Determination of the protein components of native thin filaments isolated from natural actomyosin: nebulin and alpha-actinin are associated with actin filaments.

A new method was developed for extracting natural actomyosin with full length connectin (titin) and nebulin from rabbit skeletal muscle. To determine the protein components of native thin filaments, thin filaments were isolated from natural actomyosin by sedimentation in a H2O/D2O/sucrose density gradient. Both alpha-actinin and nebulin were cosedimented with actin filaments, while connectin was not. This shows that the native thin filaments contained alpha-actinin and nebulin. This indicates that the native thin filaments were more complicated than synthetic filaments reconstituted from actin, tropomyosin, and troponin. It is known that synthetic filaments are less Ca2+ sensitive than native thin filaments. This difference in Ca2+ sensitivity may be due to the differences in components and/or the different structures of native and synthetic filaments. The newly developed methods described here for extracting natural actomyosin and for isolating native thin filaments are useful for addressing these important problems related to the structure and function of native thin filaments.