Table 2.

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

C2A domain	EC50	Hillcoeff	Absolute binding	n
WT	14  ± 0.2 μm	4.0  ± 0.2	33863  ± 227 dpm	3
D232N	5  ± 1.0 μm	1.5  ± 0.1	5461  ± 147 dpm	3
D238N	17  ± 1.3 μm	1.6  ± 0.2	3411  ± 115 dpm	3

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.