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At least 19 recordsLinked to original sources
PubMed1978

The effect of digitonin-containing fixatives on the retention of free cholesterol and cholesterol esters.

The influence of several fixation and dehydration procedures on the retention of free cholesterol and cholesterol esters was studied in filter paper preparations. The retention of free cholesterol by the filter paper proved to be decreased by the addition of digitonin to the aldehyde fixative (aqueous phase) and was only slightly enhanced by partial dehydration (alcoholic phase, up to 70% ethanol). Furthermore, digitonin or the presumably formed cholesterol-digitonide complex bound hardly any osmium oxides in glass-fibre paper. Up to 26% of the cholesterol esters was mobilized during the aqueous phase when digitonin was added to the aldehyde fixative. When the glass-fibre papers containing the digitonin cholesterol-ester-osmate complexes were stored in distilled water after fixation, the fluid became turbid. Particulate material isolated from this turbid solution showed ultrastructurally a close resemblance to the 'whorls' observed by several authors in tissue fixed by a digitonin-containing aldehyde fixative. Digitonin also changed the ultrastructural appearance of liposomes, containing lecithin: cholesterol: phosphatidic acid; in a molar ratio 7:2:1. Our observations lead to the conclusion that the use of digitonin-containing fixatives should be abandoned, because they give results which cannot be interpreted. By the use of K4 [Fe(CN)6] containing OSO4 in the post-fixation step were able to demonstrate an increase in the visualization of membranous structures (liposomes).

Cholesterol↗
PubMed1978

Freeze-fracture identification of sterol-digitonin complexes in cell and liposome membranes.

To advance our understanding of the organization of cholesterol within cell membranes, we used digitonin in freeze-fracture investigations of model lipid vesicles and tissues. Cholesterol suspensions or multilamellar liposomes composed of phosphatidylcholine with and without cholesterol were exposed to digitonin. Freeze-fracture replicas of those multilamellar liposomes containing cholesterol displayed either 50--60-nm wide intramembrane corrugations or extramembrane tubular complexes. Comparable intramembrane hemitubular scallops and extra-cellular free tubular complexes were observed in thin sections. Exposure of sperm, erythrocytes (whole and ghosts), and intact tissues (skin, liver, adrenal gland, epididymis) to digitonin produced the same types of intra- and extramembrane complexes or furrows as were formed in liposomes. The plasma membrane of guinea pig serum tail had two unfurrowed regions: the annulus and the zipper. Incubating erythrocyte membranes with digitonin resulted in rapid displacement of cholesterol, accompanied by intramembrane particle clustering and membrane faceting, a feature which we did not see in the intact epithelia studied. In freeze-fractured epithelia, we found that plasma membranes, lysosomes, and some vesicular organelles commonly furrowed, but that mitochondrial membranes and nuclear envelopes were generally spared, correlating well with their known cholesterol content. Finally, plasma membrane corrugations approached but did not impinge on either gap or tight junctions, or on coated vesicles. We conclude that freeze-fracture of membranes exposed to digitonin: (a) reveals distinctive cholesterol-digitonin structural complexes; (b) distinguishes cholesterol-rich and -poor organelle membranes; and (c) demonstrates membrane domains rich or poor in cholesterol.

Adrenal Glands↗
PubMed1976

The measurement of ouabain binding and some related properties of digitonin-treated (Na+,K+)-ATPase.

1. Digitonin treated membrane preparations purified from dog kidney lose their (Na+,K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity, but the K+-phosphatase and Na+-dependent ADP-ATP exchange activities survive and remain ouabain-sensitive. Because the enzyme preparations consist largely of pure (Na+,K+)-ATPase, these effects of digitonin must be intrinsic to the Na+ pump. 2. Concomitant with these enzymatic changes, digitonin treatment alters the sensitivity of the phosphatase and exchange activities to ouabain. 3. Attempts to measure ouabain binding by the usual centrifugation or filtration methods proved unsuccessful. A filtration method involving a double 0.01 mum filter and omitting water washes is necessary to demonstrate ouabain binding. Under these conditions, ouabain binding capacity appears to be unchanged in the presence of digitonin, but the apparent dissociation constant is doubled. 4. Ouabain binding is rendered more reversible by digitonin treatment, since washing filters with water removes a large fraction of bound ouabain without affecting the retention of exchange activity. 5. The double filter method traps essentially all of the ADP-ATP exchange activity on the filter. However, a large and somewhat variable proportion of the K+-phosphatase activity passes through the filter. Sodium dodecyl sulfate polyacrylamide gel analysis of the filtrate shows that a small amount of filtrable protein catalyzed this phosphatase activity at greatly increased turnover rates. Both subunits of the (Na+, K+)-ATPase are present in this latter protein fraction.

