| Abstract: | The initial steps by which ATP dissociates and binds to actomyosin subfragment 1 (acto-SF-1) were studied. Two techniques were used: stopped-flow (for acto-SF-1 dissociation kinetics) and rapid-flow quench with ATP chase quenching (for ATP binding kinetics). The experiments were carried out in 40% ethylene glycol-5 mM KCI, pH 8, at 15 degrees C. Under these conditions, the binding of SF-1 to actin remains very tight. As with SF-1, the ATP chase technique could be used, first, to titrate active sites and, second, to study the kinetics of ATP binding to acto-SF-1. The kinetic constants obtained were compared with those of SF-1 alone and with the acto-SF-1 dissociation kinetics under identical conditions. The kinetics of the acto-SF-1 dissociation did not vary with the actin to SF-1 ratio, but the ATP binding kinetics did, and a maximum value was reached at a mole ratio of 2.5. At high ATP (100 microM), kdiss = 300 s-1, which compares with 49 s-1 and 13 s-1 for the ATP binding kinetics for acto-SF-1 (actin to SF-1 = 1:1) and SF-1, respectively. As with SF-1, the ATP binding to acto-SF-1 follows a hyperbolic law with the ATP concentration. This suggests a rapid equilibrium (K) followed by an essentially irreversible step (k).(ABSTRACT TRUNCATED AT 250 WORDS) |
