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Methods in Molecular Biology (Clifton,... 2015Mitochondria play a key role in various modes of cell death. Analysis of mitochondrial dysfunction and the release of proteins from the intermembrane space of...
Mitochondria play a key role in various modes of cell death. Analysis of mitochondrial dysfunction and the release of proteins from the intermembrane space of mitochondria represent essential tools in cell death investigation. Here we describe how to evaluate release of intermembrane space proteins during apoptosis, alterations in the mitochondrial membrane potential, and oxygen consumption in apoptotic cells.
Topics: Apoptosis; Cell Death; Cell Line; Cell Membrane Permeability; Cell Respiration; Cytochromes c; Digitonin; Humans; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Membranes; Oxygen Consumption
PubMed: 25631030
DOI: 10.1007/978-1-4939-2257-4_33 -
Journal of Neurochemistry Feb 1988Cultured chromaffin cells were preincubated with digitonin to deplete endogenous ATP from the cell cytoplasm. Catecholamine release from these digitonin-pretreated cells...
Cultured chromaffin cells were preincubated with digitonin to deplete endogenous ATP from the cell cytoplasm. Catecholamine release from these digitonin-pretreated cells was then studied in the presence and absence of exogenous ATP to elucidate a possible involvement of the cytoplasmic ATP in the exocytotic process. The preincubation of the cells with digitonin in the ATP-free permeabilizing medium resulted in a marked decline of the releasing response to a calcium challenge. Furthermore, the declined activity of catecholamine release caused by digitonin pretreatment was restored by the presence of ATP, but not by other adenine nucleotides, and this recovery was observed in a manner dependent on the concentration of ATP. These findings, therefore, seem to indicate that a decrease in the releasing activity of the digitonin-pretreated cells may be due to the removal of endogenous ATP from the cytoplasmic space of the cells, thus suggesting that the cytoplasmic ATP may be involved in the exocytotic mechanism of catecholamine secretion.
Topics: Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Adrenal Glands; Animals; Calcium; Catecholamines; Cattle; Cell Membrane Permeability; Cells, Cultured; Chromaffin System; Digitonin; Exocytosis
PubMed: 3335864
DOI: 10.1111/j.1471-4159.1988.tb02959.x -
The Biochemical Journal Sep 1953
Topics: Digitalis; Digitonin; Humans; Hydroxysteroids; Plant Extracts; Saponins; Steroids
PubMed: 13093688
DOI: 10.1042/bj0550340 -
Archives of Histology and Cytology 1992Results obtained by extracting human fibroblast cells for the study of cytoskeletal structures are compared. Cells grown in culture were treated with digitonin in...
Results obtained by extracting human fibroblast cells for the study of cytoskeletal structures are compared. Cells grown in culture were treated with digitonin in contrast to other methods of detergent preparation using Triton and Saponin. The three dimensional intracellular network which resulted from digitonin treatment was found to be similar in appearance to the structures observed by high voltage transmission electron microscopy of untreated cells described as a microtrabecular lattice by Porter and Tucker (1981). Our results, obtained by high resolution, field emission scanning electron microscopy indicate that the microtrabecular lattice may indeed be one conformation of a dynamic cytoskeleton.
Topics: Cells, Cultured; Cytoskeleton; Digitonin; Fibroblasts; Humans; Microscopy, Electron, Scanning
PubMed: 1290677
DOI: 10.1679/aohc.55.suppl_57 -
Cellular and Molecular Neurobiology Sep 19881. Calcium-dependent exocytosis of catecholamines from intact and digitonin-permeabilized bovine adrenal chromaffin cells was investigated. 2. 45Ca2+ uptake and... (Review)
Review
1. Calcium-dependent exocytosis of catecholamines from intact and digitonin-permeabilized bovine adrenal chromaffin cells was investigated. 2. 45Ca2+ uptake and secretion induced by nicotinic stimulation or depolarization in intact cells were closely correlated. The results provide strong support for Ca2+ entry being the trigger for exocytosis. 3. Experiments in which the H+ electrochemical gradient across the intracellular secretory granule (chromaffin granule) membrane was altered indicated that the gradient does not play an important role in exocytosis. 4. Ca2+ entry into the cells is associated with activation of phospholiphase C and a rapid translocation of protein kinase C to membranes. 5. The plasma membrane of chromaffin cells was rendered permeable to Ca2+, ATP, and proteins by the detergent digitonin without disruption of the intracellular secretory granules. In this system in which the intracellular milieu can be controlled, micromolar Ca2+ directly stimulated catecholamine secretion. 6. Treatment of the cells with phorbol esters and diglyceride, which activate protein kinase C, enhanced phosphorylation and subsequent Ca2+-dependent secretion in digitonin-treated cells. 7. Phorbol ester-induced secretion could be specifically inhibited by trypsin. The experiments indicate that protein kinase C modulates but is not necessary for Ca2+-dependent secretion.
Topics: Adrenal Medulla; Animals; Calcium; Catecholamines; Cattle; Digitonin; Exocytosis; Phorbol Esters; Phosphorylation
PubMed: 3066486
DOI: 10.1007/BF00711168 -
Blut Jul 1980The kinetics of hemolysis of pig erythrocytes by digitonin was continuously monitored by a potassium selective electrode. The following minimal mechanism of hemolysis...