Adenosine Triphosphatases↗
PubMed1978

Digitonin induced alterations of the erythrocyte membrane as visible by freeze-fracturing.

Treatment of erythrocytes by low concentrations of digitonin results in the formation of elongated, bulged membrane areas free from particles and intramembraneous tubular structures emerged from these domains. At higher concentrations the tubular structures are present also outside of the membrane. A second, not bulged type of particle-free areas is more or less rounded, but mostly of rectangular shape. The rate of this type of domains increases with the concentration of digitonin. Breaks in these areas lead to a formation of sheets which results in a breakdown of the membrane structure finally into a brittle mass of many small sheets lying irregularly one upon the other. Already during the first steps of this membrane breakdown hemolysis takes place. Cross-linking of the proteins with glutaraldehyde does not entirely block up the membrane alterations. In fixed erythrocytes the formation fo elongated domains is restricted, no tubular structures are present and no dislocations of particles can be observed. Nevertheless smooth sheets localized also outside of the membrane are formed. However the investigated ghosts are not stabilized by glutaraldehyde against the effects of digitonin. Particle dislocations as well as all the other membrane alterations are present. The implications of the obtained results are: 1. The elongated domains and the tubular structures are probably not digitonin-cholesterol-complexes. 2. The formation of crystalline regions by digitonin-cholesterol-complexes destroys the membrane structure.

Digitonin↗
PubMed1978

Solubilization of bovine corpus-luteum adenylate cyclase in lubrol-PX, triton X-100 or digitonin and the stabilizing effect of sodium fluoride present in the solubilization medium.

1. Adenylate cyclase activity of the washed 600g sediment of bovine corpus-luteum homogenate was solubilized by Lubrol-PX, Triton X-100 and digitonin. Digitonin was the least destructive of NaF-stimulated activity. 2. NaF, present in the solubilization medium together with MgSO4, increased the percentage yields of soluble activity from untreated 600g sediment and 600g sediment which had been preincubated with p[NH]ppG (guanosine 5'-[betagamma-imido]triphosphate). The stabilizing influence of NaF was most marked with digitonin. However, the highest specific activities of soluble enzyme were obtained with Lubrol-PX as solubilizing agent, since digitonin solubilized more membrane protein than does Lubrol-PX, and less of the activity of the digitonin-dispersed 600g sediment was recovered in the 105000g supernatant. 3. p[NH]ppG also has a stabilizing effect when present during the solubilization, but less so than NaF. 4. Both NaF and MgSO4 alone have a stabilizing effect during solubilization. The greatest amounts of soluble activity were obtained with both agents present in the solubilization medium, there being a synergistic effect.

Adenylyl Cyclases↗
PubMed1978

Prevention of hypercholesterolemia in monkeys (Macaca fascicularis) by digitonin.

Digitonin mixed in the diet was well tolerated by rats and cynomolgus monkeys (Macaca fascicularis), and prevented the expected rise in plasma cholesterol in monkeys fed a diet containing butter and cholesterol. Both digitonin and mildly hydrolyzed digitonin precipitated cholesterol dispersed in micellar suspensions and decreased intestinal absorption of cholesterol in rats, but only digitonin bound cholesterol in vitro. The hypocholesterolemic effect of digitonin may be secondary to decreased intestinal absorption of cholesterol associated with a disturbance in the micellar suspension of cholesterol in the intestinal content. Modification of other mechanisms that may be involved in membrane transport of cholesterol through the intestinal epithelium is not ruled out by the present findings.

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↗
PubMed1975

Basolateral plasma membranes of intestinal epithelial cells. Identification by lactoperoxidase-catalysed iodination and isolation after density perturbation with digitonin.

