title: Development of a novel ectonucleotidase assay suitable for high-throughput screening.
abstract: 5'-Ectonucleotidase (NT5E) catalyzes the conversion of adenosine monophosphate to adenosine and free phosphate. The role of this ectonucleotidase and its production of adenosine are linked with immune function, angiogenesis, and cancer. NT5E activity is typically assayed either by chromatographic quantification of substrates and products using high-performance liquid chromatography (HPLC) or by quantification of free phosphate using malachite green. These methods are not suitable for robust screening assays of NT5E activity. HPLC is not readily suitable for the rapid and efficient assay of multiple samples and malachite green is highly sensitive to the phosphate-containing buffers common in various media and sample buffers. Here the development and validation of a novel high-throughput ectonucleotidase screening assay are described, which makes use of a luciferase-based assay reagent, the Promega CellTiter-Glo kit, to measure the catabolism of AMP by NT5E. This multiwell plate-based assay facilitates the screening of potential ectonucleotidase antagonists and is unaffected by the presence of contaminating phosphate molecules present in screening samples.
authors.affiliation: MedImmune, LLC, Gaithersburg, MD, USA. sachsenmeierk@medimmune.com
authors.forename: Kris F
authors.initials: KF
authors.lastname: Sachsenmeier
authors.forename: Carl
authors.initials: C
authors.lastname: Hay
authors.forename: Erin
authors.initials: E
authors.lastname: Brand
authors.forename: Lori
authors.initials: L
authors.lastname: Clarke
authors.forename: Kim
authors.initials: K
authors.lastname: Rosenthal
authors.forename: Sandrine
authors.initials: S
authors.lastname: Guillard
authors.forename: Steven
authors.initials: S
authors.lastname: Rust
authors.forename: Ralph
authors.initials: R
authors.lastname: Minter
authors.forename: Robert
authors.initials: R
authors.lastname: Hollingsworth
mesh_terms: D015720:5'-Nucleotidase / Q000032:analysis*; D000241:Adenosine / Q000096:biosynthesis*; D000249:Adenosine Monophosphate / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D058851:GPI-Linked Proteins / Q000032:analysis; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D009708:Nucleotidases / Q000032:analysis*
chemical_list: D058851:GPI-Linked Proteins; D000249:Adenosine Monophosphate; D009708:Nucleotidases; C030287:nucleotidase; D015720:5'-Nucleotidase; C548241:NT5E protein, human; D000241:Adenosine