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PubMed1976

Effects of digitonin on mitochondrial and lysosomal fractions from renal cortex.

Digitonin was studied as a means of purifying mitochondrial fractions which were contaminated by lysosmes and other membranous structures. At concentrations which did not cause loss of mitochondrial enzymes, digitonin released into the supernatant significant amounts of marker enzymes for lysosomes, plasma membranes, and other cytoplasmic organelles. Acid glycosidases followed differing patterns of release that may reflect the heterogenous organization of the lysosomal enzyme-membrane complex. While there are limitations to the digitonin method, studies of certain aspects of mitochondrial function may benefit from the digitonin pretreatment step.

Animals↗
PubMed1978

Superoxide generation by digitonin-stimulated guinea pig granulocytes. A basis for a continuous assay for monitoring superoxide production and for the study of the activation of the generating system.

Stimulation of guinea pig granolocytes by digitonin results in superoxide (O-2) generation. A continuous assay shows that there is a lag between the addition of digitonin and the onset of O-2 production. The rate of activation of the O-2 generating system is dependent upon the concentration of digitonin and the temperature. The final linear rate of O-2 production is affected by the concentration of digitonin, temperature, pH, and the presence of exogenous reduced pyridine nucleotides. Thus, factors which alter either the activation process or the activity of the O-2 generating system can affect O-2 production by stimulated granolocytes.

Animals↗
PubMed1979

Interactions of gonadotropins with corpus luteum surface membranes. V. Differential effects of digitonin on the buoyant densities of light and heavy rat ovarian membrane fractions.

Previous studies have indicated that rat luteal cells at certain stages of development can be fractionated so as to obtain two plasma membrane fractions with different densities and different profiles of marker enzymes. The light membrane fractions (density 1.13) contain the majority of hCG-binding sites and little or no cyclase enzyme, while the heavy membranes (density 1.17) contain the majority of cyclase enzyme and lesser quantities of hormone-binding sites. These membrane fractions were further compared with respect to their susceptibility to perturbation by digitonin. The buoyant density of luteal cell light membrane fractions, as marked by [125I]iodo-hCG binding, Mg2+-dependent ATPase, and 5'-nucleotidase, were highly perturbable by digotonin (delta density, greater than 0.05), while adenylate cyclase activity and phosphodiesterase activity associated with this fraction were only slightly perturbed (delta density, less than 0.02). The buoyant density of luteal cell heavy membrane fractions, as marked by adenylate cyclase, ATPase, and nucleotidase, was not significantly perturbed by digotonin. The hCG binding associated with the heavy membrane fraction was not perturbed by digitonin. From these studies, we conclude that the adenylate cyclase activity associated with light membrane fractions is due to contamination by heavy membranes, while the hCG-binding activity in heavy membrane fractions is intrinsic to that membrane. Except for the lysosomal marker (glucuronidase), which was solubilized by digitonin, the detergent had no significant effect on the density of mitochondrial, Golgi, GERL (Golgi, endoplasmic reticulum, and lysomal), or endoplasmic reticulum membranes. Plasma membranes from isolated granulosa cells and ovaries obtained 24 h after priming with PMS gonadotropin-hCG behaved as heavy membranes (density, 1.17) which contained hCG-binding sites, adenylate cyclase, nucleotidase, and Mg2+-dependent ATPase. These were not significantly perturbed by digitonin. The appearance of light membranes and the segregation of adenylate cyclase from the majority of hCG-binding sites is a development feature of the luteal cell.

Adenylyl Cyclases↗
PubMed1989

Solubilization of high affinity corticotropin-releasing factor receptors from rat brain: characterization of an active digitonin-solubilized receptor complex.

