Rozdzial M M, Neighbors B W, McIntosh J R
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder 80309-0347.
Eur J Cell Biol. 1990 Jun;52(1):27-35.
Microtubule (MT)-binding peptides have been detected in homogenates of bovine brain tissue utilizing a blot overlay assay. Blots were prepared by the electrophoretic transfer to nitrocellulose of proteins separated on polyacrylamide gels. These blots were incubated with taxol stabilized MTs or tubulin, rinsed, and then fixed by air drying. About 17 soluble MT-associated proteins (MAPs) were identified by immunodetection of bound tubulin, including MAP2, kinesin, and tau. The interaction of MTs with these peptides appears to be specific, since MT binding can be displaced by a fluorescent tubulin analog, is competitively inhibited by the addition of exogenous brain MAPs, is decreased by raising the salt concentration, and is diminished by sodium dodecyl sulfate (SDS) denaturation. Only one protein (150 kDa) appears to have an interaction with MTs that is stable in high salt. The specificity of the binding on blots is further illustrated by the interaction of MTs with the MT-binding domains of MAP2 (32-35 kDa fragments) and kinesin (64 kDa fragment). Specific MT-binding peptides or domains can thus be isolated and characterized with this method, which requires little protein and is suitable for use with proteins that are either soluble or insoluble under physiological conditions.
利用印迹覆盖分析法已在牛脑组织匀浆中检测到微管（MT）结合肽。印迹是通过将聚丙烯酰胺凝胶上分离的蛋白质电泳转移到硝酸纤维素膜上制备的。这些印迹与紫杉醇稳定的微管或微管蛋白一起孵育，冲洗，然后通过空气干燥固定。通过对结合的微管蛋白进行免疫检测，鉴定出约17种可溶性微管相关蛋白（MAPs），包括MAP2、驱动蛋白和tau蛋白。微管与这些肽的相互作用似乎具有特异性，因为微管结合可被荧光微管蛋白类似物取代，通过添加外源性脑MAPs可竞争性抑制，通过提高盐浓度而降低，并且通过十二烷基硫酸钠（SDS）变性而减弱。只有一种蛋白质（150 kDa）似乎与微管具有在高盐中稳定的相互作用。微管与MAP2（32 - 35 kDa片段）和驱动蛋白（64 kDa片段）的微管结合结构域的相互作用进一步说明了印迹上结合的特异性。因此，可用这种方法分离和表征特定的微管结合肽或结构域，该方法所需蛋白质很少，适用于在生理条件下可溶或不可溶的蛋白质。
