cDNA cloning of the key enzyme of oxalate biosynthesis and the cellular localization in the copper tolerant brown-rot fungi

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Cellular localization of isocitrate lyase(ICL) has not been clarified yet for the brown rot fungi although it has been reported to localize in peroxisomes (or glyoxysomes) in other living organisms.First, we prepared protoplast of the brown rot fungus and homogenized to obtain mitochondrial and peroxisomal fractions by the sucrose density gradient centrifugation method. Soluble proteins, mitochondrial fractions and peroxisomal fractions were recognized by assay of activities of the glyoxysomal key enzymes of ICL and malate synthase, and the peroxisomal key enzyme of catalase, and the mitochondrial key enzyme of succinate dehydrogenase in separated fractions. The enzymatic analysis clearly showed that catalase and succinate dehydrogenase were detected in the peroxisomal and mitochondrial fractions, and the gloxylate key enzymes were recognized in the same fraction as proxisomal fraction but not in the mitochondrial fraction.Second, we purified successfully IC1 from the brown rot fungus … More Fomitopsis palustris grown on a glucose medium. On the basis of the amino acid sequence analysis of the purified enzyme, we have successfully determined the cDNA sequence encoding the whole enzyme. After fragmentation of the cDNA, we obtained three fragments (ICLa, ICLb and ICLc), which were enhanced by use of PCR method to express the three corresponding polypeptides. However, we found that only the polypeptide which was encoded by ICLb is effective for preparing the useful antibody. By use of the antibody labeled with rabbit antibody -gold- we prepared samples for the electron-microscopic analysis. Alternatively, we fixed the fungal cells with a common chemical method and aminobenzidine/hydrogen peroxide to die specifically catalases localized in peroxisomes. As a result, peroxisomes were more densely died than the mitochondria. Furthermore, peroxisomes were more labeled with the antibody-gold than the mitochondria. In conclusion, we have first demonstrated that also in wood rotting basidiomycetes glyoxisomal key enzymes of ICL and MS are localized in cells. Less