Comparative Kinetics of the ATPase and Actin Sliding Velocity of Myosin Isoforms

Abstract

Myosin isoform expression varies according to demand and pathology, and the kinetics of the resultant actomyosin motor protein determine maximal sarcomere shortening velocity. Studies of muscle fibers and isolated myosin isoforms have shown that actin sliding velocity correlates with ATP hydrolysis. We studied this relationship for isoforms of actomyosin complexes and examined ATP hydrolysis and the effect of association and dissociation of myosin with actin. Sliding velocity of actin filaments was measured in motility assays with different myosin isoforms. Actin-dependent ATP hydrolysis rate of isolated myosin sub-fragments interacting with filamentous actin, or cross-linked with actin by the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were measured. ATPase activity and velocity of actin moved by myosin isoforms from rat and canine cardiac and skeletal muscle were measured at 25°, 30° and 35°C. Motility velocity plotted against ATPase activity of different myosin isoforms showed a linear correlation with a slope of 330 ±20 (nm/sec)/(ATP/sec) ( R2 = 0.98); the slope coefficient was 19% of the slope of the relationship for intact muscle described by M. Barany (J Gen Physiol, 1967) across a wide range of temperatures. Activation energy of sliding velocity (92 - 96 kJ/mol) and ATPase rate (83 - 121 kJ/mol) of different myosin isoforms were similar. Cross-linking of actomyosin complexes by EDC increased ATP hydrolysis rate 4-fold above ATPase at saturating [Actin]. This suggests that association/dissociation kinetics are rate-limiting and that ATPase is activated in maximally 25% myosin molecules interacting with actin in solution. The calculated displacement of actin filaments (D = 330 nm) per ATP hydrolyzed under the experimental conditions used here suggests that unloaded cross-bridges may displace actin over a multitude of the minimal steps of 2.7 nm that can be made by myosin along the actin filaments.