Maximal activation of skeletal muscle thin filaments requires both rigor myosin S1 and calcium.
The regulation by calcium and rigor-bound myosin-S1 of the rate of acceleration of 2'-deoxy-3'-O-(N-methylanthraniloyl)ADP (mdADP) release from myosin-mdADP-P(i) by skeletal muscle thin filaments (reconstituted from actin-tropomyosin-troponin) was measured using double mixing stopped-flow fluorescence with the nucleotide substrate 2'-deoxy-3'-O-(N-methylanthraniloyl). The predominant mechanism of regulation is the acceleration of product dissociation by a factor of approximately 200 by thin filaments in the fully activated conformation (bound calcium and rigor S1) relative to the inhibited conformation (no bound calcium or rigor S1). In contrast, only 2-3-fold regulation is due to a change in actin affinity such as would be expected by "steric blocking" of the myosin binding site of the thin filament by tropomyosin. The binding of one ligand (either calcium or rigor-S1) produces partial activation of the rate of product dissociation, but the binding of both is required to maximally accelerate product dissociation to a rate similar to that obtained with F-actin in the absence of regulatory proteins. The data support an allosteric regulation model in which the binding of either calcium or rigor S1 alone to the thin filament shifts the equilibrium in favor of the active conformation, but full activation requires binding of both ligands.
Heeley
David H
DH
Department of Biochemistry, Memorial University, St. John's, Newfoundland A1B 3X9 Canada.
Belknap
Betty
B
White
Howard D
HD
D008841:Actin Cytoskeleton / Q000737:chemistry / Q000502:physiology*; D000205:Actomyosin / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D006868:Hydrolysis; D066298:In Vitro Techniques; D009119:Muscle Contraction / Q000502:physiology; D018482:Muscle, Skeletal / Q000502:physiology*; D015879:Myosin Subfragments / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011817:Rabbits
D015879:Myosin Subfragments; D000255:Adenosine Triphosphate; D000205:Actomyosin; D002118:Calcium