Effects of EDTA treatment upon the protein subunit composition and mechanical properties of mammalian single skeletal muscle fibers

Abstract

Considerable interest has been focused on the role of myosin light chain LC2 in the contraction of vertebrate striated muscle. A study was undertaken to further our investigations of the effects of LC2 removal upon contraction in skinned fibers from rabbit psoas muscles. Isometric tension and maximum velocity of shortening, V(max), were measured in fiber segments prior to LC2 removal. The segments were then bathed at 30°C for up to 240 min in a buffer solution containing 20 mM EDTA in order to extract up to 60% of the LC2. Troponin C (TnC) was also partially removed by this procedure. Mechanical measurements were done following the EDTA extraction and the readditions of first TnC and then LC2 to the segments. The protein subunit compositions of the same fiber segments were determined following each of these procedures by SDS PAGE of small pieces of the fiber. V(max) was found to decrease as the LC2 content of the fiber segments was reduced by increasing the duration of extraction. EDTA treatment also resulted in substantial reductions in tension due mainly to the loss of TnC, though smaller reductions due to the extraction of LC2 were also observed. Reversal of the order of recombination of LC2 and TnC indicated that the reduction in V(max) following EDTA treatment was a specific effect of LC2 removal. These results strongly suggest that LC2 may have roles in determining the kinetics and extent of interaction between myosin and actin.