digitonin and Hemolysis

OSW-1 is a structurally unique steroidal saponin isolated from the bulbs of Ornithogalum saundersiae, and has exhibited highly potent and selective cytotoxicity in tumor cell lines. This study aimed to investigate the molecular mechanism for the membrane-permeabilizing activity of OSW-1 in comparison with those of other saponins by using various spectroscopic approaches. The membrane effects and hemolytic activity of OSW-1 were markedly enhanced in the presence of membrane cholesterol. Binding affinity measurements using fluorescent cholestatrienol and solid-state NMR spectroscopy of a 3-d-cholesterol probe suggested that OSW-1 interacts with membrane cholesterol without forming large aggregates while 3-O-glycosyl saponin, digitonin, forms cholesterol-containing aggregates. The results suggest that OSW-1/cholesterol interaction is likely to cause membrane permeabilization and pore formation without destroying the whole membrane integrity, which could partly be responsible for its highly potent cell toxicity.

Topics: Antineoplastic Agents, Phytogenic; beta-Cyclodextrins; Biological Transport; Cholestenones; Cholesterol; Digitonin; Dimyristoylphosphatidylcholine; Erythrocyte Membrane; Fluoresceins; Glycyrrhizic Acid; Hemolysis; Humans; Membrane Lipids; Oleanolic Acid; Ornithogalum; Phosphatidylcholines; Saponins; Unilamellar Liposomes

In the present investigation we studied the molecular mechanisms of the monodesmosidic saponin digitonin on natural and artificial membranes. We measured the hemolytic activity of digitonin on red blood cells (RBCs). Also different lipid membrane models (large unilamellar vesicles, LUVs, and giant unilamellar vesicles, GUVs) in the presence and absence of cholesterol were employed. The stability and permeability of the different vesicle systems were studied by using calcein release assay, GUVs membrane permeability assay using confocal microscopy (CM) and fluorescence correlation spectroscopy (FCS) and vesicle size measurement by dynamic light scattering (DLS). The results support the essential role of cholesterol in explaining how digitonin can disintegrate biological and artificial membranes. Digitonin induces membrane permeability or causes membrane rupturing only in the presence of cholesterol in an all-or-none mechanism. This effect depends on the concentrations of both digitonin and cholesterol. At low concentrations, digitonin induces membrane permeability while keeping the membrane intact. When digitonin is combined with other drugs, a synergistic potentiation can be observed because it facilitates their uptake.

Topics: Animals; Cell Membrane; Cell Membrane Permeability; Cholesterol; Digitonin; Erythrocytes; Fluoresceins; Hemolysis; Lipid Bilayers; Saponins; Sheep; Steroids

The cytotoxicity of Bordetella bronchiseptica to infected cells is known to be dependent on a B. bronchiseptica type III secretion system. Although the precise mechanism of the type III secretion system is unknown, BopN, BopD and Bsp22 have been identified as type III secreted proteins. In order to identify other proteins secreted via the type III secretion machinery in Bordetella, a type III mutant was generated, and its secretion profile was compared with that of the wild-type strain. The results showed that the wild-type strain, but not the type III mutant, secreted a 40-kDa protein into the culture supernatant. This protein was identified as BopB by the analysis of its N-terminal amino acid sequence. Severe cytotoxicity such as necrosis was induced in L2 cells by infection with the wild-type B. bronchiseptica. In contrast, this effect was not observed by the BopB mutant infection. The haemolytic activity of the BopB mutant was greatly impaired compared with that of the wild-type strain. The results of a digitonin assay strongly suggested that BopB was translocated into HeLa cells infected with the wild-type strain. Taken together, our results demonstrate that Bordetella secretes BopB via a type III secretion system during infection. BopB may play a role in the formation of pores in the host plasma membrane which serve as a conduit for the translocation of effector proteins into host cells.

