Superoxide radical generation in peroxisomal membranes: 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 (O-2(-)) 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 O-2(-) 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 ex-posed to the cytosolic side of the peroxisomal membrane which is essential for O-2(-) production. The 18-kDa PMP was purified by preparati iie SDS-PAGE and in the reconstituted protein the NADH-driven production of O-2(-) radicals was investigated. The isolated polypeptide showed a high generation rate of superoxide (about 300 nmol O-2(-) 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 b(5) 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 O-2(-) radical generating system of peroxisomal membranes, the 18-kDa integral membrane polypeptide, which appears to be Cyt b(5), is clearly involved in superoxide radical production.