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Journal of Colloid and Interface Science Oct 2017Digitonin is commonly used to permeabilize cell membranes and solubilize membrane components. It interacts specifically with cholesterol in the membrane which leads to...
Digitonin is commonly used to permeabilize cell membranes and solubilize membrane components. It interacts specifically with cholesterol in the membrane which leads to the formation of pores. Thus far, the mechanism by which digitonin interacts with the membrane has only been described qualitatively. We investigated this interaction in model membranes that contain little or no cholesterol with a combination of isothermal titration calorimetry, dynamic light scattering, and zeta potential measurements. Digitonin partitions fully asymmetrically into large unilamellar vesicles of phosphocholine (PC) lipid at 20°C (remaining in the outer leaflet only), with a partition coefficient of 0.22±0.04mM and ΔH of partitioning of 23.3±1.6kJmol. Beyond a digitonin/lipid ratio of ∼0.1 in the outer leaflet, digitonin micelles coexist with vesicles without solubilizing them-even at high digitonin concentrations. This "staying out" of digitonin was also observed with phosphoserine (PS), PC/PS, and PC/PS/cholesterol vesicles. The mechanism by which digitonin perturbs and solubilizes the membrane is very different when the membrane contains little or no cholesterol as opposed to 20-30mol% cholesterol. The role of digitonin should thus be carefully considered in the design of preparative protocols and experiments in studies of cellular processes and membrane proteins.
Topics: Cholesterol; Digitonin; Lipid Bilayers; Liposomes; Micelles; Phosphatidylcholines; Phosphatidylserines; Thermodynamics
PubMed: 28551523
DOI: 10.1016/j.jcis.2017.05.034 -
Digitonin concentration is determinant for mitochondrial supercomplexes analysis by BlueNative page.Biochimica Et Biophysica Acta.... Jan 2021The BlueNative page (BNGE) gel has been the reference technique for studying the electron transport chain organization since it was established 20 years ago. Although...
The BlueNative page (BNGE) gel has been the reference technique for studying the electron transport chain organization since it was established 20 years ago. Although the migration of supercomplexes has been demonstrated being real, there are still several concerns about its ability to reveal genuine interactions between respiratory complexes. Moreover, the use of different solubilization conditions generates conflicting interpretations. Here, we thoroughly compare the impact of different digitonin concentrations on the liquid dispersions' physical properties and correlate with the respiratory complexes' migration pattern and supercomplexes. Our results demonstrate that digitonin concentration generates liquid dispersions with specific size and variability critical to distinguish between a real association of complexes from being trapped in the same micelle.
Topics: Animals; Digitonin; Electron Transport Complex I; Mice; Mitochondria, Heart; Mitochondria, Liver; Mitochondrial Proteins; Native Polyacrylamide Gel Electrophoresis
PubMed: 33129827
DOI: 10.1016/j.bbabio.2020.148332 -
Biochimica Et Biophysica Acta Feb 1982We describe a radioimmunoassay for digitonin which utilizes the ability of digotonin to compete with 125I-labeled digoxigenin for binding to anti-digoxin antiserum. As...
We describe a radioimmunoassay for digitonin which utilizes the ability of digotonin to compete with 125I-labeled digoxigenin for binding to anti-digoxin antiserum. As performed using the Gammaflo automated radioimmunoassay system and commercially available reagents, the assay can detect as little as 20 micrograms/ml (15 microM) digitonin. The assay is insensitive to interference by cholesterol or other cell membrane constituents and is useable above and below the critical micelle concentration of digitonin. It should be useful for the monitoring of digitonin concentrations in solubilized biochemical preparations.
Topics: Animals; Autoanalysis; Digitonin; Erythrocyte Membrane; Micelles; Radioimmunoassay; Rats; Solubility
PubMed: 7059589
DOI: 10.1016/0005-2736(82)90032-3 -
Langmuir : the ACS Journal of Surfaces... Sep 2016In this article, we describe the effect of a highly hemolytic saponin, digitonin, on model lipids cholesterol and dipalmitoylphosphatidylcholine (DPPC) using a...
In this article, we describe the effect of a highly hemolytic saponin, digitonin, on model lipids cholesterol and dipalmitoylphosphatidylcholine (DPPC) using a combination of tensiometric (surface pressure and dilatational surface elasticity), spectroscopic (infrared reflection absorption spectroscopy, IRRAS), microscopic (fluorescence microscopy), and scattering techniques (neutron reflectivity, NR, and grazing incidence X-ray diffraction, GIXD). The monolayers of individual lipids and their 10:9 (mol/mol) mixture were exposed to an aqueous solution of digitonin (10(-4) M) by subphase exchange using a setup developed recently in our laboratory. The results confirm that digitonin can adsorb onto both bare and lipid-covered water-air interfaces. In the case of DPPC, a relatively weak interaction can be observed, but the presence of cholesterol drastically enhances the effect of digitonin. The latter is shown to dissociate the weak cholesterol-DPPC complexes and to bind cholesterol in an additional layer attached to the original lipid monolayer.
Topics: 1,2-Dipalmitoylphosphatidylcholine; Adsorption; Air; Cholesterol; Digitonin; Surface Properties; Thermodynamics; Unilamellar Liposomes; Water
PubMed: 27518122
DOI: 10.1021/acs.langmuir.6b01737 -
Langmuir : the ACS Journal of Surfaces... Apr 2017Digitonin, a steroidal saponin obtained from the foxglove plant (Digitalis purpurea), displays a wide spectrum of biological properties and is often used as a model in...
