Determination of 2-5A synthetase and 2-5A phosphodiesterase in neuroblastoma cells by analytical capillary isotachophoresis: effects of cytokines and comparison with radioenzymatic methods.
2-5A Synthetase and 2-5A phosphodiesterase were determined by analytical capillary isotachophoresis in comparison to radioenzymatic methods. By means of isotachophoretic analysis, a frequently used radioenzymatic 2-5A synthetase assay was optimized and the results of both assays were compared. Using the isotachophoretic assay the influence of interferon-related cytokines (tumor necrosis factor-alpha and interleukin-2) on 2-5A synthetase induction in neuroblastoma cells was estimated. In contrast to mononuclear blood cells, the tumor necrosis factor induced 2-5A synthetase in these cells. 2-5A Phosphodiesterase was determined using an isotachophoretic assay and a radioenzymatic method. Degradation of A2'p5'A2'p5'A (trimeric form of 2-5A core) was measured by isotachophoresis whereas degradation of a mixture of phosphorus-32 labeled 2-5A cores was registered by radioenzymatic assay. Activity of 2-5A phosphodiesterase was only insignificantly enhanced by interferon in mononuclear blood and neuroblastoma cells. In contrast to the radioenzymatic assays, an accurate determination of 2-5A synthetase as well as of 2-5A phosphodiesterase is possible using the isotachophoretic method because the reactions are followed by measuring the substrates ATP and A2'p5'A2'p5'A, respectively.
Bruchelt
G
G
Children's Hospital, University of Tübingen, Germany.
Büdenbender
M
M
Schmidt
K H
KH
Birk
A
A
Treuner
J
J
Niethammer
D
D
D015088:2',5'-Oligoadenylate Synthetase / Q000032:analysis*; D000469:Alkaline Phosphatase; D002197:Capillary Action; D002460:Cell Line; D016207:Cytokines / Q000494:pharmacology*; D004586:Electrophoresis / Q000379:methods*; D005095:Exoribonucleases / Q000032:analysis*; D006801:Humans; D007376:Interleukin-2 / Q000494:pharmacology; D007963:Leukocytes, Mononuclear / Q000201:enzymology; D009447:Neuroblastoma / Q000201:enzymology*; D010761:Phosphorus Radioisotopes; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D016207:Cytokines; D007376:Interleukin-2; D010761:Phosphorus Radioisotopes; D014409:Tumor Necrosis Factor-alpha; D015088:2',5'-Oligoadenylate Synthetase; D005095:Exoribonucleases; C029364:2'-phosphodiesterase; D000469:Alkaline Phosphatase