Kinetics of Interaction of Digitonin and Its Analogues with HEK293 Cells Studied by the Bioluminescence Method

Autor: N.N. Ugarova, A. D. Fomina, G. Yu. Lomakina
Rok vydání: 2020
Předmět:
Zdroj: Moscow University Chemistry Bulletin. 75:186-194
ISSN: 1935-0260
0027-1314
Popis: Digitonin (monodesmoid saponin) forms complexes with cholesterol of cell membranes, causing the disintegration of the membrane structure, the formation of pores, and the release of intracellular components in the reaction medium. We use HEK293 cells transiently transfected with the pcDNALuc plasmid expressing firefly luciferase to study the kinetics of the initial stage of the interaction of cell membranes with digitonin. A procedure is developed to continuously monitor this process by the bioluminescence method; it enables us to record the kinetic curves of accumulation of luciferase and ATP in the extracellular space in the presence of digitonin in real time. The concentration of digitonin in the reaction medium determined its effect on the cell membranes. At a concentration of less than 0.02 mM, a long induction period is observed during which the protein does not leak from the cells, and small pores are formed that are permeable to low-molecular-weight compounds (ATP). An increase in the concentration of digitonin to 0.05 mM shortens the induction period, and the concentration of the released luciferase increases rapidly, which indicates the formation of large pores. Digitonin is most toxic to cells at a concentration of 0.08 mM or higher for a period of seconds after the concentration of the proteins released reach the maximum and the induction period disappears in the kinetic curves. Thus, the bioluminescence method offers the possibility to study in situ changes in the permeability of cell membranes under the action of membrane-active effectors in the early stages of cells’ lysis process.
Databáze: OpenAIRE
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