Detection of local ATP release from activated platelets using cell surface-attached firefly luciferase.
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 the complete sequence for firefly luciferase (proA-luc). Expression of proA-luc in Escherichia coli and its one-step affinity purification are straightforward. Attachment to cells is demonstrated to be specific and antibody dependent using several suspended and adherent cell types. Light production by cell surface-attached luciferase is continuous, linearly related to ATP concentration, 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. Furthermore, the activity of cell-attached luciferase is relatively refractory to the inclusion of nucleotidases in the medium, arguing for its effectiveness in cell systems possessing potent ecto-ATPases.
Beigi
R
R
Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4970, USA.
Kobatake
E
E
Aizawa
M
M
Dubyak
G R
GR
D000255:Adenosine Triphosphate / Q000097:blood*; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism* / Q000502:physiology; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007158:Immunologic Techniques; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008163:Luminescent Measurements; D051379:Mice; D015539:Platelet Activation / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000235:genetics
D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D000255:Adenosine Triphosphate; D008156:Luciferases