Lactoperoxidase-catalysed iodination was used to label intestinal epithelial cell sheets with 125I. The iodination was carried out under conditions that allowed little penetration of lactoperoxidase into the cells and membrane-bound 125I therefore provided an effective marker for following plasma-membrane fragments through subcellular-fractionation procedures. 2. After homogenization and isopycnic zonal centrifugation through sucrose gradients two peaks of membrane-bound 125I were detected. One coincided with brush border enzymes such as alkaline phosphatase, disaccharidases and L-leucine B-naphthylamidase, whereas the other was coincident with the major peak of (Na++K+)-stimulated ATPase (adenosine triphosphatase), which has been thought to be concentrated in the basolateral plasma membranes of these cells. Neither peak of 125I reflected the distribution of any marker for an intracellular organelle. 3. A larger proportion of the (Na++K+)-stimulated ATPase, and thus of the basolateral plasma-membrane material, was found in a crude 'mitochondrial' fraction. It was not readiily separated from mitochondria by conventional techniques of subcellular fractionation. 4. Treatment of the 'mitochondrial' fraction with digitonin increased the density of basolateral plasma membrane but had little effect on mitochondrial density. A purified preparation of digitonin-loaded basolateral plasma membranes was isolated at a density of 1.20-1.22 by isopycnic centrifugation. 5. The enzymic composition of this preparation of basolateral plasma membranes is compared with previous preparations isolated from intestinal mucosal 'scrape' materials and from isolated cells.

Adenosine Triphosphatases↗
PubMed1977

Stabilization of cholesterol in myelin with digitonin: observation with polarized light.

Cholesterol can be preserved in tissues dehyrated for light microscopy by incorporating digitonin into glutaraldehyde fixation. Fresh and fixed frozen sections of rat sciatic nerve exhibit strong, radially positive birefringence when viewed with polarized optics. However, tissue fixed and dehydrated with standard methods for Epon embedding shows very weak radially positive birefringence. The use of digitonin with a variety of techniques generally permits retention of optical activity in embedded nerves. Such observations suggest a preservation of lipids (presumably cholesterol) in molecular orientations necessary to produce Maltese cross patterns.

Animals↗
PubMed1978

[Disintegration and fractionation of barley chloroplast membranes at different concentrations of digitonin and chloroplasts].

Chloroplasts pretreated with digitonin were fractionated by electrophoresis in order to study the arrangement of chloroplast membranes. Different types of particles resulting from the electrophoresis are shown to be due to fractionation of the chloroplast membranes of the same type rather than to the isolation of particular types from the chloroplasts of different ages. The stopped-flow measurement of quickly changing light scattering demonstrated that the membrane solubilization increased with the increase in the digitonin concentration. Meanwhile the fractionation result (the amount of particles in the electrophoretic zones and their spectral properties) remain very similar over a wide range of the detergent and chloroplast concentrations. This is explained by the fact that the increasing detergent concentrations do not cause particle destruction. Thus the method used revealed the particles which different from one another before they were isolated from the membrane. They are fractionated following the natural boundaries between them and the containing membrane. In other words, chloroplast membranes have a discrete set of submembrane particles of different types.

Cell Fractionation↗
PubMedDate not supplied

[Effect of hydrolases and digitonin on the electrophysiological characteristics of the neurons of the snail, Helix pomatia].

Studies have been made on the effect of trypsin, chymotrypsin, pronase, lipases, hyaluronidase and digitonin on electrophysiological properties of the neurons of the snail H. pomatia under external application. Proteases and lipases gradually depolarize the neuronal membrane, decrease the amplitude and prevent the onset of action potentials, initially increase and then decrease the membrane resistance. The decrease in the membrane resistance coincides with the period of maximum inhibition of resting and action potentials in the neurons. The enzymes studied do not affect the membrane capacitance. Changes in electrophysiological characteristics induced by the enzymes are partially reversible provided the preparation is soaked in Ringer's solution for a sufficient time. Digitonin rapidly and irreversibly depolarizes the membrane, decreases its resistance and blocks action potentials. Hyaluronidase does not significantly affect neuronal electrophysiological properties when applied solely, but facilitates the development of changes during subsequent effect of proteases.

