Fannon A M, Mastronardi F G, Moscarello M A
Biochemistry Research, Hospital for Sick Children, Toronto, Ontario, Canada.
Neurochem Res. 1994 Aug;19(8):1005-12. doi: 10.1007/BF00968710.
Proteolipids were isolated from 20 day old normal and jimpy mouse brain by extraction into chloroform-methanol (2:1, w/v), delipidated by size-exclusion HPLC, and analyzed by SDS-PAGE, Western blots, amino acid analyses, and N-terminal sequencing. SDS-PAGE showed that a major proteolipid from jimpy mouse brain had an apparent molecular weight of approximately 23 kDa, intermediate to that of PLP and DM-20 from normal mouse brain. Western blots with 3 different antibodies which recognize residues 200-224, 116-150, and 270-276 respectively recognized immunoreactive material in normal and jimpy PLP. Since antibody reactive with 270-276 did not recognize jimpy PLP, an altered C-terminus of the jimpy protein is suggested. These results demonstrated that a PLP can be partially purified from jimpy mouse brain. Amino acid analyses failed to show the predicted increase in cysteinyl residues (predicted from cDNA) in jimpy PLP. However, when jimpy brain proteolipids were subjected to N-terminal sequencing, Gly, Leu, Leu, Gly the first four amino acids of PLP were detected. Thus, the partial purification of a proteolipid from jimpy mouse brain, whose characteristics (apparent molecular weight, immunoreactivity, N-terminal sequence and relative net charge) strongly suggested that PLP of altered size is present in jimpy mouse brain.
通过用氯仿 - 甲醇（2:1，w/v）萃取，从20日龄正常和震颤小鼠脑中分离出蛋白脂质，通过尺寸排阻高效液相色谱法去除脂质，并通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）、蛋白质免疫印迹、氨基酸分析和N端测序进行分析。SDS - PAGE显示，震颤小鼠脑的一种主要蛋白脂质的表观分子量约为23 kDa，介于正常小鼠脑的髓磷脂蛋白（PLP）和二甲基脂蛋白20（DM - 20）之间。用分别识别200 - 224位、116 - 150位和270 - 276位残基的3种不同抗体进行蛋白质免疫印迹，在正常和震颤PLP中均识别出免疫反应性物质。由于与270 - 276反应的抗体不能识别震颤PLP，提示震颤蛋白的C端发生了改变。这些结果表明，可以从震颤小鼠脑中部分纯化出一种PLP。氨基酸分析未能显示震颤PLP中半胱氨酸残基（根据cDNA预测）的预期增加。然而，当对震颤脑蛋白脂质进行N端测序时，检测到了PLP的前四个氨基酸：甘氨酸、亮氨酸、亮氨酸、甘氨酸。因此，从震颤小鼠脑中部分纯化出了一种蛋白脂质，其特征（表观分子量、免疫反应性、N端序列和相对净电荷）强烈表明震颤小鼠脑中存在大小改变的PLP。
