Gottlieb R A, Murphy D B
J Cell Biol. 1985 Nov;101(5 Pt 1):1782-9. doi: 10.1083/jcb.101.5.1782.
We examined the microtubule-binding domain of the microtubule-associated protein (MAP), MAP-2, using rabbit antibodies that specifically bind to the microtubule-binding region ("stub") and the projection portion ("arm") of MAP-2. We found that (a) microtubules decorated with arm antibody look similar to those labeled with whole unfractionated MAP antibody, though microtubules are not labeled with stub antibody; (b) incubation of depolymerized microtubule protein with stub antibody prior to assembly partially inhibits the rate of microtubule elongation, presumably because MAPs that are complexed with antibody cannot bind to microtubules and stabilize elongating polymers; (c) the rate of appearance and amounts of 36- and 40-kD microtubule-binding peptides produced by digestion with chymotrypsin are distinct for MAPs associated with microtubules vs. MAPs free in solution. The enhanced stability of the 40-kD peptide when associated with microtubules suggests that this domain of the protein is closely associated with, or partially buried in, the microtubule surface; (d) MAP-2 is a slender, elongate molecule as determined by unidirectional platinum shadowing (90 +/- 30 nm), which is in approximate agreement with previous observations. Stub antibody labels MAP-2 in the terminal one-quarter of the extended protein, indicating an intrinsic asymmetry in the molecule.
我们使用能特异性结合微管相关蛋白（MAP）-2的微管结合区域（“残端”）和突出部分（“臂”）的兔抗体，研究了微管相关蛋白MAP-2的微管结合结构域。我们发现：（a）用臂抗体标记的微管与用未分级的完整MAP抗体标记的微管看起来相似，不过微管用残端抗体不能被标记；（b）在组装前将解聚的微管蛋白与残端抗体一起温育会部分抑制微管伸长速率，推测是因为与抗体结合的MAP不能与微管结合并稳定伸长的聚合物；（c）用胰凝乳蛋白酶消化产生的36-kD和40-kD微管结合肽的出现速率和量，对于与微管相关的MAP和溶液中游离的MAP是不同的。40-kD肽与微管结合时稳定性增强，表明该蛋白结构域与微管表面紧密相关或部分埋于其中；（d）通过单向铂阴影法测定（90±30nm），MAP-2是细长的分子，这与先前的观察结果大致相符。残端抗体标记延伸蛋白末端四分之一处的MAP-2，表明该分子存在内在不对称性。
