title: A new, sensitive ecto-5'-nucleotidase assay for compound screening.
abstract: Ecto-5'-nucleotidase (eN) is a membrane-bound enzyme that hydrolyzes extracellular nucleoside-5'-monophosphates yielding the respective nucleoside and phosphate. Increased levels of eN expression have been observed in many cancer cells. By increasing extracellular adenosine concentrations, they contribute to their proliferative, angiogenic, metastatic, and immunosuppressive effects. Therefore, eN is of considerable interest as a novel drug target for the treatment of cancer as well as of inflammatory diseases. In this study, we developed, optimized, and applied a highly sensitive radiometric assay using [³H]adenosine-5'-monophosphate (AMP) as a substrate. The reaction product [³H]adenosine was separated from [³H]AMP by precipitation of the latter with lanthanum chloride and subsequent filtration through glass fiber filters. Conditions were optimized to reproducibly collect the [³H]adenosine-containing filtrate used for quantitative determination. Validation of the assay yielded a mean Z' factor of 0.73, which demonstrates its suitability for high-throughput screening. The new assay shows a limit of detection that is at least 30-fold lower than those of common colorimetric methods (e.g., optimized malachite green assay and capillary electrophoresis-based assay procedures), and it is also superior to a recently developed luciferase-based assay.
authors.affiliation: PharmaCenter Bonn, Pharmaceutical Sciences Bonn (PSB), Pharmaceutical Institute, Pharmaceutical ChemistryI, University of Bonn, D-53121 Bonn, Germany.
authors.forename: Marianne
authors.initials: M
authors.lastname: Freundlieb
authors.affiliation: Institute of Cell Biology and Neuroscience, Molecular and Cellular Neurobiology, Goethe University, 60438 Frankfurt am Main, Germany.
authors.forename: Herbert
authors.initials: H
authors.lastname: Zimmermann
authors.affiliation: PharmaCenter Bonn, Pharmaceutical Sciences Bonn (PSB), Pharmaceutical Institute, Pharmaceutical ChemistryI, University of Bonn, D-53121 Bonn, Germany. Electronic address: christa.mueller@uni-bonn.de.
authors.forename: Christa E
authors.initials: CE
authors.lastname: Müller
mesh_terms: D015720:5'-Nucleotidase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000378:metabolism; D002138:Calibration; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D057166:High-Throughput Screening Assays; D007700:Kinetics; D015203:Reproducibility of Results; D054852:Small Molecule Libraries / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D054852:Small Molecule Libraries; D000249:Adenosine Monophosphate; D015720:5'-Nucleotidase
keywords: CD73; Cancer therapy; Ecto-5′-nucleotidase assay; Lanthanum chloride precipitation; Michaelis–Menten analysis; Radiometric enzyme assay