Royuela M, Fraile B, De Miguel M P, Cervera M, Paniagua R
Department of Cell Biology and Genetics, University of Alcalá de Henares, Madrid, Spain.
Microsc Res Tech. 1996 Nov 1;35(4):349-56. doi: 10.1002/(SICI)1097-0029(19961101)35:4<349::AID-JEMT6>3.0.CO;2-H.
The presence and distribution of titin-like proteins have been examined in transversely striated muscle of Drosophila melanogaster, in obliquely striated muscles (body wall and inner muscular layer of the pseudoheart) and smooth muscle (outer muscular layer of the pseudoheart) from the earthworm Eisenia foetida by means of Western blotting analysis, light microscopy immunohistochemistry, and electron microscopy immunogold labeling, using antibodies anti vertebrate (chicken) titin (3,000 kDa) and arthropod (D. melanogaster) mini-titin (twitchin or projectin) (700 kDa). To determine whether these antibodies immunoreact non-specifically against vertebrate titin, mouse skeletal muscle was also studied. As negative control, mouse smooth muscle was used. Immunoreaction to mini-titin was found in all the invertebrate muscles studied. For each of these muscles, Western blotting analysis of mini-titin showed a single band, at approximately 700 kDa. Electron microscopy immunolabeling to this protein was observed along the whole sarcomere length (A bands and I bands) in both transversely striated muscles of the insect and obliquely striated muscles of the earthworm, although the number of immunogold particles was more abundant in the insect muscles. Mini-titin immunolabeling was also observed in the smooth muscle cells that formed the outer layer of the earthworm pseudoheart although in lower amounts than in the obliquely striated muscle. The absence of true sarcomeres in the smooth muscle cells did not permit to determine the extension of mini-titin immunolabeling. No immunoreaction to this protein was found in the striated and smooth muscles of the mouse. Immunoreaction to titin was only observed in the mouse skeletal muscle, in which both A bands and I bands appeared immunolabeled. Present results show that mini-titin in the invertebrate muscles studied differs immunohistochemically from vertebrate titin and, in contrast with titin, mini-titin is also present in invertebrate smooth muscles.
利用蛋白质免疫印迹分析、光学显微镜免疫组织化学以及电子显微镜免疫金标记技术，使用抗脊椎动物（鸡）肌联蛋白（3000 kDa）和节肢动物（黑腹果蝇）微小肌联蛋白（肌动蛋白或投射蛋白）（700 kDa）的抗体，研究了黑腹果蝇横纹肌、蚯蚓（赤子爱胜蚓）斜纹肌（体壁和假心脏内层肌肉）和平滑肌（假心脏外层肌肉）中类肌联蛋白的存在情况和分布。为了确定这些抗体是否会与脊椎动物肌联蛋白发生非特异性免疫反应，还对小鼠骨骼肌进行了研究。作为阴性对照，使用了小鼠平滑肌。在所研究的所有无脊椎动物肌肉中均发现了对微小肌联蛋白的免疫反应。对于这些肌肉中的每一种，微小肌联蛋白的蛋白质免疫印迹分析均显示在约700 kDa处有一条单一的条带。在昆虫的横纹肌和蚯蚓的斜纹肌中，沿着整个肌节长度（A带和I带）均观察到了针对该蛋白的电子显微镜免疫标记，尽管昆虫肌肉中的免疫金颗粒数量更多。在构成蚯蚓假心脏外层的平滑肌细胞中也观察到了微小肌联蛋白免疫标记，但其含量低于斜纹肌。平滑肌细胞中不存在真正的肌节，因此无法确定微小肌联蛋白免疫标记的范围。在小鼠的横纹肌和平滑肌中均未发现对该蛋白的免疫反应。仅在小鼠骨骼肌中观察到了对肌联蛋白的免疫反应，其中A带和I带均出现了免疫标记。目前的结果表明，在所研究的无脊椎动物肌肉中，微小肌联蛋白在免疫组织化学上与脊椎动物肌联蛋白不同，并且与肌联蛋白相反，微小肌联蛋白也存在于无脊椎动物平滑肌中。
