Table 1.
Characterization of wild-type PP1 and PP1 mutants expressed in Sf9 cells
| Km | Vmax | S-D32 IC50 | I-2 IC50 | |
|---|---|---|---|---|
| Wild-type PP1 | 3.7 | 18.5 | 0.7 | 1.5 |
| D220V | 1.6 | 7.3 | 0.8 | ND |
| E256R | 5.7 | 6.6 | 0.8 | ND |
| E275R | 3.5 | 3.9 | 3.0 | ND |
| E252A:D253A | 2.0 | 3.9 | 1.8 | ND |
| E252A:D253A:E256R | 19.5 | 2.1 | ND | ND |
| Y272F | 4.4 | 0.8 | 58 | 4.9 |
| Y272A | ND | ND | 179 | 54 |
| PP1[GEFD > YRCG] | 5.6 | 0.2 | 2.3 | 155 |
| PP1[MC > KY] | 1.6 | 6.4 | 58 | 78 |
| PP1[QILK > LQFD] | 6.1 | 1.9 | 0.7 | 23 |
| PP1/PP2A Ch | 1.8 | 1.0 | 400 | 1,583 |
| PP1/PP2A Ch[YRCG > GEFD] | 6.2 | 4.3 | 857 | 345 |
| PP1/PP2A Ch[KY > MC] | 1.4 | 1.8 | 5.9 | 1,000 |
| PP1/PP2A Ch[LOFD > QILK] | 1.1 | 0.7 | 675 | 2,050 |
[32P]phosphorylase a was used as substrate. Apparent Km (μM) and Vmax (μmol/mg/min) are given. The IC50 values for thiophospho-DARPP-32 (S-D32) and inhibitor-2 (I-2) are in nM. ND, not determined. Both thiophospho-DARPP-32 and inhibitor-2 inhibit native rabbit muscle PP1 with IC50 values of ≈1 nM (5). Results are the average of two to six separate experiments.