کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1176190 1491401 2014 6 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
A new, sensitive ecto-5′-nucleotidase assay for compound screening
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
A new, sensitive ecto-5′-nucleotidase assay for compound screening
چکیده انگلیسی

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.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 446, 1 February 2014, Pages 53–58
نویسندگان
, , ,