Walker J H, Boustead C M, Witzemann V, Shaw G, Weber K, Osborn M
Eur J Cell Biol. 1985 Jul;38(1):123-33.
Treatment of the electric organ of Torpedo marmorata with Triton X-100 in the presence of 2 mM MgCl2 generated a cytoskeletal fraction in which a 54 kDa polypeptide is a major constituent. This 54 kDa polypeptide accounted for about 8% of the cellular protein when total electric organ tissue was analyzed by two-dimensional gel electrophoresis. Immunoblotting experiments showed that this protein reacts with monoclonal antibodies to desmin, the major intermediate filament protein of avian and mammalian muscle tissue. Negative stain analysis revealed that filaments of about 10 nm diameter are the major structural elements of the electric organ cytoskeleton. In the presence of Ca2+ there was a rapid degradation of the desmin-like protein and intermediate filaments due to a Ca2+-activated protease. Some of the resulting fragments retained antigenic activity against the desmin antibodies. Immunoblotting of membrane fractions enriched in acetylcholine receptor revealed desmin in addition to some actin. A further cytoskeletal component was identified from biochemical and immunological properties as a homologue of the mammalian neurofilament L-polypeptide. Thus Torpedo expresses proteins homologous to the mammalian desmin and neurofilament L-protein which can be detected using immunological approaches. Immunofluorescence microscopy was used to map the location of various cytoskeletal proteins of the cholinergic synapse on paraffin sections and on en face preparations of membranes. Desmin staining was restricted to electrocytes and in en face preparations was seen associated with both the ventral receptor-containing membrane and with the non-innervated dorsal membrane. Antibodies to neurofilament L-protein stained only the axons and not the electrocytes. Staining for fodrin, a non-erythrocyte spectrin, resulted in submembraneous decoration of both the axons and the electrocytes. Axonal staining for neurofilaments and microtubules did not extend into the ends of the nerve terminal arborizations.
在存在2 mM氯化镁的情况下，用曲拉通X-100处理斑纹电鳐的电器官，产生了一个细胞骨架组分，其中一种54 kDa的多肽是主要成分。当通过二维凝胶电泳分析整个电器官组织时，这种54 kDa的多肽约占细胞蛋白的8%。免疫印迹实验表明，这种蛋白质与抗结蛋白的单克隆抗体发生反应，结蛋白是鸟类和哺乳动物肌肉组织的主要中间丝蛋白。负染分析显示，直径约10 nm的细丝是电器官细胞骨架的主要结构成分。在钙离子存在的情况下，由于一种钙激活蛋白酶，结蛋白样蛋白和中间丝会迅速降解。一些产生的片段保留了针对结蛋白抗体的抗原活性。对富含乙酰胆碱受体的膜组分进行免疫印迹分析，除了一些肌动蛋白外，还发现了结蛋白。从生化和免疫学特性鉴定出另一种细胞骨架成分是哺乳动物神经丝L多肽的同源物。因此，电鳐表达与哺乳动物结蛋白和神经丝L蛋白同源的蛋白质，可用免疫学方法检测到。免疫荧光显微镜用于在石蜡切片和膜的正面制剂上绘制胆碱能突触各种细胞骨架蛋白的位置。结蛋白染色仅限于电细胞，在正面制剂中，可见其与含受体的腹侧膜和无神经支配的背侧膜相关。抗神经丝L蛋白抗体仅对轴突染色，而不对电细胞染色。对非红细胞血影蛋白血影蛋白进行染色，导致轴突和电细胞的膜下装饰。轴突上神经丝和微管的染色未延伸到神经末梢分支的末端。
