Purification of native myosin filaments from muscle
Department of Cell Biology
Animals; Calcium; Cattle; Centrifugation; Electrophoresis; Gelsolin; Microfilaments; Muscles; Myofibrils; Myosin Heavy Chains; Myosin Light Chains; Myosins; Spiders
Life Sciences | Medicine and Health Sciences
Analysis of the structure and function of native thick (myosin-containing) filaments of muscle has been hampered in the past by the difficulty of obtaining a pure preparation. We have developed a simple method for purifying native myosin filaments from muscle filament suspensions. The method involves severing thin (actin-containing) filaments into short segments using a Ca(2+)-insensitive fragment of gelsolin, followed by differential centrifugation to purify the thick filaments. By gel electrophoresis, the purified thick filaments show myosin heavy and light chains together with nonmyosin thick filament components. Contamination with actin is below 3.5%. Electron microscopy demonstrates intact thick filaments, with helical cross-bridge order preserved, and essentially complete removal of thin filaments. The method has been developed for striated muscles but can also be used in a modified form to remove contaminating thin filaments from native smooth muscle myofibrils. Such preparations should be useful for thick filament structural and biochemical studies.
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Citation: Biophys J. 2001 Nov;81(5):2817-26.