Kinetics of Association of Myosin Subfragment-1 to Unlabeled and Pyrenyl-labeled Actin

The kinetics of reaction of myosin subfragment-1 (S1) with F-actin have been monitored by the changes in light scattering and in pyrenyl-actin fluorescence at 20°C, pH 7.5, and physiological ionic strength. The association rate constant of S1 to F-actin decreases about 10-fold as the molar ratio of bound S1 increases from 0 to 1. This decrease in k+ is most likely due to the steric hindrance of available binding sites by initially bound S1. The apparent rate constant for association of S1 to bare filaments is 9 μM–1 s–1, a value 1 order of magnitude higher than the one previously estimated from experiments in which S1 was in excess over F-actin. The anticooperative binding kinetics of S1 to F-actin are consistent with the negative cooperativity displayed in the equilibrium binding curves of S1 to pyrenyl-F-actin.Fluorescence titration curves of partially labeled pyrenyl-F-actin by S1 are sigmoidal, consistent with a 4-fold higher affinity of S1 for unlabeled than for labeled actin. This conclusion is strengthened by kinetic data of S1 binding to partially labeled F-actin, which exhibit a biphasic behavior due to the slower dissociation of S1 from unlabeled than from labeled actin. The kinetics of reaction of myosin subfragment-1 (S1) with F-actin have been monitored by the changes in light scattering and in pyrenyl-actin fluorescence at 20°C, pH 7.5, and physiological ionic strength. The association rate constant of S1 to F-actin decreases about 10-fold as the molar ratio of bound S1 increases from 0 to 1. This decrease in k+ is most likely due to the steric hindrance of available binding sites by initially bound S1. The apparent rate constant for association of S1 to bare filaments is 9 μM–1 s–1, a value 1 order of magnitude higher than the one previously estimated from experiments in which S1 was in excess over F-actin. The anticooperative binding kinetics of S1 to F-actin are consistent with the negative cooperativity displayed in the equilibrium binding curves of S1 to pyrenyl-F-actin. Fluorescence titration curves of partially labeled pyrenyl-F-actin by S1 are sigmoidal, consistent with a 4-fold higher affinity of S1 for unlabeled than for labeled actin. This conclusion is strengthened by kinetic data of S1 binding to partially labeled F-actin, which exhibit a biphasic behavior due to the slower dissociation of S1 from unlabeled than from labeled actin.