Development of a Novel Ectonucleotidase Assay Suitable for High-Throughput Screening

被引：15

作者：

Sachsenmeier, Kris F. ^{[1
]}

Hay, Carl ^{[1
]}

Brand, Erin ^{[1
]}

Clarke, Lori ^{[1
]}

Rosenthal, Kim ^{[1
]}

Guillard, Sandrine ^{[2
]}

Rust, Steven ^{[2
]}

Minter, Ralph ^{[2
]}

Hollingsworth, Robert ^{[1
]}

机构：

[1] Medimmune LLC, Gaithersburg, MD 20878 USA

[2] MedImmune, Cambridge, England

来源：

JOURNAL OF BIOMOLECULAR SCREENING | 2012年 / 17卷 / 07期

关键词：

enzyme assay; ectonucleotidase; luciferase; antibody; antagonist;

D O I：

10.1177/1087057112443987

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

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.

引用

页码：993 / 998

页数：6

共 7 条

[1] METABOLISM OF ADENINE-NUCLEOTIDES BY ECTO-ENZYMES OF VASCULAR ENDOTHELIAL AND SMOOTH-MUSCLE CELLS IN CULTURE [J].