Cy3-ATP binding to myosin. (a) The amplitudes from stopped-flow transients measuring binding of Cy3-ATP to myosin indicate a 2:1 ratio of Cy3-ATP:myosin S1 in the absence of nucleotide regeneration. Fitted values: E_T 500 nM, S_T 278 (±38) nM, V_max = 1.96 (±0.08). (b) Using pyruvate kinase–based regeneration (inset), the binding of Cy3-ATP is seen to follow a binding isotherm consistent with a single ATP per myosin S1. Fitted values: E_T 500 nM, S_T 544 (±85) nM, V_max = 1.46 (±0.06). Each point was an average of at least three repeats.