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At least 127 records · Page 7Linked to original sources
PubMed1987

Loss of proteins from digitonin-permeabilized adrenal chromaffin cells essential for exocytosis.

Cultured chromaffin cells can be permeabilized with digitonin; the cell interior is then accessible to the cytoplasm, and addition of calcium provokes release of catecholamines. Increasing the incubation time between the permeabilization step and calcium-induced stimulation resulted in a progressive inhibition of secretion reaching 60% after 20 min. Cytosoluble proteins which leak from detergent-permeabilized cells were collected, dialyzed, and concentrated. When these proteins were added back to permeabilized cells which were unable to secrete, catecholamine release was fully restored, suggesting that certain proteins necessary for exocytosis had been dialyzed from these cells. One of the released proteins was characterized as calmodulin. However, addition of calmodulin alone was ineffective in maintaining or restoring secretory activity in digitonin-permeabilized cells, excluding calmodulin as the sole factor responsible for the loss of release. Protein kinase C was also identified as one of the leaked proteins. This enzyme is known to be retained in cells in the presence of 12-O-tetradecanoylphorbol 13-acetate (TPA). However, under TPA-dependent conditions, there was also a loss of secretory activity. The present paper shows that among the proteins leaked from digitonin-permeabilized cells, there are specific proteins crucial to the exocytotic mechanism.

Adrenal Medulla↗
PubMed1985

Use of digitonin permeabilization for characterization of calcium incorporated into beta-cells in response to glucose.

beta-Cell rich pancreatic islets were used to characterize 45Ca uptake into pools with different sensitivities to permeabilization with digitonin. The plasma and secretory granule membranes were among the membranes most sensitive to digitonin treatment whereas the mitochondria were more resistant. Most of the 45Ca incorporated in response to glucose was found in a fraction released after exposure to a high concentration of digitonin. The results suggest that glucose promotes active sequestration of calcium in mitochondria and that the uptake of 45Ca in the secretory granules is secondary to a raised cytosolic Ca2+ activity.

Animals↗
PubMed1983

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.

Adrenergic alpha-Agonists↗
PubMed1983

Different sensitivity of Zajdela hepatoma mitochondrial ATPase activity to uncouplers in digitonin-treated cells and isolated mitochondria.

Digitonin-treated Zajdela hepatoma cells and rat hepatocytes devoid of almost all cytosol but retaining intact mitochondria were found to represent a suitable system for direct measurement of mitochondrial ATPase activity. The enzyme activity in digitonin-treated Zajdela hepatoma cells in contrast to that of isolated coupled mitochondria was stimulated by uncouplers. No difference in response of mitochondrial ATPase activity to uncouplers in digitonin-treated hepatocytes and isolated liver mitochondria was found. It is concluded that uncoupler-insensitive mitochondrial ATPase activity does not occur in intact in situ tumor mitochondria but is acquired during the isolation of the organelles.

2,4-Dinitrophenol↗
PubMed1995

The acrosome reaction in digitonin-permeabilized sea urchin sperm in the absence of the natural inducer.

In many species, the acrosome reaction of sperm is an obligatory step in fertilization. Increases in [Ca2+]i and pHi, activation of adenylyl cyclase and inositol trisphosphate generation accompany the egg jelly-induced acrosome reaction of sea urchin sperm. The signaling mechanisms involved are unknown. We used digitonin, a cholesterol-complexing compound, to selectively permeabilize the plasma membrane of sea urchin sperm suspended in a medium that mimics the cytosolic ion composition. Within 6 to 8 min, 30 to 50 microM digitonin allowed incorporation of the membrane-impermeant dye Hoechst 33258 into the sperm, staining exclusively the nucleus. No alterations in sperm morphology were caused by digitonin at the concentrations used, however, it irreversibly permeabilized the plasma membrane. Permeabilized sperm retained lactate dehydrogenase and actin. When incubated in Ca(2+)-containing permeabilization buffer (pH 7.8), sperm were capable of undergoing spontaneously the acrosome reaction; this reaction was pH dependent and displayed an absolute Ca2+ requirement. Electron microscopy indicates that the acrosome reaction undergone by permeabilized sperm resembled that induced by egg jelly. Additionally, rhodaminyl-phalloidin staining of sperm reacted under permeabilizing conditions revealed a fluorescent filament in the acrosomal tubule region, demonstrating the occurrence of actin polymerization. Thus, in permeabilized sperm the machinery necessary to perform a [Ca2+]i- and pHi-sensitive acrosome reaction is functionally preserved. Permeabilized sperm offer new avenues to study the molecular bases of the sea urchin sperm acrosome reaction.

