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G. Yu. Lomakina, A. D. Fomina, N. N. Ugarova

Kinetics of interaction of digitonin and its analogues with HEK293 cells studied by bioluminescent method


Digitonin monodesmoid saponine forms complexes with cholesterol of cell membranes, which leads to the disintegration of membrane structure, pores formation and release of intracellular components into the extracellular environment. Cells of the HEK293 line, transiently transfected by pcDNALuc plasmide, expressing the firefly luciferase, were used to study the kinetics of the initial stage of cell membrane interaction with digitonin. A method of continuous monitoring of this process by bioluminescent method was developed, which allowed the real-time registration of kinetic curves for accumulations in the extracellular environment of luciferase and ATP in the presence of digitonin. It is shown that the effect of digitonin on the cell membranes is determined by its concentration in the reactionary media. At concentrations of less than 0.02 mM there is a long period of induction, during which there is no release of luciferase from the cells, but small pores are formed, permeable to low-molecular substance (ATP). As the digitonin concentration increases to 0.05 mM, the induction period is reduced, the concentration of extracellular luciferase is rapidly increasing, that indicates on the formation of large pores. The most toxic for cells are the digitonin concentrations of 0.08 mM and more, when within a few tens of seconds the maximum concentration of the luciferase released is reached, and the induction period disappears on the kinetic curves. Thus, the bioluminescent method allows to study in situ the changes in permeability of cell membranes under the influence of membrane-active effectors in the early stages of the process of cell lysis.
Key words: bioluminescence, firefly luciferase, HEK293 cells, digitonin, saponins, cytotoxicity, cellular membrane permeability.
Moscow University Chemistry Bulletin.
2020, Vol. 61, No. 3, P. 232

Copyright (C) Chemistry Dept., Moscow State University, 2002
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