Topics: Amino Acid Sequence; Animals; Bacterial Proteins; Bordetella bronchiseptica; Carrier Proteins; Cell Line; Culture Media; Digitonin; Gene Deletion; Genes, Bacterial; Hemolysis; Humans; Molecular Sequence Data; Molecular Weight; Protein Transport; Rats; Virulence Factors, Bordetella

A multitarget functional bioassay was optimized as a method for detecting substances interacting with the inflammatory process of activated neutrophil granulocytes, mainly to release elastase detected by p-nitroanilide (pNA) formation. Using this bioassay, 100 fractionated extracts of 96 plants were screened, with results presented in a manner that links recorded biological activity to phylogenetic information. The plants were selected to represent a major part of the angiosperms, with emphasis on medicinal plants, Swedish anti-inflammatory plants, and plants known to contain peptides. Of the tested extracts, 41% inhibited pNA formation more than 60%, and 3% stimulated formation. The extract of Digitalis purpurea enhanced pNA formation, and digitoxin, the active compound, was isolated and identified. Plant extracts that exhibited potent nonselective inhibition (>80% inhibition) were evaluated further for direct inhibition of isolated elastase and trypsin enzyme. The inhibitory effect of most tested extracts on the isolated enzyme elastase was similar to that of PAF- and fMLP-induced pNA formation. Compared to trypsin, inhibition of elastase by extracts of Rubus idaeus and Tabernaemontana dichotoma was significantly higher (80% and 99%, respectively). Inhibition of trypsin by the extract of Reseda luteola was high (97%). Orders such as Lamiales and Brassicales were shown to include a comparably high proportion of plants with inhibitory extracts.

Topics: alpha-Amylases; Anti-Inflammatory Agents; Benzoylarginine Nitroanilide; Biological Assay; Biological Products; Cytochalasin B; Digitalis; Digitonin; Digitoxin; Dose-Response Relationship, Immunologic; Hemolysis; Humans; Lamiaceae; Leukocyte Elastase; Magnoliopsida; N-Formylmethionine Leucyl-Phenylalanine; Neutrophil Activation; Neutrophils; Peptides; Plant Proteins; Plants, Medicinal; Platelet Activating Factor; Superoxides; Sweden; Tabernaemontana; Trypsin Inhibitors; Violaceae

The oligosaccharide chain of the monodesmosidic haemolytic saponin digitonin (1) undergoes an efficient and regioselective acylation in organic solvent by use of Novozym 435 (lipase B from Candida antarctica supported on acrylic resin) in the presence of an activated ester. With vinyl acetate, acetylation occurs at C-6 OH of glucose(II) and C-4 OH of xylose to afford the previously unreported diacetyl derivative 2 and the monoacetyl derivatives 3 and 4. With vinyl laurate only the monolauryl derivative 5 is formed. The structures of these acylated digitonins have been established using modern 2D NMR techniques, which allowed complete assignments of all proton resonances. The hemolytic activity of derivatives 2-5 is significantly reduced compared to that of digitonin.

Topics: Animals; Candida; Carbohydrate Sequence; Cattle; Digitonin; Esters; Hemolysis; In Vitro Techniques; Lipase; Magnetic Resonance Spectroscopy; Molecular Sequence Data

Non-genomic actions of progesterone have been described in the ovary, and luteal membranes of several species have been shown to possess specific binding sites for [3H]-progesterone. However, binding of radiolabelled progesterone to luteal membranes was demonstrable only in the presence of digitonin. Digitonin is a non-ionic detergent which is thought to act by forming one-to-one complexes with certain sterols. It is also a cardiotonic agent, inhibiting (Na+-K+) ATPase activity by interaction with the extracellular (ouabain/K+) binding site. We therefore investigated which properties of digitonin were responsible for its stimulatory actions on progesterone binding to bovine luteal membranes. A range of compounds with detergent, cardiotonic and or cholesterol-complexing activities were tested for their effects on [3H]-progesterone binding to bovine luteal membrane fractions, and on haemolysis of rat erythrocytes. Stimulation of progesterone binding to luteal membranes was highly specific for digitonin, and a number of ionic and non-ionic detergents, cardenolides, saponins and cholesterol-complexing reagents tested failed either to stimulate [3H]-progesterone binding to bovine luteal membranes in the absence of digitonin, or to inhibit binding specifically in the presence of digitonin. When digitonin was first reacted with excess cholesterol or pregnenolone to form the respective digitonides, stimulatory activity was greatly reduced, suggesting that the ability of digitonin to interact with (an) endogenous steroid(s) may be important in its action. High performance liquid chromatography (HPLC)-mass spectrometry of commercially available digitonin preparations indicated the presence of numerous minor impurities in most commercial digitonin preparations. Three major UV-absorbing peaks were isolated and characterised by mass spectrometry: all stimulated progesterone binding to bovine luteal membrane receptors in a dose-dependent manner, though to differing extents. Our data suggest that the unique action of digitonin on luteal membrane progesterone receptors is not related to its detergent or cardiotonic properties, but appears to be related to its ability to complex with membrane sterols.

