Iwamasa T, Nashiro K, Ohshita T, Matsuda I
Histochem J. 1986 Nov-Dec;18(11-12):613-24. doi: 10.1007/BF01675297.
From fibroblasts of two cases of Pompe's disease (acid alpha-glucosidase deficiency), one of the childhood type (RH-SF-1) and one of the adult type (RH-SF-2), and normal fibroblasts, antigenically cross-reactive material and acid alpha-glucosidase were immunoprecipitated and analysed by immunoelectrotransfer blotting. The acid alpha-glucosidase and antigenically cross-reactive material (which reacts with antibody raised against normal acid alpha-glucosidase) revealed a precursor form of molecular weight 97,000 and two major components of 79,000 and 76,000. When monensin was added to the fibroblast culture, the two major components of normal acid alpha-glucosidase were decreased, whereas the large molecular weight precursor was increased. On the other hand, the 97,000 molecular weight component of cross-reactive material in the Pompe's fibroblasts (RH-SF-1 and RH-SF-2) was only slightly increased on monensin treatment. The fibroblasts were pulse-chase labelled with [2-H3] mannose and 32Pi. The cross-reactive material and acid alpha-glucosidase were precipitated with anti acid alpha-glucosidase antibody, and after sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), fluorography was performed. The radiolabel of 3H in the cross-reactive material of RH-SF-1 and -2 was weak, and 32P in the cross-reactive material of both fibroblasts was very weak when compared with those of the acid alpha-glucosidase. The radiolabel of 32P in the cross-reactive material of RH-SF-1 was extremely weak. Immunofluorescence histochemistry revealed a granular localization of acid alpha-glucosidase in the normal fibroblast cytoplasm, and a diffuse distribution of cross-reactive material in the cytoplasm of RH-SF-1 and -2. Immuno-electron microscopic examinations showed a normal acid alpha-glucosidase localization on the inner side of the lysosomal membrane and also diffusely in the lysosome; when treated with monensin, it was present on the trans part of the Golgi apparatus. Antigenically cross-reactive material, however, was found in the cytoplasm and endoplasmic reticulum. Some lysosomal localization was observed sporadically. Even after monensin treatment, it was not demonstrated on the Golgi apparatus.
从两例庞贝氏病（酸性α-葡萄糖苷酶缺乏症）的成纤维细胞中提取样本，其中一例为儿童型（RH-SF-1），另一例为成人型（RH-SF-2），同时选取正常成纤维细胞作为对照。将抗原交叉反应物质和酸性α-葡萄糖苷酶进行免疫沉淀，并通过免疫电转移印迹法进行分析。酸性α-葡萄糖苷酶和抗原交叉反应物质（与针对正常酸性α-葡萄糖苷酶产生的抗体发生反应）显示出分子量为97,000的前体形式以及79,000和76,000的两个主要成分。当在成纤维细胞培养物中添加莫能菌素时，正常酸性α-葡萄糖苷酶的两个主要成分减少，而大分子前体增加。另一方面，庞贝氏病成纤维细胞（RH-SF-1和RH-SF-2）中交叉反应物质的97,000分子量成分在莫能菌素处理后仅略有增加。用[2-H3]甘露糖和32Pi对成纤维细胞进行脉冲追踪标记。用抗酸性α-葡萄糖苷酶抗体沉淀交叉反应物质和酸性α-葡萄糖苷酶，经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）后进行荧光自显影。与酸性α-葡萄糖苷酶相比，RH-SF-1和-2交叉反应物质中3H的放射性标记较弱，两种成纤维细胞交叉反应物质中32P的放射性标记非常弱。RH-SF-1交叉反应物质中32P的放射性标记极其微弱。免疫荧光组织化学显示正常成纤维细胞胞质中酸性α-葡萄糖苷酶呈颗粒状定位，而RH-SF-1和-2胞质中交叉反应物质呈弥漫性分布。免疫电子显微镜检查显示正常酸性α-葡萄糖苷酶定位于溶酶体膜内侧，也弥漫分布于溶酶体中；用莫能菌素处理后，它存在于高尔基体的反式部分。然而，抗原交叉反应物质存在于细胞质和内质网中。偶尔观察到一些溶酶体定位。即使在莫能菌素处理后，也未在高尔基体上显示出来。