Digitonin, a steroidal saponin obtained from the foxglove plant (Digitalis purpurea), displays a wide spectrum of biological properties and is often used as a model in mechanistic investigations of the biological activity of saponins. In the present study, Langmuir monolayers of zwitterionic (DPPC, DMPE, POPC, POPE, DSPC, DSPE, and DPPE) and ionic (DPPS and DPPG) phospholipids were employed in order to better understand the effect of digitonin on the lipid organization. For this purpose, a combination of surface pressure relaxation, infrared reflection absorption spectroscopy (IRRAS), and fluorescence microscopy measurements was used. The observed increase in surface pressure (Π) suggests that digitonin can adsorb at the air/water interface, both bare and covered with the uncompressed phospholipid monolayers. However, the detailed analysis of IRRAS and fluorescence microscopy data shows that digitonin interacts with the lipid monolayers in a very selective way, and both the headgroup and the lipid tails affect this interaction. Nevertheless, it should be noted that in no case did digitonin cause any disruptive effects on the monolayers. The DPPE and DPPS monolayers get disordered by penetration with digitonin, despite an increase in surface pressure, leading to an unprecedented LC-LE transition. Interestingly, saponin could be easily squeezed out of these monolayers by mechanical compression.
Topics: 1,2-Dipalmitoylphosphatidylcholine; Digitonin; Phospholipids; Spectrophotometry, Infrared; Surface Properties
PubMed: 28333465
DOI: 10.1021/acs.langmuir.6b04613 -
Analytical Biochemistry Dec 2005
Review
Topics: Animals; Cell Membrane Permeability; Cell Separation; Digitonin; Hepatocytes; Lipid Metabolism; Methods; Rats
PubMed: 16291302
DOI: 10.1016/j.ab.2005.03.032 -
Methods in Molecular Biology (Clifton,... 2016The high concentration of cholesterol in the plasma membrane relative to the endomembranes of eukaryotic cells allows the selective permeabilization of the plasma...
The high concentration of cholesterol in the plasma membrane relative to the endomembranes of eukaryotic cells allows the selective permeabilization of the plasma membrane with the glycoside digitonin leaving the intracellular membrane bound organelles intact. In this chapter, we describe the basic method to use digitonin permeabilized cells to reconstitute the transport of proteins containing nuclear localization signals into the nucleus. The assay requires only a target cell line that can be permeabilized with digitonin, a source of soluble transport factors, typically provided by the cytosol fraction of cultured cells, and a cargo protein of interest. No other specialized equipment is required other than a fluorescence microscope. The assay can be used to identify transport factors required to transport specific proteins, to study the regulation of protein transport, or to study nuclear protein transport under different conditions.
Topics: Active Transport, Cell Nucleus; Animals; Cell Nucleus; Digitonin; Nuclear Proteins; Permeability; Protein Transport; Rabbits; Reticulocytes
PubMed: 27147060
DOI: 10.1007/978-1-4939-3530-7_29 -
Methods in Molecular Biology (Clifton,... 2015Nucleocytoplasmic transport is crucial not only for basic cellular activities but also for the physiological adaptation of cells to various environmental stimuli that...
Nucleocytoplasmic transport is crucial not only for basic cellular activities but also for the physiological adaptation of cells to various environmental stimuli that affect development, cell-fate determination, or disease development. The basic transport mechanisms have been revealed during the past two decades through the identification and biochemical characterizations of factors mediating the transport, dissecting the transport process and examining the function of nuclear pore complexes (NPCs). In this chapter, we describe methods for a nuclear transport reconstitution assay using digitonin-permeabilized mammalian cells. The transport assay can be generally conducted in the lab without special equipment. The assay system is efficient and significantly contributes to the study of nucleocytoplasmic transport.
Topics: Active Transport, Cell Nucleus; Cell Membrane Permeability; Cell Nucleus; Cytoplasm; Digitonin; Fluorescent Antibody Technique; HeLa Cells; Humans; In Vitro Techniques; Nuclear Pore; Time-Lapse Imaging
PubMed: 25555589
DOI: 10.1007/978-1-4939-2253-6_18 -
Journal of Biochemistry Oct 1984The interaction of digitonin with membrane cholesterol was studied by using various digitonin analogs, and radioactive desglucodigitonin. The following results were...
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)
Topics: Animals; Calorimetry; Cell Membrane; Chemical Phenomena; Chemistry; Cholesterol; Digitonin; Electron Spin Resonance Spectroscopy; Erythrocyte Membrane; Granulocytes; Guinea Pigs; Hemolysis; Humans; Kinetics; Liposomes; Membrane Lipids; Phosphatidylcholines; Tritium
PubMed: 6097588
DOI: 10.1093/oxfordjournals.jbchem.a134941 -
Scientific Reports Jul 2021In higher plants, the photosynthetic process is performed and regulated by Photosystem II (PSII). Arabidopsis thaliana was the first higher plant with a fully sequenced...
In higher plants, the photosynthetic process is performed and regulated by Photosystem II (PSII). Arabidopsis thaliana was the first higher plant with a fully sequenced genome, conferring it the status of a model organism; nonetheless, a high-resolution structure of its Photosystem II is missing. We present the first Cryo-EM high-resolution structure of Arabidopsis PSII supercomplex with average resolution of 2.79 Å, an important model for future PSII studies. The digitonin extracted PSII complexes demonstrate the importance of: the LHG2630-lipid-headgroup in the trimerization of the light-harvesting complex II; the stabilization of the PsbJ subunit and the CP43-loop E by DGD520-lipid; the choice of detergent for the integrity of membrane protein complexes. Furthermore, our data shows at the anticipated MnCaO-site a single metal ion density as a reminiscent early stage of Photosystem II photoactivation.
Topics: Arabidopsis; Cryoelectron Microscopy; Digitonin; Photosystem II Protein Complex
PubMed: 34330992
DOI: 10.1038/s41598-021-94914-x