Díaz José Fernando, Barasoain Isabel, Souto André A, Amat-Guerri Francisco, Andreu José Manuel
Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, Madrid 28040, Spain.
J Biol Chem. 2005 Feb 4;280(5):3928-37. doi: 10.1074/jbc.M407816200. Epub 2004 Nov 18.
The macromolecular accessibility of the paclitaxel binding site in microtubules has been investigated using a fluorescent taxoid and antibodies against fluorescein, which cannot diffuse into the microtubule lumen. The formation of a specific ternary complex of microtubules, Hexaflutax (7-O-{N-[6-(fluorescein-4'-carboxamido)-n-hexanoyl]-l-alanyl}paclitaxel) and 4-4-20 IgG (a monoclonal antibody against fluorescein) has been observed by means of sedimentation and electron microscopy methods. The kinetics of binding of the antibody to microtubule-bound Hexaflutax has been measured. The quenching of the observed fluorescence is fast (k+ 2.26 +/- 0.25 x 10(6) m(-1) s(-1) at 37 degrees C), indicating that the fluorescein groups of Hexaflutax are exposed to the outer solvent. The velocity of the reaction is linearly dependent on the antibody concentration, indicating that a bimolecular reaction is being observed. Another fluorescent taxoid (Flutax-2) bound to microtubules has also been shown to be rapidly accessible to polyclonal antibodies directed against fluorescein. A reduced rate of Hexaflutax quenching by the antibody is observed in microtubule-associated proteins containing microtubules or in native cellular cytoskeletons. It can be concluded that the fluorescent taxoids bind to an outer site on the microtubules that is shared with paclitaxel. Paclitaxel would be internalized in a further step of binding to reach the known luminal site, this step being blocked in the case of the fluorescent taxoids. Because the fluorescent ligands are able to induce microtubule assembly, binding to the outer site should be enough to induce assembly by a preferential binding mechanism.
已使用一种荧光紫杉烷类化合物和抗荧光素抗体（其不能扩散到微管腔中）研究了微管中紫杉醇结合位点的大分子可及性。通过沉降和电子显微镜方法观察到了微管、六氟他赛（7 - O - {N - [6 - (荧光素 - 4'- 羧酰胺基)-正己酰基]-L - 丙氨酰基}紫杉醇）和4 - 4 - 20 IgG（一种抗荧光素单克隆抗体）形成的特定三元复合物。测定了抗体与微管结合的六氟他赛的结合动力学。观察到的荧光猝灭很快（37℃时k + 2.26 +/- 0.25×10(6) m(-1) s(-1)），表明六氟他赛的荧光素基团暴露于外部溶剂中。反应速度与抗体浓度呈线性相关，表明观察到的是双分子反应。另一种与微管结合的荧光紫杉烷类化合物（氟他赛 - 2）也已被证明可被抗荧光素的多克隆抗体快速接近。在含有微管的微管相关蛋白或天然细胞骨架中观察到抗体对六氟他赛的猝灭速率降低。可以得出结论，荧光紫杉烷类化合物结合到微管上与紫杉醇共有的外部位点。紫杉醇在进一步的结合步骤中会内化以到达已知的腔内部位，而在荧光紫杉烷类化合物的情况下这一步骤被阻断。由于荧光配体能够诱导微管组装，结合到外部位点应该足以通过优先结合机制诱导组装。
