Fumagalli L, Businaro R, Nori S L, Toesca A, Emmons C, De Renzis G
Departement of Cardiovascular Sciences, University "La Sapienza," Rome, Italy.
Mol Chem Neuropathol. 1996 Apr;27(3):211-23. doi: 10.1007/BF02815105.
Alpha(2)-Macroglobulin (alpha(2)M), a major serum protease inhibitor, was localized in mouse skeletal muscle by immunoperoxidase histochemistry. In all muscles examined (mm. soleus, plantaris, and extensor digitorum longus) specific immunoreactivity occurred diffusely in extracellular structures (periendomysium, blood vessel wall) as well as inside about a half of the muscle fibers. This localization pattern did not change substantially by extensively perfusing deeply anesthetized mice with phosphate buffered saline (PBS) to remove serum alpha(2)M. In release experiments on fresh (nonfixed) cryostat sections, specific immunoreactivity persisted after an extensive prewash with PBS (up to 5-6 h), but a new specific staining appeared inside those fibers that were originally negative. Western blotting experiments were negative on the soluble fraction of muscle homogenate, thus confirming that the perfusion procedure was effective in removing serum alpha(2)M. By contrast, three specific bands (185, 165, and 35 kDa) appeared in detergent-solubilized extracts (0.3% Triton X-100), indicating the occurrence of tissue-associated alpha(2)M. Confocal immunofluorescence microscopy revealed that the intracellular specific staining was associated to a longitudinal network, probably corresponding to the sarcoplasmic reticulum. A multifunctional role of alpha(2)M in skeletal muscle was hypothesized.
α（2）-巨球蛋白（α（2）M）是一种主要的血清蛋白酶抑制剂，通过免疫过氧化物酶组织化学法在小鼠骨骼肌中定位。在所有检查的肌肉（比目鱼肌、跖肌和趾长伸肌）中，特异性免疫反应在细胞外结构（肌束膜、血管壁）以及约一半的肌纤维内部弥漫性出现。通过用磷酸盐缓冲盐水（PBS）对深度麻醉的小鼠进行广泛灌注以去除血清α（2）M，这种定位模式没有实质性改变。在新鲜（未固定）的低温切片释放实验中，用PBS进行广泛预冲洗（长达5-6小时）后，特异性免疫反应仍然存在，但在那些原本阴性的纤维内部出现了新的特异性染色。肌肉匀浆可溶性部分的蛋白质印迹实验呈阴性，从而证实灌注程序有效地去除了血清α（2）M。相比之下，在去污剂溶解提取物（0.3% Triton X-100）中出现了三条特异性条带（185、165和35 kDa），表明存在与组织相关的α（2）M。共聚焦免疫荧光显微镜显示，细胞内特异性染色与一个纵向网络相关，可能对应于肌浆网。推测α（2）M在骨骼肌中具有多功能作用。
