Table 2.
Ca+2, Mg+2, actin-stimulated Mg+2 ATPase kinetics, tryptophan fluorescence and actin-sliding velocities of wild-type (PwMhc2) and R759E indirect flight muscle myosins
| Myosin | Basal Ca-ATPase ± SD (s−1) | Basal Mg-ATPase± SD(s−1) | Actin-stimulated Vmax ± SD(s−1) | Km (actin) ± SD (μM) | Peak tryptophan fluorescence (% compared to apo state ± SD) | Actin sliding velocity ± SD (μm s−1) |
|---|---|---|---|---|---|---|
| Wild-type (PwMhc2) | 10.34 ± 0.73 (n = 6) | 0.26 ± 0.02 (n = 6) | 1.86 ± 0.33 (n = 6) | 0.26 ± 0.10 (n = 6) | 115.0 ± 0.4 (ATP) 108.2 ± 0.3 (AMP-PNP) (n=3) | 6.7 ± 1.1 (n = 6) |
| R759E | 3.96 ± 0.99 (n = 6)* | 0.10 ± 0.04 (n = 6)* | 0.69 ± 0.06 (n = 6)* | 0.27 ± 0.08 (n = 6) | 99.9 ± 1.1 (ATP) 97.8 ± 1.6 (AMP-PNP) (n=3) | 4.3 ± 0.6 (n = 6)* |
Statistically different from PwMhc2 myosin for ATPase or actin sliding velocity data (p < 0.001, Student’s t test).
Myosin was isolated from dissected dorsolongitudinal indirect flight muscle31 and actin was prepared from chicken skeletal muscle.40 ATPase measurements were performed using [γ-32P] as described.31,32 Tryptophan fluorescence was measured as described in the legend to Figure 2. The percentage of peak fluorescence (330 nm) compared to apo state (in the absence of nucleotide) is reported. In vitro motility was performed as previously detailed,31 with some modifications.35