Kong L W, Shen Q, Ding X Y, Hiroshi K, Jing N H
Shanghai Institute of Biochemistry, Academia Sinica, Japan.
Sheng Li Xue Bao. 1997 Aug;49(4):361-9.
A 20-residue peptide corresponding to the C-terminal amino acid sequence of rat nestin was synthesized by the solid phase method. The anti-peptide antibody (designated Anti-Nes-2) against nestin was prepared. Western blots showed that Anti-Nes-2 recognized not only mouse nestin with a MW of 240 kD but also a band with a MW of 50 kD. N-terminal amino acid sequence showed that this 50 kD protein is alpha-tubulin. Western blots with Anti-Nes-2 and with monoclonal antibodies against alpha- and beta-tubulin revealed that this 50 kD band could only be detected in different stages of mouse brain and in the primary culture of neural precursor cells (NPCs), with higher expression during the development of mouse brain and the maturation of NPCs; whereas alpha- and beta-tubulin were expressed in different cell lines and tissues of adult mouse. Taken together, these results indicate that 50 kD protein recognized by Anti-Nes-2 is a neuron-specific alpha-tubulin and could be a neuron-specific posttranslational modification isotype of alpha-tubulin.
采用固相法合成了一段与大鼠巢蛋白C端氨基酸序列相对应的20个残基的肽段。制备了针对巢蛋白的抗肽抗体(命名为Anti-Nes-2)。蛋白质免疫印迹分析表明,Anti-Nes-2不仅能识别分子量为240 kD的小鼠巢蛋白,还能识别一条分子量为50 kD的条带。N端氨基酸序列分析表明,这条50 kD的蛋白质是α-微管蛋白。用Anti-Nes-2以及抗α-和β-微管蛋白的单克隆抗体进行蛋白质免疫印迹分析显示,这条50 kD的条带仅在小鼠脑发育的不同阶段以及神经前体细胞(NPC)的原代培养物中能检测到,在小鼠脑发育和NPC成熟过程中表达较高;而α-和β-微管蛋白在成年小鼠的不同细胞系和组织中均有表达。综上所述,这些结果表明,Anti-Nes-2识别的50 kD蛋白质是一种神经元特异性的α-微管蛋白,可能是α-微管蛋白的一种神经元特异性翻译后修饰异构体。