The binding characteristics of CRF receptors in rat frontal cerebral cortex membranes solubilized in 1% digitonin were determined. The binding of [125I]Tyro-ovine CRF ([125I]oCRF) to solubilized membrane proteins was dependent on incubation time, temperature, and protein concentration, was saturable and of high affinity, and was absent in boiled tissue. The solubilized receptors retained their high affinity for [125I] oCRF in the solubilized state, exhibiting a dissociation constant (KD) of approximately 200 pM, as determined by direct binding saturation isotherms. Solubilized CRF receptors maintained the rank order of potencies for various related and unrelated CRF peptides characteristic of the membrane CRF receptor: rat/human CRF congruent to ovine CRF congruent to Nle21,38-rat CRF greater than alpha-helical oCRF-(9-41) greater than oCRF-(7-41) much greater than vasoactive intestinal peptide, arginine vasopressin, or the substance-P antagonist. Furthermore, the absolute potencies (Ki values) for the various CRF-related peptides in solubilized receptors were almost identical to those observed in the membrane preparations, indicating that the CRF receptor retained its high affinity binding capacity in the digitonin-solubilized state. Chemical affinity cross-linking of digitonin-solubilized rat cortical membrane proteins revealed a specifically labeled protein with an apparent mol wt of 58,000 which was similar to the labeled protein in native membrane homogenates. Although solubilized CRF receptors retained their high affinity for agonists, their sensitivity for guanine nucleotide was lost. Size exclusion chromatography substantiated these results, demonstrating that in the presence or absence of guanine nucleotides, [125I]oCRF labeled the same size receptor complex. These data suggest that either the guanine nucleotide-binding protein (Ns) is tightly associated with the CRF receptor after solubilization and is insensitive to guanine nucleotides, or that high affinity binding for soluble CRF receptors is not dependent on the coupling of a guanine nucleotide-binding protein. The solubilization of CRF receptors from membranes in digitonin should allow for the more complete molecular and functional characterization of CRF-mediated events and purification of the receptor.

Animals↗
PubMed1985

Structural and functional properties of adult rat heart myocytes lysed with digitonin.

Low concentrations of digitonin disrupt the sarcolemma of adult rat heart myocytes selectively and completely. When the digitonin lysis is carried out in the presence of 10 mM Mg-ATP, the permeabilized cells retain the rod-cell morphology typical of heart cells in situ and show spontaneous phasic contractions. The rate of contraction is a function of the free Ca2+ concentration from a pCa of 7.2 to 5.2. Higher levels of free Ca2+ result in hypercontracture of the myocytes into round cells with characteristically distorted morphology. The sarcoplasmic reticulum of digitonin-lysed myocytes takes up Ca2+ in an ATP-dependent reaction that is inhibited and reversed by caffeine and strongly enhanced by procaine or ruthenium red. The Ca2+ accumulation has a Km of 0.6 microM Ca2+, depends on Pi (Km of 13 mM), and is strongly inhibited by bicarbonate ion. The hypercontracture of digitonin-lysed myocytes is a function of both the pCa and the Mg-ATP concentration of the suspending medium. Hypercontracture requires ATP. Hypercontracture due to Ca2+ overload occurs at lower Ca2+ concentrations when Mg-ATP is decreased from 10 to 1 mM. However, at low concentrations of Mg-ATP (in the range from 1 to 10 microM), hypercontracture also occurs and is essentially Ca2+-independent. Since hypercontracture of heart myocytes appears analogous to the formation of contraction bands in situ, these observations may be relevant to the phenomena of oxygen paradox and of Ca2+ paradox in intact myocardial tissue.

Adenosine Triphosphate↗
PubMed1989

Evaluation of a digitonin disk assay to discriminate between acholeplasma and mycoplasma isolates from bovine milk.

