Pedrotti B, Islam K
Department of Biology, University of Milan, Italy.
Cell Motil Cytoskeleton. 1995;30(4):301-9. doi: 10.1002/cm.970300407.
A simple procedure for the purification of MAP1B from bovine brain is described. The procedure requires two ion-exchange chromatographic steps and results in > 95% pure MAP1B with a typical recovery of about 25-30 mg/kg of brain tissue. SDS-PAGE analysis of the purified protein shows that it is composed of a high molecular mass (330kDa) heavy chain and two low molecular mass (32kDa and 18kDa) associated light chains. The estimated stoichiometry of heavy chain:light chain is 1:2 and 1:0.2 mole/mole protein for the 32kDa and 18kDa light chains respectively. Western blotting, using monospecific monoclonal antibodies, shows that only the heavy chain is recognised by the anti-MAP1B antibody and is not immunostained by either the MAP1A or MAP2 monoclonal antibodies. Purified MAP1B binds efficiently to both unpolymerised tubulin and polymerised tubulin and co-sediments with taxol-stabilised microtubules. Co-incubation experiments show that MAP2 can compete with MAP1B binding to microtubules, indicating common or overlapping sites. However, MAP1B binds to neither G-actin nor F-actin nor co-sediments with F-actin, suggesting that it is not an actin-binding protein.
本文描述了一种从牛脑中纯化微管相关蛋白1B（MAP1B）的简单方法。该方法需要两个离子交换色谱步骤，最终得到纯度大于95%的MAP1B，每千克脑组织的典型回收率约为25 - 30毫克。对纯化蛋白进行的十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）分析表明，它由一条高分子量（330kDa）的重链和两条低分子量（32kDa和18kDa）的相关轻链组成。重链与轻链的估计化学计量比分别为1:2（针对32kDa轻链）和1:0.2摩尔/摩尔蛋白（针对18kDa轻链）。使用单特异性单克隆抗体进行的蛋白质免疫印迹分析表明，抗MAP1B抗体仅识别重链，而不被MAP1A或MAP2单克隆抗体免疫染色。纯化的MAP1B能有效地与未聚合的微管蛋白和聚合的微管蛋白结合，并与紫杉醇稳定的微管共同沉降。共孵育实验表明，MAP2可以与MAP1B竞争结合微管，这表明它们具有共同或重叠的结合位点。然而，MAP1B既不与G - 肌动蛋白结合，也不与F - 肌动蛋白结合，也不与F - 肌动蛋白共同沉降，这表明它不是一种肌动蛋白结合蛋白。
