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