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

褐腐真菌的异柠檬酸裂解酶（ICL）的细胞定位尚未明确，尽管有报道称它定位于其他生物体的过氧化物酶体（或乙醛酶体）。首先，制备褐腐菌原生质体，用蔗糖密度梯度离心法对其进行匀浆，得到线粒体和过氧化物酶体组分。通过对ICL和苹果酸合成酶的glyoxysomal关键酶，过氧化氢酶的过氧化物酶体关键酶和琥珀酸脱氢酶的线粒体关键酶的活性测定，鉴定了可溶性蛋白、线粒体部分和过氧化物酶体部分。酶学分析清楚地表明，过氧化氢酶和琥珀酸脱氢酶在过氧化物酶体和线粒体组分中检测到，gloxylate关键酶在同一组分中被识别，但在线粒体组分中未被识别。其次，我们成功地从生长在葡萄糖培养基上的褐腐菌中纯化了IC1。在对纯化酶进行氨基酸序列分析的基础上，我们成功地确定了编码整个酶的cDNA序列。cDNA片段化后得到ICLa、ICLb和ICLc三个片段，利用PCR方法对其进行扩增，表达相应的三个多肽。然而，我们发现只有被ICLb编码的多肽才能有效地制备有用的抗体。用兔抗体金标记的抗体制备样品进行电镜分析。或者，我们用一种常见的化学方法固定真菌细胞，氨基联苯胺/过氧化氢使其特异性地死于过氧化物酶体中的过氧化氢酶。结果，过氧化物酶体比线粒体死亡的密度更大。此外，过氧化物酶体比线粒体更容易被抗体金标记。总之，我们首次证明了在木材腐烂的担子菌中，ICL和MS的glyoxisomal关键酶也定位在细胞中。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