A highly-sensitive high throughput assay for dynamin's basal GTPase activity
Fig 2
Detection of basal GTP hydrolysis by Dyn1.
A) Dyn1 was titrated from 0.3 nM to 5000 nM in the presence of 10 μM, 100 μM or 500 μM GTP and GTP hydrolysis was measured as ΔmP after 60 min incubation at room temperature (n = 1, measured in triplicates). B) The GTP hydrolysis by Dyn1 was linear during 60-minute incubations at room temperature (n = 4, each measured in duplicates). C) The GTP binding mutant, Dyn1S45N, shows no activity at any concentration of GTP (n = 1, measured in triplicates). D) The basal GTPase activity is proportional to Dyn1 concentration from 0 to 1000 nM, indicating no cooperativity under these concentrations (n = 3, each measured in triplicates). Data are presented as mean ± SD.