Table 1

Transmembrane voltage regulates annexin V binding to phospholipid vesicles.

[K⁺] outside (mM)

[K⁺] inside (mM)

Valino-mycin

Number of assays

EC₅₀ (mM)

Slope

pK_d (experimental)

pK_{d, app} at 1.25 mM Ca²⁺ (theoretical)

100

0

-

10

0.113 ± 0.004

8.7 ± 0.2

41.5 ± 1.0

16.2

100

0

+

11

0.116 ± 0.005

10.1 ± 0.4

46.6 ± 1.5

17.2

Difference:

+5.1*

+1.0

1

100

-

4

0.135 ± 0.003

9.2 ± 0.2

42.5 ± 0.7

15.8

1

100

+

3

0.132 ± 0.004

8.1± 0.1

38.5 ± 0.3

15.0

Difference:

-4.0*

-0.8

Binding of fluorescein-annexin V-117 to phospholipid vesicles containing 25% PS was measured by calcium titration at low membrane occupancy [15] and binding parameters EC₅₀, slope, and pK_d (log of binding affinity) were determined as described under Methods. A positive change in pK_d indicates higher-affinity binding. Assays were performed with the indicated concentrations of KCl outside and inside the vesicles; a transmembrane diffusion potential was created by the addition of 1 μM valinomycin, a potassium-selective ionophore. Uncertainties on experimental parameters are given as SEM; asterisks indicate differences that are statistically significant (p < 0.02) by two-tailed t test. The theoretical apparent pK_d at 1.25 mM calcium chloride was calculated from Equation 5.

Smith et al. BMC Biochemistry 2009 10:5   doi:10.1186/1471-2091-10-5