KINETICS OF ASSOCIATION OF MYOSIN SUBFRAGMENT-1 TO UNLABELED AND PYRENYL-LABELED ACTIN

The kinetics of reaction of myosin subfragment-1 (S-1) with F-actin ha ve been monitored by the changes in light scattering and in pyrenyl-ac tin fluorescence at 20 degrees C, pH 7.5, and physiological ionic stre ngth. The association rate constant of S-1 to F-actin decreases about 10-fold as the molar ratio of bound S-1 increases from 0 to 1. This de crease in k(+) is most likely due to the steric hindrance of available binding sites by initially bound S-1. The apparent rate constant for association of S-1 to bare filaments is 9 mu M(-1) s(-1), a value 1 or der of magnitude higher than the one previously estimated from experim ents in which S-1 was in excess over F-actin. The anticooperative bind ing kinetics of S-1 to F-actin are consistent with the negative cooper ativity displayed in the equilibrium binding curves of S-1 to pyrenyl- F-actin. Fluorescence titration curves of partially labeled pyrenyl-F- actin by S-1 are sigmoidal, consistent with a 4-fold higher affinity o f S-1 for unlabeled than for labeled actin. This conclusion is strengt hened by kinetic data of S-1 binding to partially labeled F-actin, whi ch exhibit a biphasic behavior due to the slower dissociation of S-1 f rom unlabeled than from labeled actin.