Chen Y T, He J K, Ding J H, Brown B I
Department of Pediatrics, Duke University Medical Center, Durham, NC 27710.
Am J Hum Genet. 1987 Dec;41(6):1002-15.
Type III glycogen storage disease is caused by a deficiency of glycogen debranching-enzyme activity. Many patients with this disease have both liver and muscle involvement, whereas others have only liver involvement without clinical or laboratory evidence of myopathy. To improve our understanding of the molecular basis of the disease, debranching enzyme was purified 238-fold from porcine skeletal muscle. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified enzyme gave a single band with a relative molecular weight of 160,000 that migrated to the same position as purified rabbit-muscle debranching enzyme. Antiserum against porcine debranching enzyme was prepared in rabbit. The antiserum reacted against porcine debranching enzyme with a single precipitin line and demonstrated a reaction having complete identity to those of both the enzyme present in crude muscle and the enzyme present in liver extracts. Incubation of antiserum with purified porcine debranching enzyme inhibited almost all enzyme activity, whereas such treatment with preimmune serum had little effect. The antiserum also inhibited debranching-enzyme activity in crude liver extracts from both pigs and humans to the same extent as was observed in muscle. Immunoblot analysis probed with anti-porcine-muscle debranching-enzyme antiserum showed that the antiserum can detect debranching enzyme in both human muscle and human liver. The bands detected in human samples by the antiserum were the same size as the one detected in porcine muscle. Five patients with Type III and six patients with other types of glycogen storage disease were subjected to immunoblot analysis. Although anti-porcine antiserum detected specific bands in all liver and muscle samples from patients with other types of glycogen storage disease (Types I, II, and IX), the antiserum detected no cross-reactive material in any of the liver or muscle samples from patients with Type III glycogen storage disease. These data indicate (1) immunochemical similarity of debranching enzyme in liver and muscle and (2) that deficiency of debranching-enzyme activity in Type III glycogen storage disease is due to absence of debrancher protein in the patients that we studied.
III型糖原贮积病是由糖原脱支酶活性缺乏引起的。许多患有这种疾病的患者肝脏和肌肉都受累，而其他患者仅肝脏受累，没有肌病的临床或实验室证据。为了增进我们对该疾病分子基础的理解，从猪骨骼肌中纯化出脱支酶，纯化倍数达238倍。在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中，纯化后的酶呈现出一条相对分子量为160,000的单一谱带，其迁移位置与纯化的兔肌肉脱支酶相同。用猪脱支酶在兔体内制备抗血清。该抗血清与猪脱支酶反应产生一条单一沉淀线，并显示出与粗肌肉中存在的酶以及肝脏提取物中存在的酶完全相同的反应。抗血清与纯化的猪脱支酶孵育几乎抑制了所有酶活性，而用免疫前血清进行这种处理则几乎没有效果。该抗血清还以与在肌肉中观察到的相同程度抑制猪和人粗肝脏提取物中的脱支酶活性。用抗猪肌肉脱支酶抗血清进行免疫印迹分析表明，该抗血清可检测人肌肉和人肝脏中的脱支酶。抗血清在人样品中检测到的条带大小与在猪肌肉中检测到的条带相同。对5例III型患者和6例其他类型糖原贮积病患者进行了免疫印迹分析。尽管抗猪抗血清在其他类型糖原贮积病（I型、II型和IX型）患者的所有肝脏和肌肉样品中检测到特异性条带，但在III型糖原贮积病患者的任何肝脏或肌肉样品中均未检测到交叉反应物质。这些数据表明：（1）肝脏和肌肉中脱支酶的免疫化学相似性；（2）我们研究的III型糖原贮积病患者中脱支酶活性缺乏是由于脱支酶蛋白的缺失。
