R. Beigi et al., Detection of local ATP release from activated platelets using cell surface-attached firefly luciferase, AM J P-CELL, 45(1), 1999, pp. C267-C278
We have developed a method for measuring the local concentration of ATP at
the extracellular surface of live cells. This method relies on the specific
attachment to the cell surface of a chimeric protein that consists of the
IgG-binding domain of Staphylococcus aureus protein A fused in-frame with t
he complete sequence for firefly luciferase (proA-luc). Expression of proA-
luc in Escherichia coli and its one-step affinity purification are straight
forward. Attachment to cells is demonstrated to be specific and antibody de
pendent using several suspended and adherent cell types. Light production b
y cell surface-attached luciferase is continuous, Linearly related to ATP c
oncentration, and sufficient to provide nanomolar sensitivity. The spatial
resolution of this method enables the observation of strictly local changes
in extracellular ATP during its secretion from activated platelets. Furthe
rmore, the activity of cell-attached luciferase is relatively refractory to
the inclusion of nucleotidases in the medium, arguing for its effectivenes
s in cell systems possessing potent ecto-ATPases.