Table 2.

Apparent Ca2+-binding affinities of wild-type and mutant C2A domains measured as Ca2+/phospholipid complexes

C2A domainEC50HillcoeffAbsolute bindingn
WT14  ± 0.2 μm4.0  ± 0.233863  ± 227 dpm3
D232N5  ± 1.0 μm1.5  ± 0.15461  ± 147 dpm3
D238N17  ± 1.3 μm1.6  ± 0.23411  ± 115 dpm3
  • The apparent Ca2+ affinities of the indicated purified C2A domains were measured as described in Figure 2using pulldowns of radiolabeled phospholipid vesicles composed of 30% phosphatidylserine/70% phosphatidylcholine with immobilized GST-fusion proteins containing the C2A domains. Data shown are mean ± SEMs from the number of experiments indicated (n) performed in triplicate as described in Materials and Methods. Each reaction contained ∼25 μg of protein and 4.5 μg of phospholipids. Binding constants and Hill coefficients were calculated using GraphPad software and are not accurate for the D232N and D238N mutants because of the decrease in absolute binding. “Absolute binding” gives the amount of phospholipid vesicles, expressed as counts of radiolabeled phospholipids, that are pulled down at saturating Ca2+ concentrations.