TABLE 2.
Affinity of wild-type and mutant TIMP-3 for prototypic target metalloproteinases
Wild-type TIMP-3 and its mutants K26A/K45A and K42A/K110A (0.25–25 nm) were incubated with target metalloproteinases (1 h, 37 °C), and residual activity against synthetic fluorescent substrates was determined. Equilibrium velocities were used to calculate the apparent inhibition constant Ki(app) (mean ± S.D., n = 3–5 technical replicates) using the tight binding equation. ND, too low to be determined.
|
Ki(app) |
|||
|---|---|---|---|
| Wild-type TIMP-3 | TIMP-3 K26A/K45A | TIMP-3 K42A/K110A | |
| nm | |||
| MMP-1ΔC | 1.20 ± 0.49 | 0.52 ± 0.33 | 0.60 ± 0.30 |
| MMP-2ΔC | 0.60 ± 0.32 | 0.63 ± 0.49 | 0.60 ± 0.53 |
| MMP-3ΔC | 1.20 ± 0.47 | 0.92 ± 0.19 | 1.40 ± 0.09 |
| ADAMTS-4 | 0.19 ± 0.01 | 0.12 ± 0.04 | 0.24 ± 0.07 |
| ADAMTS-5 | 1.27 ± 0.46 | 0.95 ± 0.55 | 1.12 ± 0.94 |
| ADAMTS-5 + heparin | ND | ND | 1.20 ± 0.42 |
| ADAM17 | 3.54 ± 1.19 | 3.78 ± 1.79 | 2.34 ± 1.40 |