Pardridge W M, Nowlin D M, Calaycay J, Shively J E
Department of Medicine, UCLA School of Medicine 90024.
J Neurochem. 1989 Oct;53(4):1014-8. doi: 10.1111/j.1471-4159.1989.tb07388.x.
Blood-brain barrier (BBB) function is endowed by the expression of unique proteins within the brain capillary endothelium. In the absence of knowing the function of BBB-specific proteins, one strategy for identification of these proteins is the purification and amino acid sequencing of proteins within the brain capillary that are not found in other cells. Earlier studies have shown that a 16-18K triplet of low-molecular-weight proteins in isolated brain capillaries is not found in either erythrocytes or in capillary-free preparations of synaptosomal proteins. Therefore, the present studies describe the purification of the 16-18K triplet of proteins as well as a 14K protein in isolated brain capillaries using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and C4 reverse-phase HPLC. Amino acid sequencing of the N-terminus of the 14K, 17K, and 18K proteins and of two tryptic peptides of the 16K protein showed that these proteins are alpha-globin, histone 2B, histone 3, and histone 2A, respectively. SDS-PAGE of subcellular fractions of bovine brain capillaries demonstrated that the 16-18K triplet of histone proteins migrated in the nuclear fraction. In addition, a 34K doublet and a 200K protein were localized in the nuclear pellet. Therefore, the present studies demonstrate that the predominant 14-18K proteins seen on SDS-PAGE of isolated brain capillaries are known proteins and provide a general scheme for purification of brain capillary proteins isolated following SDS-PAGE.
血脑屏障（BBB）的功能由脑毛细血管内皮细胞中独特蛋白质的表达赋予。在不了解BBB特异性蛋白质功能的情况下，鉴定这些蛋白质的一种策略是纯化脑毛细血管中其他细胞中不存在的蛋白质并进行氨基酸测序。早期研究表明，在分离的脑毛细血管中存在的一种16 - 18K的低分子量蛋白质三联体在红细胞或无毛细血管的突触体蛋白制剂中均未发现。因此，本研究描述了使用十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳（PAGE）和C4反相高效液相色谱法（HPLC）纯化分离的脑毛细血管中的16 - 18K蛋白质三联体以及一种14K蛋白质。对14K、17K和18K蛋白质的N端以及16K蛋白质的两个胰蛋白酶肽段进行氨基酸测序表明，这些蛋白质分别是α-珠蛋白、组蛋白2B、组蛋白3和组蛋白2A。牛脑毛细血管亚细胞组分的SDS-PAGE表明，组蛋白的16 - 18K三联体在核组分中迁移。此外，一个34K的双峰和一个200K的蛋白质定位于核沉淀中。因此，本研究表明，在分离的脑毛细血管的SDS-PAGE上看到的主要14 - 18K蛋白质是已知蛋白质，并提供了一种在SDS-PAGE后纯化分离的脑毛细血管蛋白质的通用方案。