Acrosome↗
PubMedDate not supplied

[Digitonin-sensitive calcium pool and its change during synchronous division of Tetrahymena pyriformis cells].

A very significant intracellular pool of calcium, sensitive to digitonin action and different from one of free cytozolic Ca2+, has been shown to exist in the cells of ciliate protozoan T. pyriformis. Calcium content in digitonin-sensitive pool NdigCa2+ has been estimated throughout the cell cycle in synchronized culture of T. pyriformis. The value of NdigCa2+ decreased in the period of time prior to and at the cell division, whereas after the division itself it increased. The possible role of the calcium content changes of digitonin-sensitive pool in regulation of cytozolic Ca2+ level and in control of cell division is discussed.

Animals↗
PubMed1969

The quantitative retention of cholesterol in mouse liver prepared for electron microscopy by fixation in a digitonin-containing aldehyde solution.

A major methodological problem in the intracellular localization of cholesterol is the nearly complete extraction of sterols during routine dehydration and embedding procedures for electron microscopy. Cholesterol digitonide (a sterol complex with digitonin), however, is qualitatively insoluble in these solvents. Mouse liver has been prepared as follows: (a) Flickinger's aldehyde fixative, 20 hr; (b) Flickinger's fixative containing 0.2% digitonin, 24 hr; (c) cacodylate wash, 24 hr; (d) 1% OsO(4), 2 hr; (e) acetone dehydration; and (f) Epon 812 infiltration under vacuum, 28 hr. After the last step, an analysis of the tissue for sterol content under optimal analytical conditions demonstrates a retention of 99% of the unesterified cholesterol present in unfixed mouse liver. Liver prepared in an identical manner except for omission of digitonin is essentially devoid of sterols. Cholesterol isolated chromatographically from liver processed as outlined above has been identified unequivocally by mass spectrometry. Liver from step (f) also has been polymerized, thin-sectioned, and examined in the electron microscope. A remarkable quality of fine-structural preservation is obtained. The major alteration encountered is the presence of small cylindrical "spicules," often occurring as tightly packed concentric lamellae, at membrane surfaces.

Aldehydes↗
PubMed1970

The relation of the 515 nanometers absorbance change to adenosine triphosphate formation in chloroplasts and digitonin subchloroplast particles.

The flash-induced absorbance changes at 515 nanometers has been studied in chloroplasts and in digitonin subchloroplast particles of lettuce. The effect of various conditions and uncouplers was tested on the decay kinetics of this absorbance change and on ATP formation in the presence of phenazine methosulphate, either by continuous or flash illumination. It has been found that in chloroplasts, carbonyl cyanide m-chloromethoxyphenylhydrazone and nigericin in the presence of K(+) accelerate the decay of the 515 change and inhibit ATP formation. However, under a variety of conditions the rate of decay of the 515 absorbance change was found to be unrelated to ATP formation. Preillumination, addition of valinomycin in the presence of K(+), addition of Na(+), or divalent cations accelerate the decay of the 515 absorbance change markedly but have no effect on ATP formation. Addition of phosphorylation reagents has no effect on the decay rate beyond that obtained by Mg(2+) and inorganic phosphate. NH(4)Cl, and to some extent atebrin, while inhibiting ATP formation, do not affect the decay of the 515 absorbance change.In digitonin subchloroplast particles the decay kinetics of the absorbance change resemble that of chloroplasts, but the magnitude of the change is smaller. The pH change in this preparation is reduced much more than the 515 absorbance change.According to the chemiosmotic hypothesis, the sum of DeltaE(membrane potential) and DeltapH is the driving force for ATP formation. The lack of an increase in DeltaE in digitonin subchloroplast particles, which are practically devoid of DeltapH and have a normal ATP-forming activity, is inconsistent with the chemiosmotic hypothesis.

Journal Article↗
PubMed1972

Isolation of mycoplasma membranes by digitonin.

The cell membrane of Mycoplasma hominis was isolated by lysing the cells with digitonin. Electron microscopy and chemical, density gradient, and electrophoretic analyses of the membrane proteins showed the membranes so obtained, like those isolated by osmotic lysis, to be relatively free of cytoplasmic contaminants. Sensitivity to digitonin lysis depended on temperature but was not affected by Mg(2+) ions and was only slightly affected by the age of the culture. Accordingly, it seems that digitonin may be used for the isolation of cell membranes from sterol-requiring mycoplasmas that tend to be fairly resistant to osmotic lysis.

Bacterial Proteins↗
PubMed2000

Simultaneous detection of mitochondrial respiratory chain activity and reactive oxygen in digitonin-permeabilized cells using flow cytometry.

