Fig. 2.

Determination of the fraction of mant nucleotides bound specifically to ATP binding sites in the fiber. A fiber was incubated in 250-μM mantATP and the fluorescence measured. ATP was then added to the concentrations shown and the fluorescence obtained was normalized to the initial fluorescence in the absence of added ATP. The data of this figure were fit to a simple competition model (solid line), which defined the fraction of mant nucleotides bound nonspecifically to the fiber as 0.41 ± 0.02, and the ratio of the apparent affinities of ATP to mantATP, as 0.57 ± 0.07. This ratio is consistent with previous observations showing tighter binding of mant nucleotides relative to their unlabeled counterparts (11). The temperature was 24°C; the myosin in the fibers was 7% phosphorylated.