Topics: Animals; Cattle; Cell Membrane; Cholesterol; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Digitonin; Female; Hemolysis; Luteal Cells; Mass Spectrometry; Pregnenolone; Progesterone; Rats; Rats, Wistar; Receptors, Progesterone; Tritium

The effects of the glycoalkaloids alpha-solanine, alpha-chaconine and alpha-tomatine on different cell types were studied in order to investigate the membrane action of these compounds. Hemolysis of erythrocytes was compared to 6-carboxyfluorescein leakage from both ghosts and erythrocyte lipid vesicles, whereas leakage of enzymes from mitochondria and the apical and baso-lateral side of Caco-2 cells was determined. Furthermore, the effects of glycoalkaloids on the gap-junctional communication between Caco-2 cells was studied. From these experiments, it was found that glycoalkaloids specifically induced membrane disruptive effects of cholesterol containing membranes as was previously reported in model membrane studies. In addition, alpha-chaconine was found to selectively decrease gap-junctional intercellular communication. Furthermore, the glycoalkaloids were more potent in permeabilizing the outer membrane of mitochondria compared to digitonin at the low concentrations used.

Topics: Adenylate Kinase; Animals; Caco-2 Cells; Cell Membrane; Cell Membrane Permeability; Cholesterol; Digitonin; Erythrocyte Membrane; Erythrocytes; Fluoresceins; Fluorescent Dyes; Gap Junctions; Hemolysis; Humans; L-Lactate Dehydrogenase; Male; Mitochondria, Liver; Rats; Rats, Wistar; Solanaceous Alkaloids

A simple and highly sensitive assay for measuring total digitonin in biological samples is described. The assay is based on the ability of digitonin to hemolyze red blood cells. The precision and reproducibility of the assay was excellent with intra- and inter-assay variabilities of less than 1% and 6%, respectively. The assay was used to evaluate several potential methods for removing digitonin from biological samples (digitonin extracts from guinea pig brain membranes). Dialysis and G-25 Sephadex chromatography were ineffective. However, protein and digitonin can be effectively separated by ammonium sulfate precipitation followed by dialysis. The kappa 1 opioid receptor survived these procedures with no change in affinity for [3H]U-69,593. In conclusion, the hemolytic assay for digitonin appears to provide a practical means for determining detergent concentrations during receptor purification and characterization and for evaluating potential methods for detergent removal. Although an in depth analysis of the assay was carried out only for digitonin, CHAPS and deoxycholate also caused 50% hemolysis at concentrations well below those commonly used for receptor solubilization and, therefore, the general assay procedures might have applicability for measurement of these and perhaps other detergents used in receptor solubilization as well.

Topics: Animals; Brain Chemistry; Chromatography, Ion Exchange; Dialysis; Digitonin; Guinea Pigs; Hemolysis; In Vitro Techniques; Membranes; Rats; Receptors, Cell Surface; Solubility

Reaction of propylene oxide with digitonin in strong aqueous alkali yielded water soluble, nontoxic solubilizers of drugs and hormones, the molecular composition of which could be fully deduced from plasma desorption mass spectrometry data. Preparations with average degrees of substitution ranging from 0.7 to 4.5 were investigated, and all the distributions of degrees of substitution observed were relatively narrow and symmetrical; thus, the reactivity of primary and secondary hydroxyls of saponin and of its conversion products had to be quite similar. The effects of the average degree of substitution of 2-hydroxypropyldigitonins on the formation of micelles and on the solubilization of four drugs into aqueous solutions were found to be minor. On the other hand, the ability to hemolyze erythrocytes decreased sharply with an increasing average degree of substitution.

Topics: Animals; Digitonin; Hemolysis; Hydrolysis; In Vitro Techniques; Mass Spectrometry; Rabbits

An effective concentration of alpha-toxin from Staphylococcus aureus Wood 46, directly from the culture supernatant, could be achieved by adsorption on digitonin-sepharose and elution with 3 mol/l sodium thiocyanate (NaSCN). The toxin was further purified by gelchromatography. The purified product yielded 1 single protein band upon SDS-polyacrylamide electrophoresis. It was nonhemolytic, but reacted with anti-alpha-toxin under complement fixation. Dialysis against 0.14 mol/l NaCl with hydrophobic amino acids partially reactivated the alpha-hemolytic activity of the toxin. Ultracentrifugal analysis yielded sedimentation coefficients for the purified toxin of approximately 3,7 S when dissolved in 3 mol/l NaSCN and of about 12 S after dialysis against 0.14 mol/l NaCl (Table 1). The spontaneous oligomerization of the alpha-toxin during dialysis against 0.14 mol/l NaCl possibly resulted from a change in configuration induced by its adsorption to digitonin-sepharose.

