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Am J Physiol Cell Physiol 276: C267-C278, 1999;
0363-6143/99 $5.00
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Vol. 276, Issue 1, C267-C278, January 1999

SPECIAL COMMUNICATION
Detection of local ATP release from activated platelets using cell surface-attached firefly luciferase

Reza Beigi1, Eiry Kobatake2, Masuo Aizawa2, and George R. Dubyak1

1 Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4970; and 2 Department of Bioengineering, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8501, Japan

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.

purinergic signal transduction; cell surface microenvironment


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