Ding J H, de Barsy T, Brown B I, Coleman R A, Chen Y T
Division of Pediatric Genetics and Metabolism, Duke University Medical Center, Durham, North Carolina 27710.
J Pediatr. 1990 Jan;116(1):95-100. doi: 10.1016/s0022-3476(05)81652-x.
To determine the tissue distribution of glycogen debranching enzyme, we used immunoblot analysis with a polyclonal antibody prepared against purified porcine muscle debranching enzyme. Debranching enzyme was identified in porcine brain, kidney, cardiac muscle, skeletal muscle, liver, and spleen; and in human liver, skeletal muscle, lymphocytes, lymphoblastoid cells, skin fibroblasts, cultured chorionic villi, and amniocytes. In each of these tissues the debranching enzyme band was 160 kd. To determine the molecular basis for glycogen storage disease type III at the protein level, tissues from 41 patients with glycogen storage disease type III were also subjected to immunoblot analysis. Three patients having isolated transferase deficiency with retention of glucosidase activity (type IIID disease) had nearly normal amounts of cross-reactive material. In the remaining patients (both transferase and glucosidase deficiency), debranching enzyme was either absent or greatly reduced. These latter patients included 31 with disease that appeared to involve both liver and muscle (type IIIA), four with disease that was present only in the liver (type IIIB), and three with unknown muscle status. In patients with both type IIIA and type IIIB disease, debranching enzyme protein was absent in skin fibroblasts, lymphoblastoid cells, and lymphocytes. The parents of two patients with type IIIA disease had an intermediate level of debranching enzyme protein, consistent with their presumed heterozygote state. An immunoblot analysis of cultured amniotic fluid cells from a woman whose fetus was at risk for type IIIA disease predicted an unaffected fetus; the prediction was confirmed postnatally. Thus Western blot analysis offers an alternate method of prenatal diagnosis for the most common form of glycogen storage disease type III.
为了确定糖原脱支酶的组织分布，我们使用针对纯化的猪肌肉脱支酶制备的多克隆抗体进行免疫印迹分析。在猪脑、肾、心肌、骨骼肌、肝脏和脾脏中鉴定出了脱支酶；在人肝脏、骨骼肌、淋巴细胞、淋巴母细胞、皮肤成纤维细胞、培养的绒毛膜绒毛和羊水中也鉴定出了脱支酶。在这些组织中的每一个中，脱支酶条带均为160kd。为了在蛋白质水平上确定III型糖原贮积病的分子基础，还对41例III型糖原贮积病患者的组织进行了免疫印迹分析。3例仅有转移酶缺乏而保留葡糖苷酶活性的患者（IIID型疾病）具有几乎正常量的交叉反应物质。在其余患者（转移酶和葡糖苷酶均缺乏）中，脱支酶要么缺失，要么大大减少。这些患者包括31例似乎同时累及肝脏和肌肉的疾病（IIIA型）、4例仅存在于肝脏的疾病（IIIB型）和3例肌肉状况不明的患者。在IIIA型和IIIB型疾病患者中，皮肤成纤维细胞、淋巴母细胞和淋巴细胞中不存在脱支酶蛋白。两名IIIA型疾病患者的父母具有中等水平的脱支酶蛋白，这与其推测的杂合子状态一致。对一名胎儿有患IIIA型疾病风险的妇女的培养羊水细胞进行免疫印迹分析，预测胎儿未受影响；产后证实了这一预测。因此，蛋白质印迹分析为最常见的III型糖原贮积病提供了一种产前诊断的替代方法。
