Measurement of nucleotide exchange kinetics with isolated synthetic myosin filaments using flash photolysis

The kinetics of nucleotide release have been measured at the level of isolated synthetic myosin filaments. This was achieved by displacing a rhodamine nucleotide analog from a filament by flash photolysis of caged-ATP with selective observation using total internal reflectance fluorescence microscopy. The procedure gave improved time resolution over previously used flow methods. Kinetics were measured both in the presence of the ADP analog and during steady-state hydrolysis of the ATP analog, These studies open the way for kinetic measurements during actin filament sliding on myosin tracks.