Structural changes in myosin during contraction and the state of ATP in the intact frog muscle.

The reactivity of myosin to [14C]-labeled N-ethylmaleimide ([14C]NEM) or to tritium was determined in functionally different frog muscles. The incorporation of [14C]NEM into myosin decreased during isotonic or isometric contractions, as compared to resting muscle. The cysteine residues which were protected during contraction were not involved in the ATPase activity or the actin-binding ability of myosin. Peptide mapping revealed that several residues were protected simultaneously. The incorporation of tritium into the peptide N-H groups of myosin was also decreased during muscle activity. These data support the idea that activation and subsequent contraction of muscle are correlated with structural changes in the myosin molecule. The reactivity of myosin to [14C]NEM was increased when the muscle was stretched to 140% rest length and treated with iodoacetate to deplete ATP. Based on in vitro experiments and on literature data, it is suggested that in the resting muscle myosin contains bound MgATP which decreases the rate of incorporation of [14C]NEM into myosin and that upon the irreversible loss of ATP the rate increases. 31P nuclear magnetic resonance signals from a number of phosphates were detected in the intact frog muscle. The data indicated that the minimum concentration of ATP in the muscle is 3 mM, a value which agrees with that of chemical determination. The characteristic chemical shifts, coupling constants, and line widths of ATP in the muscle were considerably altered from that of either free ATP in aqueous solutions or ATP in perchloric acid extracts of muscle.