Etlinger J D, Zak R, Fischman D A
J Cell Biol. 1976 Jan;68(1):123-41. doi: 10.1083/jcb.68.1.123.
The localization of high-molecular-weight (80,000-200,000-daltons) proteins in the sarcomere of striated muscle has been studied by coordinated electron-microscopic and sodium dodecyl sulfate (SDS) gel electrophoretic analysis of native myofilaments and extracted and digested myofibrils. Methods were developed for the isolation of thick and thin filaments and of uncontracted myofibrils which are devoid of endoproteases and membrane fragments. Treatment of crude myofibrils with 0.5% Triton X-100 results in the release of a 110,000-dalton component without affecting the myofibrillar structure. Extraction of uncontracted myofibrils with a relaxing solution of high ionic strength results in the complete disappearance of the A band and M line. In this extract, five other protein bands in addition to myosin are resolved on SDS gels: bands M 1 (190,000 daltons) and M 2 (170,000 daltons), which are suggested to be components of the M line; M 3 (150,000 daltons), a degradation product; and a doublet M 4, M 5 (140,000 daltons), thick-filament protein having the same mobility as C protein. Extraction of myofibrils with 0.15% deoxycholate, previously shown to remove Z-line density, releases a doublet Z 1, Z 2 (90,000 daltons) with the same mobility as alpha-actinin, as well as proteins of 60,000 daltons and less, and small amounts of M 1, M 2, M 4, and M 5; these proteins were not extracted with 0.5% Triton X-100. The C, M-line, and Z-line proteins and/or their binding to myofibrils are very sensitive to tryptic digestion, whereas the M 3 (150,000 daltons) component and an additional band at 110,000 daltons are products of proteolysis. Gentle treatment of myofibrils with an ATP relaxing solution results in the release of thick and thin myofilaments which can be pelleted by 100,000-g centrifugation. These myofilaments lack M-and Z-line structure when examined with the electron microscope, and their electrophoretograms are devoid of the M 1, M 2, Z 1, and Z 2 bands. The M 4, M 5 (C-protein doublet), and M 3 bands, however, remain associated with the filaments.
通过对天然肌丝以及提取和消化后的肌原纤维进行电子显微镜和十二烷基硫酸钠（SDS）凝胶电泳协同分析，研究了高分子量（80,000 - 200,000道尔顿）蛋白质在横纹肌肌节中的定位。已开发出用于分离粗细肌丝以及不含内蛋白酶和膜片段的未收缩肌原纤维的方法。用0.5% Triton X - 100处理粗肌原纤维会释放出一种110,000道尔顿的成分，而不影响肌原纤维结构。用高离子强度的松弛溶液提取未收缩的肌原纤维会导致A带和M线完全消失。在该提取物中，除肌球蛋白外，SDS凝胶上还分辨出另外五条蛋白带：M1带（190,000道尔顿）和M2带（170,000道尔顿），推测它们是M线的成分；M3带（150,000道尔顿），一种降解产物；以及一个双重带M4、M5（140,000道尔顿），其迁移率与C蛋白相同的粗丝蛋白。用0.15%脱氧胆酸盐提取肌原纤维（先前已证明可去除Z线密度）会释放出迁移率与α - 辅肌动蛋白相同的双重带Z1、Z2（90,000道尔顿），以及60,000道尔顿及以下的蛋白质，还有少量的M1、M2、M4和M5；这些蛋白质不能用0.5% Triton X - 100提取。C线、M线和Z线蛋白和/或它们与肌原纤维的结合对胰蛋白酶消化非常敏感，而M3（150,000道尔顿）成分和110,000道尔顿的另一条带是蛋白水解产物。用ATP松弛溶液温和处理肌原纤维会释放出粗细肌丝，这些肌丝可通过100,000克离心沉淀。用电镜检查时，这些肌丝缺乏M线和Z线结构，其电泳图谱中没有M1、M2、Z1和Z2带。然而，M4、M5（C蛋白双重带）和M3带仍与肌丝相关。
