Fujii T, Koizumi Y
Department of Imagination Science (Kansei Engineering), Faculty of Textile Science and Technology, Shinshu University, Ueda, Nagano, 386-8567, Japan.
J Biochem. 1999 May;125(5):869-75. doi: 10.1093/oxfordjournals.jbchem.a022362.
Calponin is a basic smooth muscle protein capable of binding to actin, calmodulin, tropomyosin, and phospholipids. We have found that the basic calponin interacted with brain tubulin under polymerized and unpolymerized conditions in vitro [Fujii, T., Hiromori, T., Hamamoto, M., and Suzuki, T. (1997) J. Biochem. 122, 344-351]. We examined the calponin-binding site on the tubulin molecule by sedimentation, limited digestion, chemical-cross linking, immunoblotting, and delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometric (DE MALDI-TOF) analyses. Calponin interacts with both the alpha and beta tubulins and only slightly with the tyrosinated and acetylated form of alpha tubulin. The binding of calponin to microtubules was blocked by adding poly(L-aspartic acid) (PLAA) or MAP2. After digestion of microtubule proteins with subtilisin, the amount of calponin binding to alphabetas microtubules was reduced compared to native microtubules, but no further reduction was observed in the case of alphasbetas microtubules. The chemical cross-linked products of calponin and synthesized peptides (KDYEEVGVDSVEGE; alpha-KE) derived from the C-terminal region of alpha tubulin and (YQQYQDATADEQG; beta-YG) and (GEFEEEGEEDEA; beta-GA) from that of beta tubulin were detected by mass spectrometry. One kind of calponin-peptide complex was formed in the presence of alpha-KE or beta-YG, while five complexes (calponin:peptide = 1:1-5) were generated in the presence of beta-GA. Peptides alpha-KE and beta-GA inhibited the binding of calponin to tubulin produced by EDC in a concentration-dependent manner. These findings suggest that basic calponin interacts with both tubulin subunits and that their C-terminal regions, which also contain the binding sites of MAP2, tau, and kinesin, may be involved in calponin-binding.
钙调蛋白是一种碱性平滑肌蛋白，能够与肌动蛋白、钙调蛋白、原肌球蛋白和磷脂结合。我们发现，碱性钙调蛋白在体外聚合和未聚合条件下均能与脑微管蛋白相互作用[藤井，T.，平森，T.，滨本，M.，和铃木，T.（1997年）《生物化学杂志》122卷，344 - 351页]。我们通过沉降、有限消化、化学交联、免疫印迹以及延迟提取基质辅助激光解吸电离飞行时间质谱（DE MALDI - TOF）分析，研究了微管蛋白分子上的钙调蛋白结合位点。钙调蛋白与α和β微管蛋白均有相互作用，而与酪氨酸化和乙酰化形式的α微管蛋白仅有微弱相互作用。通过添加聚（L - 天冬氨酸）（PLAA）或微管相关蛋白2（MAP2）可阻断钙调蛋白与微管的结合。用枯草杆菌蛋白酶消化微管蛋白后，与天然微管相比，钙调蛋白与αβ微管的结合量减少，但在αβ微管的情况下未观察到进一步减少。通过质谱检测到了钙调蛋白与源自α微管蛋白C末端区域的合成肽（KDYEEVGVDSVEGE；α - KE）以及源自β微管蛋白C末端区域的（YQQYQDATADEQG；β - YG）和（GEFEEEGEEDEA；β - GA）的化学交联产物。在α - KE或β - YG存在的情况下形成了一种钙调蛋白 - 肽复合物，而在β - GA存在的情况下产生了五种复合物（钙调蛋白：肽 = 1:1 - 5）。肽α - KE和β - GA以浓度依赖的方式抑制了钙调蛋白与由1 - 乙基 - 3 -（3 - 二甲基氨基丙基）碳二亚胺（EDC）产生的微管蛋白的结合。这些发现表明碱性钙调蛋白与两种微管蛋白亚基均有相互作用，并且它们的C末端区域（该区域也包含MAP2、微管相关蛋白tau和驱动蛋白的结合位点）可能参与了钙调蛋白的结合。
