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Analytical Biochemistry Jan 1998A quick subcellular fractionation procedure using differential centrifugation, which is applicable to isolated and cultured cells, is presented. This technique was...
A quick subcellular fractionation procedure using differential centrifugation, which is applicable to isolated and cultured cells, is presented. This technique was developed for studying the subcellular localization of phosphorylated proteins in isolated liver cells after various stimuli, but is also applicable to many other situations. The main difference with the usual techniques is that by including digitonin in the homogenization buffer, the procedure is greatly shortened. Furthermore, because the soluble fraction is separated from the particulate fraction very early in the fractionation procedure, subcellular organelles are not exposed to phosphatases and other soluble enzymes such as esterases and proteases during the fractionation. The entire procedure is carried out in an Eppendorf centrifuge, which allows isolation of the cytosolic fraction in less than 1 min, a washed nuclear fraction in about 4 min, a mitochondrial fraction in less than 10 min, and a washed light mitochondrial L fraction in about 40 min. Judging by the behavior of marker enzymes and the morphology of the fractions, the method is highly comparable to classical procedures.
Topics: Animals; Cell Fractionation; Cells, Cultured; Centrifugation; Digitonin; Indicators and Reagents; Liver; Organelles; Rats; Subcellular Fractions
PubMed: 9451511
DOI: 10.1006/abio.1997.2453 -
Experimental Cell Research Jan 1959
Topics: Chemical Fractionation; Digitalis; Digitonin; Liver; Mitochondria; Mitochondria, Liver; Plant Extracts
PubMed: 13639943
DOI: 10.1016/0014-4827(59)90199-5 -
PloS One Jan 2010Most medicinal plants contain a mixture of bioactive compounds, including chemicals that interact with intracellular targets and others that can act as adjuvants to...
BACKGROUND
Most medicinal plants contain a mixture of bioactive compounds, including chemicals that interact with intracellular targets and others that can act as adjuvants to facilitate absorption of polar agents across cellular membranes. However, little is known about synergistic effects between such potential drug candidates and adjuvants. To probe for such effects, we tested the green tea compound epigallocatechin gallate (EGCG) and the membrane permeabilising digitonin on Plasmodium sporozoite motility and viability.
METHODOLOGY/PRINCIPAL FINDINGS
Green fluorescent P. berghei sporozoites were imaged using a recently developed visual screening methodology. Motility and viability parameters were automatically analyzed and IC50 values were calculated, and the synergism of drug and adjuvant was assessed by the fractional inhibitory concentration index. Validating our visual screening procedure, we showed that sporozoite motility and liver cell infection is inhibited by EGCG at nontoxic concentrations. Digitonin synergistically increases the cytotoxicity of EGCG on sporozoite survival, but shows an additive effect on sporozoite motility.
CONCLUSIONS/SIGNIFICANCE
We proved the feasibility of performing highly reliable visual screens for compounds against Plasmodium sporozoites. We thereby could show an advantage of administering mixtures of plant metabolites on inhibition of cell motility and survival. Although the effective concentration of both drugs is too high for use in malaria prophylaxis, the demonstration of a synergistic effect between two plant compounds could lead to new avenues in drug discovery.
Topics: Animals; Catechin; Digitonin; Drug Synergism; Liver; Plasmodium berghei
PubMed: 20072627
DOI: 10.1371/journal.pone.0008682 -
The Journal of Pharmacology and... Aug 1964
Topics: Cell Death; Chemical Phenomena; Chemistry; Digitalis Glycosides; Digitonin; Erythrocytes; Glycosides; Hemolysis; Holothurin; Mucoproteins; Quillaja; Research; Saponins
PubMed: 14214417
DOI: No ID Found -
European Journal of Cell Biology May 1995In 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...
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.
