Superoxide Radical Generation in Peroxisomal Mimbranes: Evidence for the Participation of the 18-kDa Integral Membrane Polypeptide

Peroxisomes were isolated from pea (Pisum sativum L.) leaves and the peroxisomal membranes were purified by treatment with Na2CO3. The production of superoxide radicals (O2) induced by NADH was investigated in peroxisomal membranes from intact organelles incubated with proteases (pronase E and proteinase K). Under isoosmotic conditions, in the presence of pronase E, the production of O2-. radicals was inhibited by 80%. SDS-PAGE of peroxisomal membranes after protease treatment demonstrated a decrease in the 18-kDa PMP. This suggests that this polypeptide has a small fragment exposed to the cytosolic side of the peroxisomal membrane which is essential for O2-. production. The 18-kDa PMP was purified by preparative SDS-PAGE and in the reconstituted protein the NADH-driven production of O2-. radicals was investigated. The isolated polypeptide showed a high generation rate of superoxide (about 300 nmol O2-. x mg-1 protein x min-1) which was completely inhibited by 50 mM pyridine. The 18-kDa PMP was recognized by a polyclonal antibody against Cyt b5 from human erythrocytes. The presence of b-type cytochrome in peroxisomal membranes was demonstrated by difference spectroscopy. Results obtained show that in the NADH-dependent O2-. radical generating system of peroxisomal membranes, the 18-kDa integral membrane polypeptide, which appears to be Cyt b5, is clearly involved in superoxide radical production.