4] Hybridization and reconstitution of the thin filament

Abstract

This chapter presents procedure for hybridization and reconstitution of the thin filament. Reconstitution of the thin filament is conveniently carried out using pure actin and a complex of troponin and tropomyosin as starting materials. The complex, Ebashi's native tropomyosin, is prepared from rabbit back and leg muscle by dissociating it from the thin filaments at low ionic strength. The thin filament is reassembled by polymerizing actin in the presence of an excess of the regulatory proteins. To purified G-actin (1-5 mg/ml) is added twice its weight of the troponin-tropomyosin complex plus sufficient buffer, KC1, MgCl 2 , and ATP to bring the final mixture to 0.1 M KCl, 2 mM MgCl 2 , 0.1 mM ATP, pH 7.0. The mixture is stirred briefly, then incubated without stirring at 15 ° for 45 min. The thin filaments are collected by centrifugation at 145,000g for 2 hr. The supernatant is decanted, and the pellets and tubes are rinsed with buffer and allowed to drain. To resuspend the thin filaments evenly, homogenization will be necessary. However, the quality of the final product is sensitive to the mechanical stresses brought to bear upon it as these pellets are resuspended. For best results, the pellets should be covered with the resuspension medium and allowed to swell and soften for a few hours before gentle homogenization with a loose-fitting glass-Teflon homogenizer.