Pathogenesis of Pompe's Disease

我们从人胎盘和猪肝中提纯了酸性α-葡萄糖苷酶。基本上按照前面所述的提纯程序进行。最近我们发现该酶对Sephacryl S-200凝胶的亲和力具有异质性。该酶被分离为两个主要组分(S1和S2)。通过SDS-PAGE和层析聚焦进一步将每个组分分离为76 KDa和67 KDa两个组分。研究了这四个组分(S1和S2组分的76 KDa和67 KDa)的组织分布。肝脏、心脏和肾脏的酶是由这四种成分组成的。而巨噬细胞酶仅含有S176 KDa组分。糖原、麦芽糖、异麦芽糖和呋喃糖对4-甲基伞形花环状α-葡萄糖苷酶活性均有竞争性抑制作用，说明酸性α-葡萄糖苷酶具有与这些底物相同的单一底物结合部位。根据氨基酸序列a…进一步分析，这四种成分被认为是相同的基因产物。用凝集素-硝酸纤维素单层法对这四个组分和Pompe‘s病的免疫交叉反应物质CRM(与抗酸性α-葡萄糖苷酶反应)进行糖链分析。S_1、S_2组分和CRM中含有甘露糖。ConA在Endo H、Endo F和PNGase消化前后的染色图谱表明，可能存在少量不同于富含甘露糖的N-链的其他糖链。此外，Pompe‘s病的CRM糖链中含有少量唾液酸。用转导SV-40大T DNA的Pompe病成纤维细胞也进行了上述实验。目前对Pompe病成纤维细胞的cDNAs和mRNAs进行了研究。从细胞病理学的角度来看，溶酶体储积症可分为A和B两组。在庞贝氏病中，肝细胞和肌肉中储存了大量的糖原。酸性α-葡萄糖苷酶主要定位于肝细胞、肌细胞等实质细胞。在B组病例中，与Hurler病的病例一样，在巨噬细胞中也发现了储存物质。我们从人胎盘和猪肝中纯化了α-L艾杜糖醛酸酶。免疫组织化学显示巨噬细胞内有α-L艾杜糖苷酶的表达。我们现在正在研究溶酶体疾病，A组和B组，从病理学、生化和遗传学角度进行研究。较少

We purified acid alpha-glucosidase from human placenta and pig liver. The purification procedures were followed essentially as described previously. Recently we found that the enzyme showed the heterogeneity in its affinity to Sephacryl s-200 gel. The enzyme was separated into two major components (S1 and S2). Each component was further separated into two (76 KDa and 67 KDa components) by SDS-PAGE and also chromatofocusing. The tissue distribution of these four components (76 KDa and 67 KDa of S1 and S2 components) was investigated. The liver, heart and kidney enzymes were consisted of these four components. However, the macrophage enzyme contained only S1 76 KDa component. The 4-methylumbelliferyl alpha-glucosidase activities of these four components were all inhibited competitively by glycogen, maltose, isomaltose and turanose, which suggests that the acid alpha-glucosidase has a single substrate binding site which is common to those substrates. According to the amino acid sequence a … More nalysis, the four components were considered to be the same gene product. Sugar chain analysis of these four components and CRM (immunologically cross-reactive material which reacts with anti acid alpha-glucosidase) from Pompe's disease was performed by the lectin-nitrocellulose sheet method. The S1 and S2 components and CRM contained the mannose. Con A staining patterns before and after digestion with Endo H, Endo F and PNGase indicated that there may be small amounts of other sugar chain different from mannose-rich N-linked chain. In addition, CRM from Pompe's disease contains small amounts of sialic acid in its sugar chains. These experiments were also carried out using Pompe's disease fibroblasts which were transfected with SV 40 large T DNA. cDNA and mRNA from Pompe's disease fibroblasts are now investigating.From a cytopathological view point, lysosomal storage disease may be classified into two groups : A and B. In the disease of A group, the storage substances are mainly observed in the parenchymal cells. In the Pompe's disease, large amounts of glycogen were stored in the hepatocytes and muscle. The acid alpha-glucosidase localized mainly in the parenchymal cells such as hepatocytes and muscle cells. On the other hand, in the diseases of group B, the storage substances were found in the macrophages as in the case of Hurler disease. We purified alpha-L-iduronidase from human placenta and pig liver. alpha-L-Iduronidase was demonstrated immunohistochemically in the macrophages. We are now investigating the lysosomal diseases, group A and B, pathologically, biochemically and genetically. Less