A sensitive assay for the IFN-regulated 2-5A synthetase enzyme.
2-5A synthetase is the central enzyme of the 2-5A system, an important mediator of interferon action. An assay capable of detecting low, yet biologically important levels of 2-5A synthetase enzyme activity is described. The purification of enzyme reaction products on SepPak C-18 cartridges resulted in a significant reduction in background, when a high specific activity substrate was used to label the 2-5A. Quantitation of labeled 2-5A by chromatography and scintillation counting provided a means of detecting femptomolar amounts of 2-5A. The combination of these procedures accounts for a 3-4 log increase in sensitivity over existing assays. This degree of sensitivity should permit a more accurate determination of the 2-5A synthetase activity in vivo leading to a better understanding of the role of the 2-5A system in virus infection and other cellular processes.
Hassel
B A
BA
Department of Biochemistry, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, MD 21205.
Ts'o
P O
PO
D015088:2',5'-Oligoadenylate Synthetase / Q000302:isolation & purification*; D004789:Enzyme Activation; D005354:Fibrosarcoma / Q000201:enzymology* / Q000473:pathology; D006801:Humans; D007372:Interferons / Q000378:metabolism; D014407:Tumor Cells, Cultured
D007372:Interferons; D015088:2',5'-Oligoadenylate Synthetase