Homogenization, isolated smooth muscle

The key step in LC isolation is the preparation of pure smooth muscle myosin (Hasegawa et al., 1988). This is usually executed by homogenizing the muscle mince with salt solutions (60-100 mM ionic strength) to remove the cytoplasmic proteins and to a certain extent the thin filament proteins. A crude actomyosin is then extracted form the well-washed muscle residue with salt solutions containing ATP and the actin component is removed by ultracentrifugation. The LCs are... 

The use of saponin, a plant glycoside, for permeabilization was introduced by Endo and coworkers (1977). Saponin removes the surface membrane without impairment of the functions of SR. The plasma membrane, unlike that of triton skinned smooth muscle (Spedding, 1983), appears fairly intact in electron microscopic pictures of cross sections of permeabilized cells. However, when patches of isolated membranes were viewed face on in homogenates of permeabilized cells, numerous 70- to 80-A holes were visible (Kargacin and Lay, 1987). 

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