BACKGROUND: Increased mitochondrial generation of reactive oxygen intermediates (ROI) due to defective respiratory chain activity has been implicated in physiological processes such as apoptosis, in the pathogenesis of mitochondrial diseases, and as part of the normal aging process. Established methods addressing activity of the respiratory chain complexes have been limited to bulk assays for single parameters. This study describes a flow cytometry-based method and its validation for the detection of respiratory chain function in single cells permeabilized by digitonin. METHODS: Flow cytometry was used to measure mitochondrial membrane potential (DeltaPsi(m)) and reactive oxygen generation under differing conditions of respiration. This was brought about by the addition of substrates and inhibitors to digitonin-permeabilized cells. This method was validated by measurement of oxygen consumption and ATP production and by confocal microscopy. RESULTS: Activity of the respiratory chain complexes assessed by DeltaPsi(m) responded to substrates and inhibitors as predicted from assessment by oxygen consumption and ATP synthesis. In addition, the flow cytometry method allows the simultaneous assessment of mitochondrial ROI generation. This was confirmed by the localization of the ROI probe, carboxy-DCF, to the same site as the mitochondrial probe observed by confocal microscopy. CONCLUSIONS: This method allows the functional integrity of the respiratory chain complexes to be studied at the single-cell level, thus addressing the relationship between disordered function of respiratory chain complexes and mitochondrial ROI generation.

Adenosine Triphosphate↗
PubMed1986

Separation of the components of commercial digitonin using high-performance liquid chromatography and centrifugal countercurrent chromatography. Identification of the products by californium-252 plasma desorption mass spectrometry.

Commercial digitonin has been separated into its components using high-performance liquid chromatography and centrifugal countercurrent chromatography. The individual glucosides have been identified by californium-252 plasma desorption mass spectrometry, occasionally supplemented by hydrolysis and analysis of the liberated sugars and aglycones. Mass spectra of commercial digitonin that have appeared in the literature are discussed in the light of their complex nature. A new glycoside of molecular weight 1035 is described and a structure proposed.

Californium↗
PubMed1989

Vasopressin rapidly stimulates protein kinase C in digitonin-permeabilized Swiss 3T3 cells: involvement of a pertussis toxin-insensitive guanine nucleotide binding protein.

Guanine nucleotides and pertussis toxin were used to test for the involvement of a guanine nucleotide binding protein in the vasopressin V1 receptor-mediated stimulation of protein kinase C activity in Swiss 3T3 cells. Addition of vasopressin in the presence of [gamma-32P]ATP and digitonin caused a marked and rapid increase (8 +/- 1-fold after 1 min) in the phosphorylation of an Mr = 80,000 cellular protein (80K), a specific marker for protein kinase C activation. This phosphorylation was selectively blocked by the V1 receptor antagonist Pmp1-0-Me-Tyr2 [Arg8] vasopressin, indicating that the effect was mediated through the vasopressin V1 receptor. Down regulation of protein kinase C by prior prolonged pretreatment of intact cells with phorbol 12,13-dibutyrate (PBt2) blocked the ability of vasopressin to stimulate the phosphorylation of 80K in digitonin-permeabilized cells. Addition of a submaximal concentration of vasopressin together with the GTP analogue GTP-gamma-S caused a synergistic stimulation of 80K phosphorylation. The GDP analogue GDP-beta-S caused a 50% inhibition of the phosphorylation of 80K induced by a saturating concentration of vasopressin and shifted the vasopressin dose-response curve to the right. GDP-beta-S had no effect on the dose-response for the stimulation of 80K phosphorylation induced by PBt2. Prior incubation of intact quiescent cultures of Swiss 3T3 cells with pertussis toxin did not impair either vasopressin-induced increase in cytosolic [Ca2+] or activation of protein kinase C. These findings provide functional evidence for the involvement of a pertussis toxin-insesitive G protein in the vasopressin V1 receptor-mediated stimulation of protein kinase C in Swiss 3T3 cells.

Animals↗
PubMed1999

Use of digitonin-permeabilized cells in studies of steroid receptor subnuclear trafficking.

The application of a cell permeabilization technique to the analysis of nuclear import has led to many major breakthroughs in our understanding of this trafficking pathway. Digitonin permeabilization maintains the nucleus in a state competent for faithful, signal-dependent translocation through the nuclear pore complex. This system has also been used to probe the mechanism of hormone-regulated nuclear import through the use of glucocorticoid receptors (GR) as a model substrate. In this report we provide detailed descriptions of the digitonin-permeabilized cell system for use in studies of GR nuclear import. In addition, we present several novel applications that expand the utility of this system to probe for mechanisms of nuclear protein export and subnuclear trafficking.

Cell Membrane Permeability↗
PubMed1988

Orthogonal arrays of particles in non-pigmented cells of rat ciliary epithelium: relation to distribution of filipin- and digitonin-induced alterations of the basolateral membrane.

