Kinetic analysis of site-specific synapsin I dephosphorylation by purified protein phosphatases
| PP1c | PP2Ac | Calcineurin | |||||||
|---|---|---|---|---|---|---|---|---|---|
| Km (μm) | kcat (sec−1) | kcat/Km (1/sec μm) | Km (μm) | kcat (sec−1) | kcat/Km (1/sec μm) | Km (μm) | kcat (sec−1) | kcat/Km (1/sec μm) | |
| P-Site 1 | * | * | * | 11.1 | 12000 | 1081 | 55 | 0.9 | 0.023 |
| P-Site 2,3 | 1.8 | 21 | 11.7 | 6.6 | 8780 | 1330 | 31 | 0.6 | 0.016 |
| P-Site 4,5,6 | * | * | * | * | * | * | 8.3 | 2.9 | 0.3 |
| P-Site 6 | * | * | * | * | * | * | 7.4 | 9.8 | 1.35 |
The activities of purified PP1c, PP2Ac, and calcineurin were measured under initial rate conditions for 5 min using various concentrations of 32P-labeled phospho-synapsin I as described (see Materials and Methods). Phospho-substrates incubated in the absence of phosphatases served as control. Kinetic parameters were calculated from linear regression of Lineweaver–Burk plots, each representing the mean of two independent experiments, each done in duplicate.
↵* Indicates activities too low for accurate kinetic measurements.