Spectrophotometric pyrophosphate assay of 2′,5′-ollgoadenylate synthetase

✍ Scribed by Just Justesen; Niels Ole Kieldgaard

• Publisher: Elsevier Science
• Year: 1992
• Tongue: English
• Weight: 346 KB
• Volume: 207
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(92)90506-3

✦ Synopsis

A nonradioactive multiwell spectrophotometric assay for the interferon-induced enzyme 2',5'-oligoadenylate synthetase measuring the inorganic pyrophosphate produced during oligoadenylate synthesis has been developed. A coupled enzymatic reaction results in a mole to mole formation of NADPH compared to the inorganic pyrophosphate through the use of the three enzymes UDP-Glc pyrophosphorylase (EC2.7.7.9), phosphoglucomutase (EC5.4.2.2), and glucose-6-phosphate dehydrogenase (EC1.1.1.49). The assay is at least as sensitive for measurements of 2',5'-oligoadenylate synthetase activity as the conventional assays using radioactive nucleotides as substrates. Even higher sensitivity of the assay can be obtained by taking advantage of the strong fluorescence of NADPH.