Myosin-free ghosts of single fibers and an attempt to re-form myosin filaments in the ghost fibers

Abstract

With the final aim of replacing myosin in a single muscle fiber, a technique for removing myosin almost completely from single fibers was developed and an attempt to "re-form" thick filaments in the myosin-free ghosts of single fibers was made. Complete removal of myosin from single glycerol-treated rabbit psoas fibers with Hasselbach-Schneider solution was difficult. However, when skinned glycerol-treated fibers were used and 1% (v/v) Triton X-100 was added to the Hasselbach-Schneider solution, almost complete removal of myosin was possible. The myosin-free ghosts of skinned single fibers were very fragile but retained the overall structure. In the ghost fibers, Z-membranes and thin filaments remained. The ghost fibers, after irrigation with myosin, underwent contraction upon addition of Mg-ATP. In the myosin-irrigated fibers, thick filaments were re-formed in lengths from one Z-membrane to the other Z-membrane of a sarcomere, running parallel to the thin filaments. The packing of these two filaments was not good. The isometric tension developed by the irrigated fibers upon addition of mg-ATP was about 10% of the tension developed by untreated fibers. The weak tension developed by irrigated fibers is probably due to the irregular packing of the thick and thin filaments in the fibers. The ghost fibers also contracted, though only slightly, upon addition of Mg-ATP after irrigation with heavy meromyosin.