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

在褐腐菌中，异柠檬酸裂解酶(ICL)的细胞定位尚不清楚，但已有报道定位于其他生物体的过氧化物体(或乙氧基体)中。首先，我们制备了褐腐菌的原生质体，并通过蔗糖密度梯度离心法获得线粒体和过氧化物体的组份。对分离出的蛋白质组分、线粒体组分和过氧化氢酶关键酶、线粒体琥珀酸脱氢酶关键酶进行活性测定，识别出可溶性蛋白质、线粒体组份和过氧化物体组份。酶分析表明，过氧化氢酶和琥珀酸脱氢酶在过氧化物体和线粒体组分中均可检测到，谷氨酸关键酶与过氧化物体和线粒体组分相同，但不在线粒体组分中。第二，我们成功地从褐腐菌…中分离纯化了IC1更多的沼泽壳霉生长在葡萄糖培养基上。在对纯化的酶进行氨基酸序列分析的基础上，我们成功地测定了全酶的编码序列。片段后，我们得到了3个片段(IclA、ICLb和ICLc)，并用聚合酶链式反应的方法增强表达了相应的3个多肽。然而，我们发现只有ICLb编码的多肽才能有效地制备出有用的抗体。用兔抗体金标记的抗体制备了用于电子显微镜分析的样品。或者，我们用一种普通的化学方法和氨基联苯胺/过氧化氢固定真菌细胞，使其死亡，过氧化氢酶定位于过氧化物体中。结果，过氧化物体的死亡密度比线粒体更大。此外，过氧化物体比线粒体更容易被抗体金标记物所标记。综上所述，我们首次证明了在木材腐烂担子菌中，ICL和MS的关键酶ICL和MS也定位于细胞内。较少

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