A study was undertaken to evaluate effectiveness of a digitonin disk inhibition test to discriminate between Acholeplasma laidlawii and Mycoplasma sp. isolated from bovine milk. The test measured zone diameters of growth inhibition surrounding a digitonin-containing disk on solid medium. Zones of inhibition for 20 isolates of A. laidlawii, ranging from 8-14 mm, did not overlap those of 261 isolates of Mycoplasma sp., ranging from 16 to 38 mm. Examination of variation in zone diameters for M. bovis found that inhibition was not appreciably affected by agar dehydration. Zones of inhibition increased with increasing dilutions of stock culture and decreased with increasing incubation time. Analysis of variance and Fisher's least significant difference test of logn zone diameters revealed that differences in mean logn zone diameters were different at the 0.01 level of significance between some of the six species of mycoplasma examined, indicating that growth among some species of mycoplasma was effected differently by digitonin. The digitonin test was found to clearly discriminate between A. laidlawii and Mycoplasma sp. indicating that the test would be useful as a practical screening test of individual-cow and bulk tank milk for mycoplasmas.

Acholeplasma laidlawii↗
PubMed1985

Ascorbic acid and catecholamine release from digitonin-treated chromaffin cells.

The subcellular localization of catecholamines and ascorbic acid in cultured bovine adrenal chromaffin cells was studied by permeabilizing the cells with digitonin, a steroid glycoside. Catecholamine release from permeabilized chromaffin cells was dependent on the free calcium concentration and the temperature of the incubation mixture. By contrast, [14C]ascorbic acid, preloaded into the cells, was released by digitonin treatment in a manner independent of the concentration of free calcium and with only moderate regard to the incubation temperature. The sensitivity of ascorbic acid release to digitonin treatment was identical to that of calcium-dependent catecholamine release. These results thus suggest that ascorbic acid preloaded into the cells may directly efflux from the cell cytoplasm as a result of the permeabilization of the plasma membrane. Dimethylepinephrine, a permanently positively charged catecholamine analog which is known to be excluded from vesicular fractions, was also released by digitonin treatment in a manner independent of calcium. The time course of dimethylepinephrine release was very similar to that of ascorbic acid release. Thus, newly accumulated ascorbic acid in chromaffin cells may be localized to a free pool in the cell cytoplasm rather than in a vesicular compartment.

Adrenal Medulla↗
PubMed1981

Effects of digitonin and glutaraldehyde on the preservation of erythrocyte membrane structures.

In erythrocyte membranes digitonin induces different structural alterations. Crosslinking of the membrane proteins by a pretreatment with glutaraldehyde cannot suppress these alterations completely. The bulged, elongated and the flat, rectangular particle-free areas are by their appearance two types of lipid domains. The other structures are derived from these domains, as supported by the observed transition stages. An analysis of the behaviour of the structures by different digitonin concentrations, after a pretreatment of the intact erythrocytes and ghosts with glutaraldehyde, and after an incubation at 37 degrees C or at 2 degrees C, results in a differentiation between "phase-separation" and "phase-formation". "Phase-separation" occurs at a low digitonin concentration, is suppressed to a high extent by glutaraldehyde pretreatment and is stabilized by low temperature. "Phase-formation" occurs at higher digitonin concentrations, is not suppressed by glutaraldehyde pretreatment and can be suppressed by low temperature.

Aldehydes↗
PubMed1997

Suspension of smooth muscle cells treated with digitonin as a model for studying the myometrial endoplasmic reticulum calcium pump.

The effect of the nonionic detergent digitonin on ruthenium red-insensitive, oxalate-stimulated, and thapsigargin-suppressed accumulation of Ca2+ by suspension of myometrial cells from ATP- and Mg(2+)-containing incubation medium was studied using passive loading with 45Ca2+. The dependence of Ca2+ accumulation by the cells on the concentration of digitonin is bell-shaped, the maximal Ca2+ uptake being observed at 0.05-0.1 mg/ml digitonin. Myocytes chemically skinned with digitonin represent a suitable experimental model for studying kinetic properties of the uterine myocyte endoplasmic reticulum Mg2+, ATP-dependent Ca2+ pump and assessing sensitivity of this Ca(2+)-transporting system to various effectors including the uterotonic octapeptide oxytocin.

Adenosine Triphosphate↗
PubMed1985

Studies with digitonin-treated rat hepatocytes (nude cells).

