Cell-surface-localized ATP detection with immobilized firefly luciferase.
We demonstrate the application of an assay technique for the detection of ATP efflux from the cell surface. Until recently, the firefly luciferase assay has conventionally been used to detect ATP release because of its high sensitivity and wide detection ability. However, since this technique can be used only to infer the amount of diffused ATP in bulk solutions, it does not accurately reflect the dynamic ATP flux at the cell membrane. We therefore constructed luciferase fusion proteins that could be immobilized onto the cell surface. However, the low activities and limited application ranges of these proteins prompted us to use biotinylated luciferase given its attributes of strong and stable localization. Furthermore, luciferase can be immobilized strongly onto the biotinylated cell surface via streptavidin-biotin binding and can thus be used to determine the dynamic release of ATP near the cell surface.
Nakamura
Makiko
M
Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan.
Mie
Masayasu
M
Funabashi
Hisakage
H
Yamamoto
Kimiko
K
Ando
Joji
J
Kobatake
Eiry
E
D000255:Adenosine Triphosphate / Q000032:analysis* / Q000378:metabolism; D000345:Affinity Labels; D000818:Animals; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D049409:Luciferases, Firefly / Q000235:genetics / Q000378:metabolism*; D008163:Luminescent Measurements / Q000379:methods*; D008954:Models, Biological; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013997:Time Factors
D000345:Affinity Labels; D004800:Enzymes, Immobilized; D011993:Recombinant Fusion Proteins; D001710:Biotin; D000255:Adenosine Triphosphate; D049409:Luciferases, Firefly