It has been suggested that orthogonal arrays of particles may increase the rigidity of plasma membrane, as does cholesterol. Therefore, using freeze-fractured non-pigmented ciliary epithelium, the distribution of such arrays was compared to the distribution of membrane deformations induced by the sterol-probes filipin and digitonin in different domains of the basolateral plasma membrane. The distribution of orthogonal arrays of particles was homogeneous between different regions of the basolateral membrane of the non-pigmented ciliary epithelium, while the number of filipin-induced alterations was nearly 4 times higher in the membrane domains not in contact with the basal lamina than in domains in contact with it. Contrary to the homogeneous distribution of arrays, digitonin-induced deformations also differed markedly in these two basolateral membrane domains. Considering that a marked positive response to sterol probes implies a high sterol content, we conclude that orthogonal arrays of particles can occur in plasma membrane regions well-provided with cholesterol and not in direct contact with the basal lamina. Other possible roles of these arrays are discussed.

Animals↗
PubMed1983

Digitonin-precipitable sterols as a measure of cholesterol biosynthesis: contradictory results.

In experiments with 4 different types of cells, we evaluated the cholesterogenic activity by incorporation of 14C-acetic acid into cholesterol and digitonin-precipitable sterols. In every case, the cholesterogenesis appeared considerably faster when expressed as digitonid than when expressed as real cholesterol production, and sometimes the data obtained by the 2 methods were contradictory. Detailed analysis of both digitonid components and nonprecipitable radioactive metabolites showed that a very variable fraction of methyl sterols (including bifunctional methyl sterols) co-precipitates with the C-27 sterols. In cholesterol regulation studies and particularly when the cells exhibit a low cholesterogenesis, the digitonin method is unsuitable and can lead to erroneous interpretations.

Acetates↗
PubMed1984

Isolation of mitochondria from ascites tumor cells permeabilized with digitonin.

A new, improved procedure for isolating mitochondria from ascites tumor cells is described. The unique feature of this technique is the use of digitonin to make the cells susceptible to disruption by Teflon pestle/glass vessel homogenization. The yield and respiratory control ratios of mitochondria isolated by this method from murine Ehrlich ascites tumor cells and rat AS30-D ascites hepatoma cells are significantly better than those obtained for mitochondria isolated by the commonly employed Nagarse method, which involves the use of proteolytic enzymes. Moreover, mitochondria isolated by this new procedure from three different lines of tumors exhibit respiratory control ratios with both adenosine diphosphate and a respiratory uncoupler comparable to those obtained with mitochondria present in situ within digitonin-permeabilized tumor cells.

Animals↗
PubMed1988

In situ assay of intracellular enzymes of yeast (Kluyveromyces fragilis) by digitonin permeabilization of cell membrane.

The yeast, Kluyveromyces fragilis was permeabilized to a number of low-molecular-weight substrates using digitonin. The activities of intracellular yeast enzymes, viz., alcohol dehydrogenase (ADH), beta-galactosidase, glucose-6-phosphate dehydrogenase, aspartase, and hexokinase were found to be much higher in the permeabilized cells than the untreated cells. The optimum conditions for permeabilization with reference to ADH were 0.1% digitonin at 37 degrees C for 15 min. The ADH activity in permeabilized cells was several-fold higher than that in cell free extracts prepared by either physical or chemical methods.

Alcohol Dehydrogenase↗
PubMed1987

Properties of digitonin-solubilized calmodulin-dependent guanylate cyclase from the plasma membranes of Tetrahymena pyriformis NT-1 cells.

Calmodulin-dependent guanylate cyclase from Tetrahymena plasma membranes was solubilized in about a 22% yield by using digitonin in the presence of 0.2 mM CaCl2 and 20% glycerol. The detergent, when present in the assay at concentrations above 0.05%, diminished the basal and calmodulin-stimulated activity of the enzyme. Guanylate cyclase solubilized with digitonin was eluted from DEAE-cellulose with 200 mM KCl in a yield of 50%. Properties of the solubilized enzyme were similar to those of the native membrane-bound enzyme. The Kms for Mg-GTP and Mn-GTP were 140 and 30 microM, respectively. The enzyme required Mn2+ for maximum activity, the relative activity in the presence of Mg2+ being 30% of the activity with Mn2+. The solubilized enzyme retained the ability to be activated by calmodulin, with its extent being reduced as compared to the membrane-bound enzyme. The presence of a Ca2+-dependent calmodulin-binding site on the solubilized enzyme was shown by the Ca2+-dependent retention of the enzyme on a calmodulin-Sepharose-4B column.

Animals↗