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
PubMed: 7407424
DOI: 10.1007/BF01039875 -
Journal of Natural Products May 1999The oligosaccharide chain of the monodesmosidic haemolytic saponin digitonin (1) undergoes an efficient and regioselective acylation in organic solvent by use of Novozym...
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
PubMed: 10346941
DOI: 10.1021/np9803068 -
Journal of Immunological Methods Feb 2021Lymph nodes (LNs) are essential secondary immune organs where the adaptive immune response is generated against most infections and vaccines. We recently described the...
Lymph nodes (LNs) are essential secondary immune organs where the adaptive immune response is generated against most infections and vaccines. We recently described the use of live ex vivo LN slices to study the dynamics of adaptive immunity. However, when working with reactive lymph nodes from vaccinated animals, the tissues frequently became dislodged from the supportive agarose matrix during slicing, leading to damage that prevented downstream analysis. Because reactive lymph nodes expand into the surrounding adipose tissue, we hypothesized that dislodging was a result of excess lipids on the collagen capsule of the LN, and that a brief wash with a mild detergent would improve LN interaction with the agarose without damaging tissue viability or function. Therefore, we tested the use of digitonin on improving slicing of vaccinated LNs. Prior to embedding, LNs were quickly dipped into a digitonin solution and washed in saline. Lipid droplets were visibly removed by this procedure. A digitonin wash step prior to slicing significantly reduced the loss of LN during slicing from 13 to 75% to 0-25%, without substantial impact on viability. Capture of fluorescent microparticles, uptake and processing of protein antigen, and cytokine secretion in response to a vaccine adjuvant, R848, were all unaffected by the detergent wash. This novel approach will enable ex vivo analysis of the generation of adaptive immune response in LNs in response to vaccinations and other immunotherapies.
Topics: Animals; Antigens; Cytokines; Detergents; Digitonin; Lymph Nodes; Mice; Mice, Inbred C57BL; Vaccination
PubMed: 33333059
DOI: 10.1016/j.jim.2020.112943 -
Journal of Cyclic Nucleotide and... 1983Three independent perturbants of the human platelet membrane were found to differentially interfere with modulation of platelet alpha 2-adrenergic receptor-agonist...
N-ethylmaleimide, elevated temperature, and digitonin solubilization eliminate guanine nucleotide but not sodium effects on human platelet alpha 2-adrenergic receptor-agonist interactions.
Three independent perturbants of the human platelet membrane were found to differentially interfere with modulation of platelet alpha 2-adrenergic receptor-agonist interactions by guanine nucleotides and Na+. Treatment of intact platelets with 1mM N-ethylmaleimide, exposure of particulate preparations to 60 degrees or digitonin solubilization of platelet membranes all eliminated guanine nucleotide regulation of alpha 2-receptor-agonist interactions without interfering with Na+-promoted decreases in receptor affinity for epinephrine. The observations with N-ethylmaleimide and elevated temperature are consistent either with separate membrane components conferring sensitivity to Na+ and guanine nucleotides or with different domains of a single protein component mediating effects of Na+ and GTP on alpha 2-receptor affinity for agonists. However, the observation that Na+ modulation of alpha 2-receptor-agonist interactions is retained in digitonin-solubilized preparations is persuasive evidence that distinct membrane components confer Na+ and GTP sensitivity to alpha 2-adrenergic receptor-agonist interactions, since previous studies (Smith, S.K. and Limbird, L.E. Proc. Natl. Acad. Sci. USA 78:4026-4030, 1981) have demonstrated that the GTP-binding protein modulating alpha 2-receptor affinity for agonists is dissociated from the alpha 2-receptor upon digitonin solubilization unless the receptor is occupied by an agonist at the time of exposure to detergent. Thus, although GTP and Na+ have similar phenomenological effects to permit or enhance alpha-adrenergic attenuation of adenylate cyclase activity and to decrease receptor affinity for agonists, these agents nonetheless appear to mediate their regulatory effects via different membrane components or at least distinct subunits of perhaps shared multisubunit proteins.
Topics: Adrenergic alpha-Agonists; Blood Platelets; Cell Membrane; Digitonin; Ethylmaleimide; Guanosine Triphosphate; Hot Temperature; Humans; In Vitro Techniques; Receptors, Adrenergic, alpha; Sodium
PubMed: 6142062
DOI: No ID Found -
Zeitschrift Fur... 1974The present study demonstrates an altered distribution pattern of anionic sites of the human erythrocyte glycocalyx. The findings prove a significant rearrangement of...
The present study demonstrates an altered distribution pattern of anionic sites of the human erythrocyte glycocalyx. The findings prove a significant rearrangement of membrane constituents induced by a digitonin treatment of aldehyde fixed red blood cells. The effect is not confined to lipids and, presumably, it causes the loss of lipidic and nonlipidic membrane components. It can be deduced that the fixation with a mixture of formaldehyde and glutaraldehyde insufficiently stabilizes the membrane structure.
Topics: Anions; Binding Sites; Cell Membrane; Digitalis Glycosides; Digitonin; Erythrocytes; Humans
PubMed: 4467565
DOI: No ID Found