Interaction of Digitonin and Its Analogs with Membrane Cholesterol1

Abstract

The interaction of digitonin with membrane cholesterol was studied by using various digitonin analogs, and radioactive desglucodigitonin. The following results were obtained concerning the effect of digitonin on erythrocytes, granulocytes and liposomes. Digitonin and its analogs showed activity to induce hemolysis, granulocyte activation and liposomal membrane damage. The activity was affected by change of the carbohydrate residue of the molecule; the order of hemolytic activity was digitonin greater than or equal to desglucodigitonin much greater than glucosyl-galactosyl-digitogenin greater than galactosyl-digitogenin, digitogenin. The relative activities of these compounds to induce granulocyte activation and liposomal membrane damage were similar to those observed in the hemolysis. [3H]Desglucodigitonin could bind to cholesterol in liposomes. The binding was stoichiometric and the ratio of desglucodigitonin bound to liposomes/cholesterol in liposomes was close to 1, irrespective of the cholesterol content in liposome. Damage to liposomes was, however, induced by desglucodigitonin only when they contained more than 0.2 molar ratio of cholesterol to phospholipid. Addition of digitonin as well as desglucodigitonin to preformed liposomes deprived of cholesterol affected the anisotropic molecular motion of spin-labeled phosphatidylcholine incorporated into the liposomes, suggesting that the molecules could be inserted into the lipid bilayer free of cholesterol. Molecules of desglucodigitonin in the lipid phase may, however, be equilibrated with those in the aqueous phase, unless they form a complex with cholesterol, since no appreciable amount of [3H]desglucodigitonin could be detected in the liposome fraction after separation by column chromatography. Digitonin decreased the order parameter of spin-labeled phosphatidylcholine when liposomes contained equimolar cholesterol.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of Highly Curved Structures in Relation to Membrane Permeabilization and Budding by the Triterpenoid Saponins, α- and δ-Hederin

“…12B), ␣-hederin would interact with cholesterol, inducing a marked decrease of GP ex values at physiological temperatures in DMPC/Chol (3:1). The formation of equimolecular complexes or aggregates composed of cholesterol and saponins could disorganize the phospholipid/cholesterol matrix and change the thermotropic behavior of membranes (54,55). Our results from DHE spectroscopy support such interaction between cholesterol and ␣-hederin.…”

Facile transfer of [2Fe-2S] clusters from the diabetes drug target mitoNEET to an apo-acceptor protein

“…HEK293 cells were labeled for 15-min at 37°C with 1 μM RPA and/or 0.1 μM CalG-AM in DMEM medium containing 10 mM HEPES buffer and supplemented with 10 μM desferrioxamine (DFO) to prevent quenching of the probe by contaminant iron from the medium. After washing with DMEM-HEPES medium and HEPES-buffered saline (HBS) buffer, the cells were permeabilized in HBS buffer pH 7.4 for 180 s with 25 μM digitonin (49). The permeabilized cells were washed with permeabilization buffer (100 mM KCl, 5 mM phosphate buffer, Eagle's MEM-amino acids mix diluted 1∶500, 10 mM HEPES, 1 μM CaCl 2 , 1 mM MgSO 4 , pH 7.2) and taken to fluorescence microscopy measurements in permeabilization buffer containing 1 mM succinate.…”

Characterization of Arabidopsis NEET Reveals an Ancient Role for NEET Proteins in Iron Metabolism

“…After washing with DMEM-HEPES medium and HBS, the cells were permeabilized in HBS buffer (37°C HEPES-buffered saline, pH 7.4) for 180 s with 25 µM digitonin (Nishikawa et al, 1984). The permeabilized cells were washed with permeabilization buffer (100 mM KCl, 5 mM Na 2 HPO 4 , Eagle's minimal essential medium-amino acids mix diluted 1:500, 10 mM HEPES, 1 µM CaCl 2 , and 1 mM MgSO 4 , pH 7.2) and used for fluorescence microscopy measurements in permeabilization buffer containing 1 mM succinate.…”