Table 2.
Binding analysis of various 4.1R80 and pyruvate kinase constructs and of importin β to human importin α2 Rch 1
| Protein | ka (1/M·s) | kd (1/s) | K(D)kin (nM) |
|---|---|---|---|
| 4.1R80 | |||
| Wild type | 7.7 ± 0.5 × 104 | 2.3 ± 0.1 × 10−3 | 30 |
| ΔE16 | 1.2 ± 0.1 × 104 | 1.0 ± 0.3 × 10−2 | 833 |
| mutKKK | 1.4 ± 0.2 × 104 | 8.3 ± 0.5 × 10−3 | 592 |
| ΔE5 | 2.2 ± 0.2 × 104 | 7.2 ± 0.7 × 10−3 | 327 |
| mutEED | 2.2 ± 0.3 × 104 | 4.9 ± 0.8 × 10−3 | 221 |
| r30kDa | |||
| Full length | 2.3 ± 0.2 × 104 | 5.5 ± 0.5 × 10−3 | 239 |
| ΔE5 | No binding | No binding | No binding |
| GST + E5 | 8.4 ± 0.2 × 104 | 8.6 ± 0.5 × 10−3 | 102 |
| Controls | |||
| Importin β | 1.2 ± 0.1 × 105 | 8.3 ± 0.2 × 10−3 | 69 |
| PK + SV40 NLS | 9.7 ± 0.8 × 104 | 5.4 ± 0.1 × 10−3 | 56 |
| PK | No binding | No binding | No binding |
| GST | No binding | No binding | No binding |
Recombinant histidine-tagged or GST fusion proteins were expressed and purified as described in MATERIALS AND METHODS. The binding assay was carried out in PBS containing 0.05% Tween 20. From the binding curves obtained by resonant mirror detection method, ka, kd, and K(D)kin were determined using the software package FAST-Fit.