A single-fiber in vitro motility assay. In vitro sliding velocity of F-actin vs. unloaded shortening velocity in skinned muscle fibers.

摘要: We describe an approach that allows us to form a micro in vitro motility assay with as little myosin can be retrieved from short ( ∼ 10 mm) segment of single skinned skeletal muscle fiber (diameter some 100 μm). Myosin is directly extracted the by high ionic strength solution presence MgATP, and immediately applied miniaturized flow cell has been pretreated BSA. The observed sliding velocities fluorescently labeled F-actin are essentially identical those reported literature. Since at level most fibers contain only heavy chain isoform this determine without additional purification steps, velocity driven myosins different isoforms. In addition, used correlate under experimental conditions unloaded shortening measured segments extraction same fibers. Such direct correlation was performed isoforms well temperatures strengths. Under all studied, 4- 8-fold faster than even temperature (22∘ C) strengths >50 mM. This suggests limited factors beyond thought limit One such factor might unspecific interactions between substrate resulting somewhat higher sensitivity for assay. become special relevance when using assessing functional consequences mutations involving charged residues actin or myosin.