Topics: Acrosome; Animals; Calcium; Cell Membrane Permeability; Digitonin; Hydrogen-Ion Concentration; Male; Microscopy, Electron; Phalloidine; Sea Urchins; Spermatozoa; Staining and Labeling
PubMed: 7543846
DOI: No ID Found -
Biochimica Et Biophysica Acta Dec 1962
Topics: Antifibrinolytic Agents; Chloroplasts; Digitonin; Photosynthesis; Spinacia oleracea; Vitamin K
PubMed: 14000081
DOI: 10.1016/0006-3002(62)90476-6 -
Journal of Neurochemistry Nov 1992Exposure of chromaffin cells to digitonin causes the loss of many cytosolic proteins. Here we report that scinderin (a Ca(2+)-dependent actin-filament-severing protein),...
Exposure of chromaffin cells to digitonin causes the loss of many cytosolic proteins. Here we report that scinderin (a Ca(2+)-dependent actin-filament-severing protein), but not gelsolin, is among the proteins that leak out from digitonin-permeabilized cells. Chromaffin cells that were exposed to increasing concentrations (15-40 microM) of digitonin for 5 min released scinderin into the medium. One-minute treatment with 20 microM digitonin was enough to detect scinderin in the medium, and scinderin leakage levelled off after 10 min of permeabilization. Elevation of free Ca2+ concentration in the permeabilizing medium produced a dose-dependent retention of scinderin. Results were confirmed by immunofluorescence microscopy of digitonin-permeabilized cells. Subcellular fractionation of permeabilized cells showed that scinderin leakage was mainly from the cytoplasm (80%); the remaining scinderin (20%) was from the microsomal fraction. Other Ca(2+)-binding proteins released by digitonin and also retained by Ca2+ were calmodulin, protein kinase C, and calcineurins A and B. Scinderin leakage was parallel to the loss of the chromaffin cell secretory response. Permeabilization in the presence of increasing free Ca2+ concentrations produced a concomitant enhancement in the subsequent Ca(2+)-dependent catecholamine release. The experiments suggest that: (1) scinderin is an intracellular target for Ca2+, (2) permeabilization of chromaffin cells with digitonin in the presence of micromolar Ca2+ concentrations retained Ca(2+)-binding proteins including scinderin, and (3) the retention of these proteins may be related to the increase in the subsequent Ca(2+)-dependent catecholamine release observed in permeabilized chromaffin cells.
Topics: Adrenal Medulla; Animals; Calcium; Catecholamines; Cattle; Cells, Cultured; Cytosol; Digitonin; Dose-Response Relationship, Drug; Gelsolin; Microfilament Proteins; Subcellular Fractions
PubMed: 1402916
DOI: 10.1111/j.1471-4159.1992.tb11003.x -
Biochimica Et Biophysica Acta Jun 1980Treatment of a purified (NA+ + 5+)-ATPase preparation from dog kidney with digitonin reduced enzymatic activity, with the (Na+ + k+)-atpase reaction inhibited more than...
Treatment of a purified (NA+ + 5+)-ATPase preparation from dog kidney with digitonin reduced enzymatic activity, with the (Na+ + k+)-atpase reaction inhibited more than the K+-phosphatase reaction that is also catalyzed by this enzyme. Under the usual assay conditions oligomycin inhibits the (Na+ + k+)-atpase reaction but not the K+-phosphatase reaction; however, treatment with digitonin made the K+-phosphatase reaction almost as sensitive to oligomycin as the (Na+ + k+)-atpase reaction. The non-ionic detergents, Triton X-100, Lubrol WX and Tween 20, also conferred sensitivity to oligomycin on the K+-phosphatase reaction (in the absence of oligomycin all these detergents, unlike digitonin, inhibited the K+-phosphatase reaction more than the (Na+ + k+)-atpase reaction). Both digitonin and Triton markedly increased the K0.5 for K+ as activator of the K+-phosphatase reaction, with little effect on the K0.5 for K+ as activator of the (Na+ + k+)-ATpase reaction. In contrast, increasing the K0.5 for K+ in the K+-phosphatase reaction by treatment of the enxyme with acetic anhydride did not confer sensitivity to oligomycin. Both digitonin and Triton also increased the inhibition of the K+-phosphatase reaction by ATP and increased the inhibition by inorganic phosphate and vanadate. These observations are interpreted as digitonin and Triton favoring the E1 conformational state of the enzyme (manifested by sensitivity to oligomycin and a greater affinity for ATP at the low-affinity substrate sites), as opposed to the E2 state (manifested by insensitivity to oligomycin, greater sensitivity to phosphate and vanadate, and a lower K0.5 for K+ in the K+-phosphatase reaction). In addition, digitonin blocked activation of the phosphatase reaction by Na+ plus CTP. This effect is consistent with digitonin dissociating the catalytic subunits of the enzyme, the interaction of which may be essential for activation by Na+ plus nucleotide.
