Kinetics of the Initial Steps of Rabbit Psoas Myofibrillar ATPases Studied by Tryptophan and Pyrene Fluorescence Stopped-Flow and Rapid Flow-Quench. Evidence That Cross-Bridge Detachment Is Slower than ATP Binding

The kinetics of the tryptophan fluorescence enhancement that occurs when myofibrils (rabbit psoas) are mixed with Mg-ATP were studied by stopped-flow in different solvents (water, 40% ethylene glycol, 20% methanol) at 4 °C. Under relaxing conditions (low Ca2+) in water (μ = 0.16 M, pH 7.4) and at high ATP concentrations, the transient was biphasic, giving a k fast max of 230 s-1 and a k slow max of 15 s-1. The kinetics of the two phases were compared with those obtained by chemical sampling using [γ-32P]ATP and quenching in acid (Pi burst experiments: these give unambiguously the ATP cleavage kinetics), or cold Mg-ATP (cold ATP chase: ATP binding kinetics). k slow is due to ATP cleavage, as with S1. Interestingly, k fast is slower than the ATP binding kinetics. Instead, this constant appears to report ATP-induced cross-bridge detachment from actin because (1) it was identical to the fluorescence transient obtained on addition of ATP to pyrene-labeled myofibrils; (2) when the initial filament overlap in the myofibrils was decreased, the amplitude of the fast phase decreased; (3) there was no fluorescent enhancement upon the addition of ADP to myofibrils. This is different from the situation with S1 or actoS1 where there was also a fast fluorescent ATP-induced transient but whose kinetics were identical to those of the tight ATP binding. To increase the time resolution and to confirm our results, we also carried out transient kinetics in ethylene glycol and methanol. We interpret our results by a scheme in which a rapid equilibrium between attached (AM*·ATP) and detached (M*·ATP) states is modulated by the fraction of myosin heads in rigor (AM) during the time of experiment.