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. 2011 Apr 20;100(8):1969–1976. doi: 10.1016/j.bpj.2011.02.061

Figure 3.

Figure 3

Determination of the fraction of mant nucleotides bound specifically to the ATP binding sites in both skeletal muscle fibers (□, dashed line) and cardiac cells (●, solid line). The cells were incubated in 250 μM mantATP with varied concentrations of ATP. The fluorescence obtained was normalized to the fluorescence in the absence of added ATP. The data were fit to a simple competition model. (Solid and dashed lines) Intensity = INS + (1 − INS)/([ATP]•KappATP /[mATP]•KappmATP + 1), where INS is the intensity due to nonspecific binding. This defines the fraction of mant-nucleotides nonspecifically bound to the cell bundles, and the ratio of the apparent affinities of the two nucleotides for the myosin binding site, Kapp. The equation was derived assuming that both nucleotides are present in excess of their Km values, which is true for the conditions used. The nonspecific fraction was 0.41 ± 0.02 for skeletal fibers (data from Stewart et al. (10)) and 0.52 ± 0.03 for cardiac cells. The ratio of the affinities of ATP and mantATP was 0.56 ± 0.07 for skeletal fibers and 0.67 ± 0.18 for cardiac cells.