Rauvala H, Pihlaskari R
Department of Medical Chemistry, University of Helsinki, Finland.
J Biol Chem. 1987 Dec 5;262(34):16625-35.
Fractionation of octyl glucoside-solubilized proteins from young rat brain was monitored using rat brain neurons, which were cultured in microwells coated with various protein fractions to be studied. An adhesive protein that promotes neurite outgrowth in rat brain neurons was isolated by chromatography on heparin-Sepharose followed by Affi-Gel blue. The apparent molecular mass of the protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions was about 30 kilodaltons (p30). Under nonreducing conditions a closely spaced doublet band was observed corresponding to 27-28-kilodalton size. Gel filtration in the presence of 4 M urea indicated the molecular size of 58 kilodaltons suggesting a dimeric structure. Western blotting experiments using affinity-purified rabbit antibodies detected p30 as an immunochemically distinct protein in brain and in N18 neuroblastoma cells. The p30 protein was also detected in the N18 cells by lactoperoxidase-catalyzed cell surface iodination. Western blotting of heparin-binding proteins solubilized from brains of rats of various age groups indicated that p30 is clearly more abundant in perinatal brain as compared to adult tissue. The neuron-binding and neurite outgrowth-promoting properties of p30 as well as the developmental regulation of its content in brain tissue suggest a role in neuronal growth.
使用大鼠脑神经元监测了从幼鼠脑中分离出的辛基葡糖苷增溶蛋白的分级分离情况，这些神经元培养在涂有各种待研究蛋白级分的微孔板中。通过肝素-琼脂糖层析，随后用Affi-Gel蓝进行分离，得到了一种能促进大鼠脑神经元轴突生长的黏附蛋白。在还原条件下，该蛋白在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中的表观分子量约为30千道尔顿（p30）。在非还原条件下，观察到一条紧密间隔的双峰带，对应于27 - 28千道尔顿的大小。在4 M尿素存在下进行凝胶过滤表明其分子大小为58千道尔顿，提示为二聚体结构。使用亲和纯化的兔抗体进行的蛋白质印迹实验检测到p30是脑中以及N18神经母细胞瘤细胞中一种免疫化学上独特的蛋白质。通过乳过氧化物酶催化的细胞表面碘化也在N18细胞中检测到了p30蛋白。对从不同年龄组大鼠脑中溶解的肝素结合蛋白进行蛋白质印迹分析表明，与成年组织相比，p30在围产期脑中明显更为丰富。p30的神经元结合和促进轴突生长的特性以及其在脑组织中含量的发育调控表明它在神经元生长中发挥作用。
