低分子量神经丝蛋白(NF-L)的体外组装

THE ASSEMBLY OF LOW MOLECULAR WEIGHT NEUROFILAMENT PROTEIN (NF-L)IN VITRO

利用超速离心和离子交换层析技术,从牛脊髓中分离纯化了神经丝蛋白三组分:NF-L,NF-M和NF-H。应用电镜负染色和金属投影方法研究神经丝的形态结构与NF-L的体外组装,结果表明:神经丝由10nm的核心纤维与外周的丝状突起组成;在近似生理条件下,NF-L可在60min内组装成10nm纤维,纤维由4根亚丝缠绕而成;在碱性缓冲液中,NaCl能促进NF-L装配成短纤维,这种10nm的短纤维无法连接成长纤维。

Neurofilaments were isolated from bovine spinal cords by ultra-speed centrifugation and examined by negative staining. The neurofilament triplet proteins :NF-L,NF-M and NF-H were purified by DE-52 anion exchange chromatography in the presence of 6 mol/L urea. The reassembly of NF-L under controlled conditions was studied. NF-L can reassemble into 10 nm width filaments within 60 minutes at physiological condition of around 0. 15mol/L NaCl, 2mmol/L MgCl2, neutral pH(pH6. 8) and 37℃. In 6 mol/L urea,NF-L was examined as 12 nm-diameter particle by low angle rotary shadowing. When dialyzed against reassembly buffer for 20 minutes,some irregular filaments were formed. Further dialyzed for another 40 minutes , the long smooth filaments appeared. Some filaments were unraveled at the end regions , where existed 2 - 4 subfilaments. Four subfilaments were more often observed. That is to say, the 10nm-width filament was composed of 4 subfilaments. While dialyzed against the alkaline buffer containing 0. 15 mol/L NaCl, NF-L reconstituted into 45 - 180nm-long, lOnm-width filaments, which were not able to elongate into long filaments.