In vitro motility of skeletal muscle myosin and its proteolytic fragments.
We have compared actin-activated myosin ATPase activity, myosin binding to actin, and the velocity of myosin-induced actin sliding in order to understand the mechanism of myosin motility. In our in vitro assay, F-actin slides at a constant velocity, regardless of length. The F-actin could slide over myosin heads at KCl concentrations below a critical value (60 mM with myosin and HMM, 100 mM with S-1), and the sliding velocities were quite similar below the critical KCl concentration. However, at KCl concentrations close to the critical value, the sliding F-actin is attached to only one or a few particular points on the surface, each of which perhaps consists of a single head of myosin. The KATPase values for actin-activated ATPase were approximately 300 microM for S-1 and approximately 200 microM with HMM below the critical KCl concentration, and approximately 5,000 microM above the critical KCl concentration. This increase in KATPase is due to a drastic reduction in the binding affinity of myosin heads to F-actin, as determined by a proteolytic digestion method and direct observation by fluorescence microscopy. We also show that the Vmax of actin-activated myosin ATPase activity decreases steadily with increasing KCl concentration, even though the velocity of F-actin sliding remains unchanged. This result provides evidence that the ATPase activity is not necessarily linked to motility. We discuss possible models that do not require a tight coupling between myosin ATPase and motility.
Takiguchi
K
K
Department of Molecular Biology, Faculty of Science, Nagoya University, Aichi.
Hayashi
H
H
Kurimoto
E
E
Higashi-Fujime
S
S
D008841:Actin Cytoskeleton / Q000201:enzymology / Q000502:physiology* / Q000648:ultrastructure; D000199:Actins / Q000378:metabolism / Q000502:physiology*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D003599:Cytoskeleton / Q000502:physiology*; D004789:Enzyme Activation; D066298:In Vitro Techniques; D007700:Kinetics; D009119:Muscle Contraction; D009132:Muscles / Q000201:enzymology / Q000502:physiology / Q000648:ultrastructure; D009218:Myosins / Q000378:metabolism*; D011817:Rabbits
D000199:Actins; D000251:Adenosine Triphosphatases; D009218:Myosins