Structural changes induced in Ca2+-regulated myosin filaments by Ca2+ and ATP.
We have used electron microscopy and proteolytic susceptibility to study the structural basis of myosin-linked regulation in synthetic filaments of scallop striated muscle myosin. Using papain as a probe of the structure of the head-rod junction, we find that this region of myosin is approximately five times more susceptible to proteolytic attack under activating (ATP/high Ca2+) or rigor (no ATP) conditions than under relaxing conditions (ATP/low Ca2+). A similar result was obtained with native myosin filaments in a crude homogenate of scallop muscle. Proteolytic susceptibility under conditions in which ADP or adenosine 5'-(beta, gamma-imidotriphosphate) (AMPPNP) replaced ATP was similar to that in the absence of nucleotide. Synthetic myosin filaments negatively stained under relaxing conditions showed a compact structure, in which the myosin cross-bridges were close to the filament backbone and well ordered, with a clear 14.5-nm axial repeat. Under activating or rigor conditions, the cross-bridges became clumped and disordered and frequently projected further from the filament backbone, as has been found with native filaments; when ADP or AMPPNP replaced ATP, the cross-bridges were also disordered. We conclude (a) that Ca2+ and ATP affect the affinity of the myosin cross-bridges for the filament backbone or for each other; (b) that the changes observed in the myosin filaments reflect a property of the myosin molecules alone, and are unlikely to be an artifact of negative staining; and (c) that the ordered structure occurs only in the relaxed state, requiring both the presence of hydrolyzed ATP on the myosin heads and the absence of Ca2+.
Frado
L L
LL
Department of Cell Biology, University of Massachusetts Medical School, Worcester 01655.
Craig
R
R
D008841:Actin Cytoskeleton / Q000032:analysis / Q000187:drug effects* / Q000648:ultrastructure; D000199:Actins / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D003599:Cytoskeleton / Q000187:drug effects*; D008854:Microscopy, Electron; D015394:Molecular Structure; D008974:Mollusca; D009218:Myosins / Q000032:analysis* / Q000378:metabolism; D010206:Papain / Q000494:pharmacology
D000199:Actins; D000255:Adenosine Triphosphate; D010206:Papain; D009218:Myosins; D002118:Calcium