Morita-Fujimura Y, Kurachi M, Tashiro H, Komiya Y, Tashiro T
Laboratory for Photo-Biology, Institute of Physical and Chemical Research (RIKEN), Sendai, Japan.
Biochem Biophys Res Commun. 1996 Aug 14;225(2):462-8. doi: 10.1006/bbrc.1996.1195.
Based on video-enhanced differential interference contrast (DIC) microscopy, we developed a small-scale method which is capable of measuring both the lengths and the number densities of microtubules (MTs) assembled in vitro. With this method, effect of dephosphorylation on the activity of bovine brain tau protein to promote the assembly of tubulin at physiological concentration (15 microM) was quantitatively analyzed. The MT number density was selectively reduced when tau isolated directly in the presence of phosphatase inhibitors (N-tau) was dephosphorylated in vitro (DP-tau), without significant changes in the mean MT length or the binding affinity toward preformed MTs. Tau obtained from brain MTs (MT-tau) also exhibited lower nucleation activity in spite of its high MT-binding affinity. The results indicate that nucleation, elongation and MT-binding are distinct aspects of tau function which are differentially affected by the phosphorylation state of tau.
基于视频增强型微分干涉差（DIC）显微镜技术，我们开发了一种小规模方法，该方法能够测量体外组装的微管（MTs）的长度和数量密度。利用此方法，我们定量分析了去磷酸化对牛脑tau蛋白在生理浓度（15微摩尔）下促进微管蛋白组装活性的影响。当直接在磷酸酶抑制剂存在下分离得到的tau（N-tau）在体外去磷酸化（DP-tau）时，MT数量密度选择性降低，而平均MT长度或对预先形成的MTs的结合亲和力无显著变化。从脑MTs中获得的tau（MT-tau）尽管具有高MT结合亲和力，但也表现出较低的成核活性。结果表明，成核、伸长和MT结合是tau功能的不同方面，它们受到tau磷酸化状态的不同影响。