Isolated rat hepatocytes were treated with digitonin to strip the plasma membrane. The effect of digitonin concentration and exposure time on the recovery of marker enzymes for cell organelles was examined. Hepatocytes treated at room temperature for 1-2 min with 1 mg/ml of digitonin lose some 40% of their protein but retain over 95% of their intact mitochondria and peroxisomes, 90-95% of their endoplasmic reticulum, and about 80% of their lysosomal enzymes. There is little loss of the mitochondrial intermembrane content, and both oxygen uptake and phosphorylation are unimpaired by the treatment. Electron microscopy reveals a complete loss of the plasma membrane, in spite of limited loss of marker enzymes for this membrane. Scanning electron microscopy revealed the interior of the cells to be made up of a dense network of fibers and lamellae attached to the nucleus, mitochondria, and small organelles. The treated cells were stable for many hours when kept in 0.25 M sucrose containing 25 mM monovalent salts. In salt-free sucrose the cells broke up very rapidly into nuclei and other single organelles. Addition of 5 mM NaCl or KCl retards breakup, and 15-20 min were required for dissolution. Intermediate stages, illustrated by scanning electron micrographs, show structure and chains made up mainly of mitochondria held together by a lamellar network. The rapid breakdown occurred at a pH above 7.5 in an oxygen atmosphere and in the presence of phosphate and apparently is an energy-requiring process. It is slow below a pH of 7.2, and at a pH of 6.8 the treated cells remain completely stable in salt-free sucrose. Our results suggest that endoplastic reticulum is a major component of the cytostructure holding together nuclei and organelles.

Animals↗
PubMed1995

Oxygraphic evaluation of mitochondrial function in digitonin-permeabilized mononuclear cells and cultured skin fibroblasts of patients with chronic progressive external ophthalmoplegia.

For quantitative elucidation of maximal mitochondrial oxidation capacities in human mononuclear cells, cultured human skin fibroblasts and human thrombocytes the optimal amount of digitonin for plasma membrane permeabilization was determined to be 5, 10, and 0.1 micrograms/10(6) cells, respectively. Using these concentrations the rate of respiration of permeabilized cells with the mitochondrial substrates succinate (+ rotenone) or glutamate + malate can be stimulated between two- and fourfold by ADP and inhibited by carboxyatractyloside. The maximal respiratory activities of well-characterized preparations of permeabilized mononuclear cells of five patients with chronic progressive external ophthalmoplegia were compared to healthy controls and a 30 to 50% decrease of the ADP-stimulated respiration rates with glutamate + malate and succinate + rotenone was detected. This is an indication for the presence of the mitochondrial defect in respiratory active blood cells. Additionally, for two of these patients the mitochondrial defects were proven to be detectable by the determination of maximal oxygen consumption rates of digitonin-permeabilized cultured skin fibroblasts. Therefore, the determination of maximal oxidation capacities of a well-defined cell population using strictly standardized conditions of digitonin permeabilization is judged as a useful and sensitive method for the elucidation of mitochondrial function in extramuscular tissue.

Adolescent↗
PubMed1990

Ultrastructural changes of liver parenchyma following digitonin-pulse perfusion of rat liver.

It has been shown that pulse perfusion of rat liver with a digitonin-containing medium results in a highly zonated hepatocyte permeabilization, allowing selective sampling of cytosolic constituents from periportal and perivenous (centrolobular) hepatocytes "in situ". In the present paper we provide an ultrastructural evaluation of the perfusion method. Identical changes in hepatocytes from affected periportal and perivenous zones are found. Affected hepatocytes appear light (electron-lucent) in electron micrographs with a sharp transition to normal hepatocytes. The most conspicuous ultrastructural findings are: (1) transformation of the sinusoidal part of the light hepatocytes, the lipocyte processes and the endothelium of affected zones apparently unifying into a continuous layer dominated by disrupted plasma membranes and 7-nm filaments; (2) deposition of osmiophilic digitonin-cholesterol complexes along the sinusoidal plasma membranes of affected zones; and (3) reduction of the cytoplasmic matrix (cytosol) in the light hepatocytes, a dilation of the mitochondrial intermembrane space with a preserved mitochondrial matrix, and a dilation of cisternae of the granular endoplasmic reticulum. The ultrastructural findings are consistent with marker-enzyme activity measured in eluates from digitonin-perfused livers, except that lysosomes appear intact, apparently contrasting with the observed eluation of amyloglucosidase (Quistorff et al. 1985).

