Effects of MgATP, MgADP, and Pi on actin movement by smooth muscle myosin.
To test the idea that the in vitro motility assay is a simplified model system for muscle contraction, the MgATP-dependent movement of actin filaments by thiophosphorylated smooth muscle myosin was characterized in the presence of the products MgADP and inorganic phosphate. The dependence of actin filament velocity on MgATP concentration was hyperbolic with a maximum velocity of 0.6 micron/s and an apparent Km = 40 microM (30 degrees C). MgADP competitively inhibited actin movement by MgATP with a Ki = 0.25 mM. Inorganic phosphate did not affect actin filament velocity in the presence of 1 mM MgATP, but competitively inhibited movement in the presence of 50 microM MgATP with a Ki = 9.5 mM. The effects of ADP and Pi on velocity agree with fiber mechanical studies, confirming that the motility assay is an excellent system to investigate the molecular mechanisms of force generation and shortening in smooth muscle. The rate at which rigor cross-bridges can be recruited to move actin filaments was observed by initiating cross-bridge cycling from rigor by flash photolysis of caged MgATP. Following the flash, which results in a rapid increase in MgATP concentration, actin filaments experienced a MgATP-dependent delay prior to achieving steady state velocity. The delay at low MgATP concentrations was interpreted as evidence that motion generating cross-bridges are slowed by a load due to a transiently high percentage of rigor cross-bridges immediately following MgATP release.
Warshaw
D M
DM
Department of Physiology and Biophysics, University of Vermont, Burlington 05405.
Desrosiers
J M
JM
Work
S S
SS
Trybus
K M
KM
D000199:Actins / Q000502:physiology*; D000244:Adenosine Diphosphate / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000502:physiology*; D000818:Animals; D009130:Muscle, Smooth / Q000502:physiology*; D009218:Myosins / Q000502:physiology*; D010710:Phosphates / Q000502:physiology*; D010782:Photolysis; D013379:Substrate Specificity; D014422:Turkeys
D000199:Actins; D010710:Phosphates; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D009218:Myosins