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Biophys J, November 2001, p. 2817-2826, Vol. 81, No. 5

Purification of Native Myosin Filaments from Muscle

Carlos Hidalgo,*dagger Raúl Padrón,dagger Rachel Horowitz,* Fa-Qing Zhao,* and Roger Craig*

 *Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655 USA;  dagger Departamento de Biología Estructural, Instituto Venezolano de Investigaciones Científicas, Caracas 1020A, Venezuela

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 Ca2+-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.

Biophys J, November 2001, p. 2817-2826, Vol. 81, No. 5
© 2001 by the Biophysical Society   0006-3495/01/11/2817/10  $2.00


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