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Abstract
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- × mg-1 protein × 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 ery-throcytes. 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.
