Determination of 2—5A synthetase and 2—5A phosphodiesterase in neuroblastoma cells by analytical capillary isotachophoresis: Effects of cytokines and comparison with radioenzymatic methods

Abstract

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.

Proposal for the use of an inhalation drug containing 2-5 oligoadenylates for treatment of COVID-19

“…The trimeric and tetrameric 2-5 oligoadenylates are biologically highly active (with respect to activation of RNaseL) [25] . The different 2-5 oligoadenylates can be characterized by isotachophoresis [20] , [21] , [22] , [23] , or by standard procedures like HPLC or capillary electrophoresis.…”

“…MNBC are isolated by Ficoll-separation and incubated with IFN. OAS activity is measured according to the protocols described [21] , [23] .…”

Recent application and developments of capillary isotachophoresis

“…Mercapto acids and neutral thiols were analyzed in an anionic system (L: 0.01 M HC1 + P-alanine, pH 3.0, T: glutamic acid) whereas for aminothiols a cationic system was used (L: 0.01 M Na' + acetic acid, pH 4.7, T: tetrabutylammonium). 2-5A synthetase and 2-5A phosphodiesterase were determined in neuroblastoma cells by anionic CITP (L: 0.01 M HCl + P-alanine, pH 3.65, T: caproic acid) in the Tachophor instrument [38]. The ITP essay allowed an exact calculation of the turnover rate of the respective substrates which was impossible by other, e.g., radioenzymatic, essays.…”