Endo F, Motohara K, Indo Y, Matsuda I
Department of Pediatrics, Kumamoto University Medical School, Japan.
Pediatr Res. 1987 Dec;22(6):627-33. doi: 10.1203/00006450-198712000-00002.
Prolidase was highly purified from human liver and erythrocytes. NaDodSO4/acrylamide gel electrophoresis revealed that these preparations contained a major protein with MW = 56,000. The mass of prolidase was estimated on gel filtration to be MW = 97,000, for both enzyme preparations. A monoclonal antibody was raised against the liver enzyme and a specific antiserum against the erythrocyte enzyme. The monoclonal antibody (EP-2) recognized prolidase from erythrocytes and liver, in equal proportions. The antiserum also recognized the enzyme from erythrocytes and liver. Immunoprecipitation studies with these antibodies suggested only a single species of prolidase in erythrocytes and liver. Using an immobilized monoclonal antibody (EP-2) as an immunoadsorbent, prolidase was partially purified from crude extracts, and the protein of the partially purified enzyme was identified by immunoblotting using antiserum. A protein band with a MW = 56,000 was demonstrated specifically when crude extracts from the liver and erythrocytes were examined using NaDodSO4/acrylamide gel electrophoresis. The subunit protein was absent in erythrocytes from a patient with prolidase deficiency. We propose that the absence of the subunit is one cause of the prolidase deficiency.
从人肝脏和红细胞中高度纯化了脯氨酰肽酶。十二烷基硫酸钠/丙烯酰胺凝胶电泳显示，这些制剂含有一种主要蛋白质，其分子量为56,000。两种酶制剂在凝胶过滤法中测得的脯氨酰肽酶分子量均为97,000。制备了针对肝脏酶的单克隆抗体和针对红细胞酶的特异性抗血清。单克隆抗体（EP - 2）对来自红细胞和肝脏的脯氨酰肽酶的识别比例相同。抗血清也能识别来自红细胞和肝脏的该酶。用这些抗体进行的免疫沉淀研究表明，红细胞和肝脏中仅有一种脯氨酰肽酶。使用固定化单克隆抗体（EP - 2）作为免疫吸附剂，从粗提物中部分纯化了脯氨酰肽酶，并使用抗血清通过免疫印迹法鉴定了部分纯化酶的蛋白质。当使用十二烷基硫酸钠/丙烯酰胺凝胶电泳检测肝脏和红细胞的粗提物时，特异性显示出一条分子量为56,000的蛋白带。脯氨酰肽酶缺乏症患者的红细胞中不存在该亚基蛋白。我们认为该亚基的缺失是脯氨酰肽酶缺乏症的一个原因。