Topics: Bacterial Toxins; Chromatography, Affinity; Chromatography, Gel; Complement Fixation Tests; Digitonin; Electrophoresis, Polyacrylamide Gel; Hemolysin Proteins; Hemolysis; Sepharose; Ultracentrifugation

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

A rapid centrifugation procedure following cell lysis with either digitonin or short hypotonic shock in EDTA containing solutions was evaluated, applicable for investigations of enzyme and metabolite compartmentations in intact rabbit reticulocytes. The application of the digitonin disruption seems to be restricted to cell suspensions containing up to 40% reticulocytes only, whereas the hypotonic lysis can be used with practically pure reticulocytes, too. The distribution of markers revealed that an almost complete cell disruption, sufficient separation into pellet and supernatant fraction and satisfactory preservation of mitochondrial intactness could be achieved under appropriate conditions. The suitability of the proposed method in studies on reticulocyte energy metabolism is further supported by the almost insignificant ATP splitting during the entire procedure.

Topics: Adenosine Triphosphate; Anemia; Animals; Cell Fractionation; Digitonin; Edetic Acid; Glutamate Dehydrogenase; Hemolysis; Hypotonic Solutions; Rabbits; Reticulocytes

The kinetics of hemolysis of pig erythrocytes by digitonin was continuously monitored by a potassium selective electrode. The following minimal mechanism of hemolysis was postulated, based upon kinetic measurements: 2 D + E (1)in equilibrium E x D2 (2)leads to (E x D2) (3)leads to (E x D2) where D denotes a digitonin molecule and E a specific digitonin binding-site on the membrane. The first step (1) represents a rapid reversible combining of digitonin with specific binding-sites on the membrane. The second step (2) is prelytic, related to the time required for bound digitonin molecules to alter the membrane structure so much that hemolysis may take place; this step has a transition temperature at 26 degrees C, probably related to the "melting" of specific membrane structures at that temperature. The third step (3) is hemolytic, and comprises the changes within the digitonin-altered membrane during hemolysis; it is stongly influenced by temperature. Lowering of temperature slows down the rate of hemolysis and increases the quantity of digitonin required to obtain a fixed extent of hemolysis. It appears that two molecules of digitonin combine with a single binding-site on the outer face of the membrane in a digitonin-membrane complex.

Topics: Animals; Binding Sites; Digitonin; Erythrocytes; Hemolysis; Mathematics; Membranes; Swine; Temperature; Time Factors

Aqueous phase and butanol phase extracts of group A1, O, M, N, P1 and P2 human erythrocytes perpared by extraction of red cell stromata with n-butanol were examined for the presence of blood group substances in inhibition of agglutination experiments using antisera and test cells of human origin. Group A substance was recovered in both aqueous phase and butanol phase extracts, M and N substance in aqueous phase extracts only, and P1 substance only in butanol phase extracts. Use of high concentrations of extract resulted in detection of P1 activity in aqueous phase extracts and of N specific inhibition in the aqueous phase extracts of group M erythrocytes. Distilled water appeared to be superior to digitonin and hypotonic phosphate buffer solutions for preparation of stromata for the recovery of group A and M substances in the butanol extracts but not for group N and P1 substances.

Topics: ABO Blood-Group System; Blood Group Antigens; Buffers; Butanols; Digitonin; Erythrocyte Membrane; Erythrocytes; Hemagglutination Inhibition Tests; Hemolysis; Humans; MNSs Blood-Group System; P Blood-Group System; Water

Topics: Animals; Cattle; Digitonin; Erythrocytes; Glucuronates; Glycyrrhiza; Hemolysis; Lethal Dose 50; Plants, Medicinal; Rats; Saponins

The unesterified cholesterol content of plasma samples can be evaluated from the extent of inhibition of lucensomycin-induced hemolysis. The test measures, however, only the fraction of cholesterol which is available for interaction with lucensomycin, this availability being adversely affected by high phospholipid-cholesterol ratios.

