Takei N, Kondo J, Nagaike K, Ohsawa K, Kato K, Kohsaka S
Department of Neurochemistry, National Institute of Neuroscience, Tokyo, Japan.
J Neurochem. 1991 Oct;57(4):1178-84. doi: 10.1111/j.1471-4159.1991.tb08277.x.
Neuronal survival factors in the central nervous system were investigated by using a primary culture of embryonic rat neocortical neurons. Bovine hippocampus was homogenized, and the supernatant from high-speed centrifugation was used as the starting material. At the step of DE-52 ion-exchange chromatography, neuronal survival activity was recovered in two fractions, fraction 14 (F14) and fraction 23 (F23). Antisera to the crude F14 and F23 fractions were raised in rabbits. These two antisera completely inhibited the neurotrophic activity of both fractions. Western blotting analysis revealed that anti-F14 antiserum recognized mainly a 30-kDa protein in F14 and anti-F23 antiserum recognized mainly a 44-kDa protein in F23. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis of F23, the 44-kDa protein was cut out from the gel and partial amino acid sequences of the protein fragments were determined. A GenBank data bank indicated that the amino acid sequence of the fragment was identical to that of neuron-specific enolase (NSE). In our assay system, commercially available NSE itself possessed neuronal survival activity for the cultured neocortical neurons. The effects of NSE and F23 were inhibited completely by anti-NSE polyclonal antibody. Furthermore, highly purified NSE supported the survival of cultured neurons in a dose-dependent manner, and the neurotrophic effect was inhibited by monoclonal antibody to the NSE. These results strongly suggest that NSE is one of the neuronal survival factors in the central nervous system.
利用原代培养的胚胎大鼠新皮质神经元，对中枢神经系统中的神经元存活因子进行了研究。将牛海马匀浆，高速离心后的上清液用作起始材料。在DE - 52离子交换色谱步骤中，神经元存活活性在两个组分中得到恢复，即组分14（F14）和组分23（F23）。用兔制备了针对粗制F14和F23组分的抗血清。这两种抗血清完全抑制了两个组分的神经营养活性。蛋白质印迹分析显示，抗F14抗血清主要识别F14中的一种30 kDa蛋白，抗F23抗血清主要识别F23中的一种44 kDa蛋白。对F23进行十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳后，从凝胶中切出44 kDa蛋白，并测定了该蛋白片段的部分氨基酸序列。GenBank数据库表明，该片段的氨基酸序列与神经元特异性烯醇化酶（NSE）的氨基酸序列相同。在我们的检测系统中，市售的NSE本身对培养的新皮质神经元具有神经元存活活性。NSE和F23的作用被抗NSE多克隆抗体完全抑制。此外，高度纯化的NSE以剂量依赖的方式支持培养神经元的存活，并且神经营养作用被针对NSE的单克隆抗体抑制。这些结果强烈表明NSE是中枢神经系统中的神经元存活因子之一。
