Table 5.
Effect of G- and F-actin on the steady-state kinetic parameters of the reaction of human MICAL-MO. The steady-state kinetic parameters of the reaction catalyzed by human MICAL-MO in the absence and presence of G- and F-actin were measured at 25 °C in 20 mM Hepes/KOH buffer, pH 7.0 (Hepes), G-buffer (5 mM Tris/HCl, pH 8.0, 0.2 mM CaCl2, 0.2 mM ATP, 1 mM DTT) or and F-buffer (9 mM Tris/HCl, pH 8.0, 0.18 mM CaCl2, 1.1 mM ATP, 1.3 mM DTT, 45 mM KCl, 1.8 mM MgCl2). The substrates concentrations or concentration ranges are indicated. The KM is that calculated for the varied substrate. Data are from [70].
| NADPH, μM | MICAL, nM | Buffer | Actin, μM | KM, μM | kcat, s−1 | kcat/Km, s−1 μM−1 |
|---|---|---|---|---|---|---|
| 10–300 | 30 | Hepes | - | 19 ± 2 | 3.1 ± 0.1 | 0.163 ± 0.018 |
| 10–300 | 30 | G-buffer | - | 102 ± 8 | 3.4 ± 0.1 | 0.034 ± 0.003 |
| 10–300 | 75 | G-buffer | G, 3.3 | 96 ± 28 | 4.5 ± 0.6 | 0.047 ± 0.015 |
| 10–300 | 120 | F-buffer | - | 555 ± 118 | 2.6 ± 0.4 | 0.005 ± 0.001 |
| 10–300 | 20 | F-buffer | F, 2.4 | 11 ± 3 | 12.3 ± 0.5 | 1.12 ± 0.31 |
| 300 | 20 | F-buffer | F, 7–85 | 4.7 ± 1 | 19.5 ± 1 | 4.2 ± 0.9 |