FIGURE 3.

ATP-induced dissociation of embryonic S1 from pyrene-labeled actin. A, traces of 50 nm WT emb-S1 and the three mutants preincubated with equimolar pyrene-labeled actin and then rapidly mixed with 10 μm ATP. Each trace has been normalized and offset by the previous one by 0.2% fluorescence. At 10 μm ATP, WT MyHC-emb, R672C, and T178I were best described by a single exponential fit resulting in a k_obs = 70 s⁻¹ (amplitude = 27%), 12 s⁻¹ (amplitude = 46%), and 24 s⁻¹ (amplitude = 45%) for WT MyHC-emb, R672C, and T178I, respectively. R672H was best described by a double exponential resulting in a fast and slow phase, giving k_obs = 36 s⁻¹ (amplitude = 32%) and k_obs = 4.4 s⁻¹ (amplitude = 3.8%), respectively. At all [ATP], WT and R672C were best described by a single exponential, as was T178I at [ATP] <50 μm. At ≥50 μm ATP, the T178I mutation was best described by a double exponential, whereas the R672H had a double exponential transient at all [ATP]. B, the dependence of k_obs on [ATP] yielded K′₁k′₊₂ = 8.5, 5.2, 1.8, and 3.1 μm⁻¹ s⁻¹ for WT MyHC-emb (black filled squares), R672H (red filled circles), R672C (green filled triangles), and T178I (blue filled diamonds). The maximum rate yielded a k′₊₂ = 806 s⁻¹ for WT MyHC-emb; 439 and 36 s⁻¹ for the R672H fast and slow (red open circles) phases, respectively; 262 s⁻¹ for R672C; and 1007 and 49 s⁻¹ for the T178I fast and slow (blue open diamonds) phases, respectively. Values given here are for a single assay.