Characterization of membrane polypeptides from pea leaf peroxisomes involved in superoxide radical generation

The production of superoxide radicals (O-2(-.)) and the activities of ferricyanide reductase and cytochrome c reductase were investigated in peroxisomal membranes from pea (Pisum sativum L.) leaves using NADH and NADPH as electron donors. The generation of O-2(-.) by peroxisomal membranes was also assayed in native polyacrylamide gels using an in situ staining method with NitroBlue Tetrazolium (NBT). When peroxisomal membranes were assayed under native conditions using NADH or NADPH as inducer, two different O-2(-.)-dependent Formazan Blue bands were detected. Analysis by SDS/PAGE of these bands demonstrated that the NADH-induced NBT reduction band contained several polypeptides (PMP32, PMP61, PMP56 and PMP18, where PMP is peroxisomal membrane polypeptide and the number indicates molecular mass in kDa), while the NADPH-induced band was due exclusively to PMP29. PMP32 and PMP29 were purified by preparative SDS/PAGE and electroelution. Reconstituted PMP29 showed cytochrome c reductase activity and O-2(-.) production, and used NADPH specifically as electron donor. PMP32, however, had ferricyanide reductase and cytochrome c reductase activities, and was also able to generate O-2(-.) with NADH as electron donor, whereas NADPH was not effective as an inducer. The reductase activities of, and O-2(-.) production by, PMP32 were inhibited by quinacrine. Polyclonal antibodies against cucumber monodehydroascorbate reductase (MDHAR) recognized PMP32, and this polypeptide is likely to correspond to the MDHAR reported previously in pea leaf peroxisomal membranes.