Dahl D
Biochim Biophys Acta. 1976 Jan 20;420(1):142-54. doi: 10.1016/0005-2795(76)90353-6.
Compared with human material glial fibrillary acidic protein isolated from bovine, rat and mouse brain was remarkably homogeneous and migrated as a single band at 54 000 mol. wt. on sodium dodecyl sulfate gel electrophoresis. The protein was extremely susceptible to proteolysis and lower molecular weight components were invariably isolated together with the major species when the brain was not rapidly frozen. Further degradation of the 54 000 mol wt. polypeptide in bovine tissues incubated at 24 degrees C resulted in preparations essentially identical to those previously isolated from human autopsy material and separating into a series of immunologically active polypeptides ranging in molecular weight from 54 000 to approximately 40 500. The gel band pattern obtained after progressively longer periods of autolysis suggested that small fragments were cleaved from the original polypeptide in successive steps of degradation. As in human brain, the lower molecular weight products in the 45 000-40 500 range were more resistant to proteolysis and still present after prolonged periods of tissue autolysis. The effect of the pH and of proteinase inhibitors on degradation was studied in homogenates of bovine brain stem incubated at 37 degrees C. At pH 8.0 PROTEOLYSIS OF The glial fibrillary acidic protein followed essentially the same pattern as in tissue. Cleavage of the major species was not prevented by the addition of proteinase inhibitors. At pH 6.0 and 6.5 a different type of degradation was observed, with rapid breakdown of the protein and loss of immunological activity. Increased solubility in buffer solutions was another effect of autolysis. Compared with cerebral cortex and brain stem, where most of the protein was water soluble, only a small fraction was extracted with buffer from bovine white matter. However, the solubility markedly increased following incubation and comparable amounts were extracted in buffer and in 6 M urea.
与从牛、大鼠和小鼠大脑中分离出的人源胶质纤维酸性蛋白相比，其具有显著的均一性，在十二烷基硫酸钠凝胶电泳上以54000分子量的单一条带迁移。该蛋白极易被蛋白酶水解，当大脑未迅速冷冻时，较低分子量的成分总是与主要成分一起被分离出来。在24℃下孵育的牛组织中，54000分子量的多肽进一步降解，产生的制剂与先前从人类尸检材料中分离出的制剂基本相同，并分离成一系列分子量从54000到约40500的免疫活性多肽。自溶时间逐渐延长后获得的凝胶条带模式表明，小片段在连续的降解步骤中从原始多肽上裂解下来。与人类大脑一样，分子量在45000 - 40500范围内的较低分子量产物对蛋白酶水解更具抗性，在组织长时间自溶后仍然存在。在37℃孵育的牛脑干匀浆中研究了pH值和蛋白酶抑制剂对降解的影响。在pH 8.0时，胶质纤维酸性蛋白的蛋白酶水解基本上与在组织中的模式相同。添加蛋白酶抑制剂并不能阻止主要成分的裂解。在pH 6.0和6.5时，观察到不同类型的降解，蛋白质迅速分解并丧失免疫活性。自溶的另一个影响是在缓冲溶液中的溶解度增加。与大脑皮层和脑干相比，其中大部分蛋白质是水溶性的，从牛白质中用缓冲液仅提取出一小部分。然而，孵育后溶解度显著增加，在缓冲液和6M尿素中提取的量相当。
