Agrawal H C, O'Connell K, Randle C L, Agrawal D
Biochem J. 1982 Jan 1;201(1):39-47. doi: 10.1042/bj2010039.
When rat brain myelin was examined by sodium dodecyl sulphate/polyacrylamideslab-gel electrophoresis followed by fluorography of the stained gel, it was found that a host of proteins of rat brain myelin were labelled 2, 4 and 24h after the intracerebral injection of H(3) (32)PO(4). Among those labelled were proteins migrating to the positions of myelin-associated glycoprotein, Wolfgram proteins, proteolipid protein, DM-20 and basic proteins. The four basic proteins with mol.wts. 21000, 18000 (large basic protein), 17000 and 14000 (small basic protein) were shown to be phosphorylated after electrophoresis in both acid-urea- and sodium dodecyl sulphate-containing gel systems followed by fluorography. The four basic proteins imparted bluish-green colour, after staining with Amido Black, which is characteristic of myelin basic proteins. The four basic proteins were purified to homogeneity. Fluorography of the purified basic proteins after re-electrophoresis revealed the presence of phosphorylated high-molecular-weight ;polymers' associated with each basic protein. The amino acid compositions of the phosphorylated large basic protein and small basic proteins are compatible with the amino acid sequences. Proteins with mol.wts. 21000 and 17000 gave the expected amino acid composition of myelin basic proteins. Radiolabelled phosphoserine and phosphothreonine were identified after partial acid hydrolysis of the four purified basic proteins. The [(32)P]phosphate-protein bond in the basic protein was stable at an acidic pH but was readily hydrolysed at alkaline pH, as would be expected of phosphoester bonds involving both serine and threonine residues. Double-immunodiffusion analysis demonstrated that the four phosphorylated proteins showed complete homology when diffused against antiserum to a mixture of small and large basic proteins. Since the four basic proteins of rat brain myelin were phosphorylated both in vivo and in vitro it is postulated that the same protein kinase is responsible for their phosphorylation in both conditions.
当用十二烷基硫酸钠/聚丙烯酰胺平板凝胶电泳对大鼠脑髓磷脂进行检测，随后对染色凝胶进行荧光自显影时，发现在脑内注射H(3)(32)PO(4)后2小时、4小时和24小时，大鼠脑髓磷脂的许多蛋白质都被标记。被标记的蛋白质中包括迁移到髓磷脂相关糖蛋白、沃尔夫拉姆蛋白、蛋白脂蛋白、DM - 20和碱性蛋白位置的蛋白质。在含酸尿素和十二烷基硫酸钠的凝胶系统中进行电泳，随后进行荧光自显影后，发现分子量分别为21000、18000（大碱性蛋白）、17000和14000（小碱性蛋白）的四种碱性蛋白被磷酸化。用酰胺黑染色后，这四种碱性蛋白呈现蓝绿色，这是髓磷脂碱性蛋白的特征。这四种碱性蛋白被纯化至同质。重新电泳后对纯化的碱性蛋白进行荧光自显影，结果显示存在与每种碱性蛋白相关的磷酸化高分子量“聚合物”。磷酸化的大碱性蛋白和小碱性蛋白的氨基酸组成与氨基酸序列相符。分子量为21000和17000的蛋白质给出了髓磷脂碱性蛋白预期的氨基酸组成。对四种纯化的碱性蛋白进行部分酸水解后，鉴定出放射性标记的磷酸丝氨酸和磷酸苏氨酸。碱性蛋白中的[(32)P]磷酸 - 蛋白键在酸性pH下稳定，但在碱性pH下很容易水解，这与涉及丝氨酸和苏氨酸残基的磷酸酯键预期的情况一致。双向免疫扩散分析表明，当这四种磷酸化蛋白与针对小碱性蛋白和大碱性蛋白混合物的抗血清进行扩散时，显示出完全同源性。由于大鼠脑髓磷脂的四种碱性蛋白在体内和体外都被磷酸化，因此推测在这两种情况下，相同的蛋白激酶负责它们的磷酸化。