Animals↗
PubMed1988

Effects of trypsin on secretion stimulated by micromolar Ca2+ and phorbol ester in digitonin-permeabilized adrenal chromaffin cells.

1. Catecholamine secretion from digitonin-treated chromaffin cells is stimulated directly by micromolar Ca2+ in the medium. The permeabilized cells are leaky to proteins. 2. In this study trypsin (30-50 micrograms/ml) added to cells after digitonin treatment completely inhibited subsequent Ca2+-dependent catecholamine secretion. The same concentrations of trypsin did not inhibit secretion from permeabilized cells if trypsin was present only prior to cell permeabilization. 3. The data indicate that trypsin entered digitonin-treated chromaffin cells which were capable of undergoing secretion and that an intracellular, trypsin-sensitive protein is involved in secretion. Chymotrypsin was less potent but had effects similar to those of trypsin. 4. The enhancement of Ca2+-dependent secretion from permeabilized chromaffin cells induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was inhibited by trypsin added simultaneously with Ca2+ to permeabilized cells at concentrations (3-10 micrograms/ml) which had little or no effect on Ca2+-dependent secretion from cells untreated with TPA. Ca2+-dependent secretion in TPA-treated cells was reduced by trypsin only to the level that would have occurred in cells not treated with TPA. Trypsin reduced the large TPA-induced increment of membrane-bound protein kinase C.

Adrenal Medulla↗
PubMed1988

Studies on the relationship between the inner and outer membranes of rat liver mitochondria as determined by subfractionation with digitonin.

The present investigation has attempted to define in rat liver mitochondria the distribution of outer membrane proteins in relation to the inner membrane by fractionation with digitonin and phospholipase A2. Porin, the channel-forming protein in the outer membrane, was measured quantitatively by immunological methods. Neither monoamine oxidase nor porin could be released by phospholipase A2 treatment, but both were released by digitonin, at the same detergent concentration. Thus, the release of monoamine oxidase and porin requires the disruption of the cholesterol but not the phospholipid domains of the membrane and the two polypeptides exist in the same, or similar, membrane environment with regard to cholesterol. Changes in the energy state, or binding of brain hexokinase to rat liver mitochondria prior to fractionation with digitonin, did not alter the release patterns of porin and monoamine oxidase. The uptake of Ca2+, however, resulted in the concomitant release of the outer membrane markers together with the matrix marker, malate dehydrogenase. The present findings with liver differ from those obtained recently with brain mitochondria (L. Dorbani et al. (1987) Arch. Biochem. Biophys. 252, 188-196) in which two populations of porin were located in two different cholesterol domains. The significance of these differences in the location of porin in liver and brain mitochondria is discussed.

Animals↗
PubMed1980

Effect of streptolysin O and digitonin on egg lecithin/cholesterol vesicles.

