SUPEROXIDE RADICAL GENERATION IN PEROXISOMAL MEMBRANES - EVIDENCE FORTHE PARTICIPATION OF THE 18-KDA INTEGRAL MEMBRANE POLYPEPTIDE

Peroxisomes were isolated from pea (Pisum sativum L.) leaves and the p eroxisomal membranes were purified by treatment with Na2CO3. The produ ction of superoxide radicals (O-2(-)) induced by NADH was investigated in peroxisomal membranes from intact organelles incubated with protea ses (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 de monstrated a decrease in the 18-kDa PMP. This suggests that this polyp eptide has a small fragment ex-posed to the cytosolic side of the pero xisomal membrane which is essential for O-2(-) production. The 18-kDa PMP was purified by preparati iie SDS-PAGE and in the reconstituted pr otein 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 polyc lonal antibody against Cyt b(5) from human erythrocytes. The presence of b-type cytochrome in peroxisomal membranes was demonstrated by diff erence 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 clearl y involved in superoxide radical production.