Animals↗
PubMed1977

In vivo sterol biosynthesis by pea aphid symbiotes as determined by digitonin and electron microscopic autoradiography.

Pea aphid primary symbiotes have previously been shown to synthesize cholesterol in vitro. Two electron microscopic techniques were used here to determine whether the symbiotes also synthesize cholesterol in vivo and whether this cholesterol is made available to the aphid. We also inquired into a possible role of secondary symbiotes in chosesterol biosynthesis. Treatment of aphids with digitonin resulted in significant alteration of ultrastructural sites in primary and secondary symbiote membranes. We concluded that these sites are areas of high cholesterol concentration in the symbiotes. Electron microscopic autoradiography with 3H-mevalonate precursor indicated that both primary and secondary symbiotes synthesize cholesterol; in both cases, the majority of grains were associated with the symbiote membranes. While the frequency of grains on the symbiotes remained constant, irrespective of incubation time in labelled media, the frequency of grains over surrounding tissues increased exponentially as the time of incubation was increased from 30 min to 8 h, indicating that symbiote cholesterol is transported to other tissues. High voltage electron microscopic autoradiography permitted thick section autoradiography, reducing the time of emulsion exposure from 54 days (thin section) to 12 days (0.5 mum sections).

Animals↗
PubMed1979

Composition and spectral properties of a cytochrome-enriched fraction obtained from chloroplasts by digitonin treatment.

A cytochrome-enriched fraction obtained from chloroplasts after treatment with the detergent digitonin contained cytochromes f, b-559LP and b-563 in the approximate proportions 1:1:2, close to those observed in unfractionated chloroplasts. The spectrum of cytochrome b-563 at temperature of liquid N2 showed a single asymmetrical alpha-band with a maximum at 561 nm.

Chloroplasts↗
PubMed1978

Superoxide production by digitonin-stimulated guinea pig granulocytes. The effects of N-ethyl maleimide, divalent cations; and glycolytic and mitochondrial inhibitors on the activation of the superoxide generating system.

N-ethylmaleimide, divalent cations, ethylene glycol bis (beta aminoethyl ether) N,N,N',N',-tetraacetate, 2-deoxyglucose, cyanide, and dinitrophenol were examined for their effect on the ability of guinea pig granulocytes to generate superoxide (O(2) (-)) when stimulated by digitonin. N-ethylmaleimide (1 mM) inhibits only when added before complete activation of the O(2) (-) generating system, and at lower concentrations (0.05-0.2 mM) slows the activation process. Ca(++) is required for maximum O(2) (-) generation, and Mg(++) decreases the amount of Ca(++) required. Ethylene glycol bis (beta aminoethyl ether) N,N,N',N',-tetraacetate (10 mM) inhibits only if added before complete activation. Incubation of cells in 2-DOG causes a time- and concentration-dependent inhibition of O(2) (-) generation. It also increases the time required for activation of this system. Cyanide and dinitrophenol increase the rate of O(2) (-) production. However, when these compounds are added to cells whose O(2) (-) production is partially inhibited by incubation in 2-deoxyglucose, complete inhibition results. If cyanide or dinitrophenol is added after activation of 2-deoxyglucose-treated cells, no further inhibition occurs. On the basis of the above results, we conclude that the activation of the O(2) (-) generating system is N-ethylmaleimide sensitive, Ca(++) dependent, and energy requiring, but that the activity of the enzyme system in the cell is not.

Animals↗
PubMed1978

Kinetic study of photoregeneration process of digitonin-solubilized squid rhodopsin.

In the photoregeneration process of squid rhodopsin, an intermediate has been found at neutral pH values (phosphate buffer) with a flash light (lambda greater than 540 nm). An intermediate R430, with the 11-cis retinal as chromophore, is produced from metarhodopsin in light and is converted to rhodopsin through the processes R430 leads to P380 and P380 leads to rhodopsin. The pH dependence of the velocity of the conversions suggests that processes R430 leads to P380 and P380 leads to rhodopsin involve a protolytic reaction and that the ionized group is a histidine residue of opsin. Kinetic parameters show that the largest conformational change in opsin occurs in the conversion of R430 leads to P380.

Animals↗