.