Spectrophotometric pyrophosphate assay of 2',5'-oligoadenylate synthetase.
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.
Justesen
J
J
Department of Molecular Biology, Aarhus University, Denmark.
Kjeldgaard
N O
NO
D015088:2',5'-Oligoadenylate Synthetase / Q000032:analysis*; D011756:Diphosphates; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005470:Fluorometry / Q000379:methods; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet / Q000379:methods
D011756:Diphosphates; D009243:NAD; D009249:NADP; D015088:2',5'-Oligoadenylate Synthetase