Topics: Animals; Antifungal Agents; Cattle; Cholesterol; Cholesterol Esters; Digitonin; Erythrocytes; Hemolysis; Humans; Liposomes; Lucensomycin; Phosphatidylcholines; Phospholipids

Topics: Animals; Cattle; Digitonin; Erythrocytes; Escin; Glucosidases; Glycyrrhiza; Hemolysis; In Vitro Techniques; Lactones; Plants, Medicinal; Quaternary Ammonium Compounds; Saponins; Tomatine

Topics: Autoanalysis; Centrifugation; Digitonin; Erythrocytes; Glucosephosphate Dehydrogenase; Glucosephosphate Dehydrogenase Deficiency; Hemoglobins; Hemolysis; Humans; Mass Screening; Methemoglobin; NADP; Phosphogluconate Dehydrogenase

Topics: Animals; Cell Membrane; Chromatography, Thin Layer; Digitonin; Erythrocytes; Glycosides; Hemolysis; Kinetics; Rats; Saponins; Time Factors

Topics: Amino Acids; Animals; Blood Proteins; Cattle; Cell Membrane; Cholesterol; Chromatography, Gas; Chromatography, Gel; Chromatography, Thin Layer; Digitonin; Erythrocytes; Fatty Acids; Hemolysis; Lipids; Medicago sativa; Phospholipids; Saponins; Serum Albumin, Bovine; Sheep

Inhibition of streptolysin O activity by cholesterol depends on the latter being in free form. The normal esterified and protein-bound cholesterol fractions in serum do not influence streptolysin O activity. However, high cholesterol levels in rabbits fed cholesterol with cholic acid were associated with an increased antistreptolysin O effect. It is suggested that this occurs when all available protein sites are saturated and where a true ;free' cholesterol fraction is present. Splitting the esterified cholesterol fraction of human sera with raised cholesterol levels, by means of pancreatin, produced an increased antistreptolysin O effect, again presumably because of saturation of protein-binding sites. Similarly, removal of non-esterified cholesterol from sera of cholesterolfed rabbits, by means of digitonin, reduced antistreptolysin O activity of the sera. Evidence is presented that combination of bovine serum albumin and streptolysin O has a steric hindrance effect on attachment of cholesterol to streptolysin O. The method described for the estimation of free cholesterol is extremely sensitive, being capable of detecting concentrations of less than 1.0 mug/ml.

Topics: Animals; Antistreptolysin; Bile Acids and Salts; Cattle; Cholesterol; Digitonin; Esters; Hemolysis; Humans; Methods; Pancreatin; Protein Binding; Rabbits; Serum Albumin, Bovine; Streptolysins

Topics: Cell Death; Chemical Phenomena; Chemistry; Digitalis Glycosides; Digitonin; Erythrocytes; Glycosides; Hemolysis; Holothurin; Mucoproteins; Quillaja; Research; Saponins

Topics: Cell Death; Digitalis; Digitalis Glycosides; Digitonin; Hemolysis; Plant Extracts

Topics: Animals; Bees; Cell Death; Cell Differentiation; Digitalis; Digitalis Glycosides; Digitonin; Hemolysis; Lecithins; Phosphatidylcholines; Plant Extracts; Snake Venoms; Venoms

The relations between lysin concentration, percentage hemolysis at the moment at which the lysin concentration is reduced by dilution, and the amount of hemolysis which follows the dilution as a result of the reaction being "progressive" point to there being an "internal" phase at the red cell surfaces, in which the lysin is less affected by the dilution than in the system as a whole. A second possibility, i.e. that the combination of lysin molecules with certain components of the cell surface has an ultimate effect on neighboring components which depend on the former for their stability cannot, however, be ruled out. In systems containing digitonin or sodium taurocholate, this internal phase, once formed, seems to be almost unaffected by the dilution of the system; i.e., these lysins are very firmly held at the cell surfaces. In systems containing saponin the lysin is less firmly attached, so that dilution of the system affects its concentration appreciably.

Topics: Cell Death; Digitonin; Hemolysis; Kinetics; Mucoproteins; Saponins; Taurocholic Acid

Topics: Cell Death; Digitalis; Digitalis Glycosides; Digitonin; Hemolysis; Plant Extracts

Topics: Cholesterol; Digitalis; Digitalis Glycosides; Digitonin; Glycosides; Hemolysis; Humans; Plant Extracts