Artificial membrane vesicles (liposomes) have been used to study the lytic mechanism of the bacterial toxin, streptolysin O, compared to that of the well-known plant glycoside, digitonin. Two types of vesicle were prepared: large unilamellar vesicles and multilamellar liposomes. The vesicles were prepared with varying molar ratios of egg lecithin and cholesterol and loaded with the water-soluble spin label, TEMPO-choline chloride. Lysis of the vesicles was registered as release of spin label and monitored by change in the electron spin resonance (ESR) spectrum. In this system digitonin was able to lyse both large unilamellar vesicles and multilamellar liposomes. The effectiveness of lysis increased by increasing the percentage of cholesterol, but even at 0% cholesterol a significant level of lysis was observed by addition of a large enough concentration of digitonin. In contrast, no lysis was detected from multilamellar liposomes after exposure to streptolysin O, even when they consisted of 50 mol% cholesterol. On the other hand, large unilamellar vesicles could be lysed by streptolysin O, provided the cholesterol content was greater than 33%. At 67 mol% cholesterol in the membranes, the degree of lysis was diminished compared to 50%, which appeared to be optimal. This is the first demonstration of liposome lysis by streptolysin O and demonstrates the cholesterol specificity which has previously been shown by inhibition studies.

Cholesterol↗
PubMed1984

Interactions between digitonin and bilayer membranes.

The morphology of interactions between digitonin and cholesterol has been investigated. When precipitated from ethanolic solutions, digitonin-cholesterol complexes form in flat lamellar sheets. In contrast, when the complex is formed in a bilayer membrane, the membrane is deformed into corrugations of hemitubules. The polarity of the deformations formed in bilayer membranes is highly correlated with the direction of entry of digitonin into the membrane. We suggest that the morphology of digitonin/cholesterol hemitubules is dependent upon the complex being formed within a bilayer and, in addition, is not correlated with asymmetry of cholesterol concentration across the membrane.

Animals↗
PubMed1989

Investigation of rat striatal dopamine D-1 receptors solubilized by digitonin with a precipitation method.

[3H]SCH 23390 binding sites solubilized from rat striatal membranes by the detergent digitonin were investigated by using a polyethylene glycol precipitation method to separate the bound [3H]SCH 23390 from the free [3H]SCH 23390. The binding of [3H]SCH 23390 to the solubilized preparations was specific and saturable with a KD of 4.99 +/- 0.03 nM and a Bmax of 619 +/- 13 fmol/mg protein. The rank order of potency of dopamine agonists and antagonists for competing with [3H]SCH 23390 binding for the solubilized preparations was appropriate for dopamine D-1 receptors. The competition of SCH 23390 and S(-)-SCH 23388 with [3H]SCH 23390 binding for the solubilized preparations was stereoselective. However, the sensitivity of the dopamine agonist high-affinity binding to guanine nucleotide GTP was almost lost upon digitonin solubilization. Preincubating the membranes with dopamine preserved the guanine nucleotide sensitivity of agonist binding for membranes in solubilized preparations. These results proved that the polyethylene glycol precipitation method can be used for assay of digitonin-solubilized dopamine D-1 receptors in rat striatum.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed1983

Comparison of glycolysis in intact and digitonin-permeabilized bloodstream trypomastigotes of Trypanosoma brucei.

Digitonin has been used to permeabilize bloodstream trypomastigotes of Trypanosoma brucei. Such permeabilized parasites revealed a fully-functional glycolytic pathway which catabolized glucose and some phosphorylated glycolytic intermediates. Glucose-starved bloodstream trypomastigotes revealed saturation kinetics with a glucose Km = 0.6 mM and Vmax = 150 natom O/min per 10(8) for intact parasites; Km = 4 mM and Vmax = 100 natom O2/min per 10(8) for permeabilized parasites. Glucose oxidation in intact parasites was stimulated 40% by addition of 3 micrograms digitonin/10(8) parasites. Higher concentrations of digitonin than this inhibited the glucose oxidation. Ten millimolar phosphoenolpyruvate (PEP) inhibited the rate of O2 consumption by permeabilized trypanosomes respiring on glucose under aerobic conditions by 50%. It is proposed that glucose oxidation is apparently limited by transport across trypanosomal plasma membrane, and phosphofructokinase is regulated by PEP levels. It is concluded that permeabilization of trypanosomes with digitonin might offer a closer physiological condition for the study of the regulation of glycolysis by using glycolytic intermediates and other chemical compounds which would otherwise not be transported across the membrane(s).

Animals↗