Topics: Animals; Digitonin; Dogs; Kidney; Kinetics; Octoxynol; Oligomycins; Osmolar Concentration; Polyethylene Glycols; Potassium Chloride; Sodium-Potassium-Exchanging ATPase
PubMed: 6248111
DOI: 10.1016/0005-2736(80)90034-6 -
Neuroscience Letters Oct 2002To determine if calcium could release Cytochrome c (Cyt c) from brain mitochondria without activating the permeability transition (mPT), brain mitochondria were prepared...
To determine if calcium could release Cytochrome c (Cyt c) from brain mitochondria without activating the permeability transition (mPT), brain mitochondria were prepared in two different ways. Digitonin was used to lyse synaptosomes and release synaptosomal mitochondria or a Percoll gradient was used to separate non-synaptosomal mitochondria from the synaptosomes. In gradient-purified mitochondria, low levels of added digitonin produced swelling and Cyt c release. Digitonin augmented Ca(2+)-induced Cyt c release that was insensitive to the mPT inhibitors, cyclosporin A CsA and ADP. Similarly, in mitochondria prepared with digitonin, these inhibitors also failed to prevent Ca(2+)-induced Cyt c release. Thus the mPT-independent, Ca(2+)-induced Cyt c release pathway was attributable to alteration of the permeability properties of the outer mitochondrial membrane by digitonin.
Topics: Animals; Brain Chemistry; Calcium; Culture Media; Cytochrome c Group; Digitonin; In Vitro Techniques; Mannitol; Membrane Potentials; Mitochondria; Mitochondrial Swelling; Rats; Sucrose
PubMed: 12384218
DOI: 10.1016/s0304-3940(02)00948-5 -
Histochemistry 1989The plasma membrane permeabilization obtained by exposure of hepatocytes to digitonin is utilized in the so-called digitonin-pulse perfusion of rat liver (Quistorff and...
The plasma membrane permeabilization obtained by exposure of hepatocytes to digitonin is utilized in the so-called digitonin-pulse perfusion of rat liver (Quistorff and Grunnet 1987). Brief pulses of digitonin applied with antegrade and retrograde perfusion of the liver caused selective elution of cytosolic enzymes and metabolites from the periportal and the perivenous zone of the same liver. In the present study a light microscopical examination of the liver fixed immediately after the digitonin pulse confirmed the very high zonal selectivity of the method inferred from the marker enzyme pattern of the eluates: Only cells around the port of entry of digitonin were affected and the borderline between affected and non-affected cells was always sharp. The typical periportal lesion was triangular in shape, enclosing the portal space, while the perivenous lesion was roughly circular, concentric with the hepatic vein. Assuming that the digitonin lesion reflects the microcirculatory flow pattern these findings seem to be at variance with the acinar model of Rappaport (Rappaport et al. 1954). The lesion in the lobuli near the surface of the liver as reflected by the discoloration pattern observed on the surface was the same as the lesion of deeper lobuli. The conducting vessels of the liver were only insignificantly affected by digitonin. At the cellular level only the sinusoidal luminal surface of the hepatocytes was affected. The cytoplasmic matrix of the cells including glycogen appeared thinned. All cell types of the liver parenchyma seemed to be equally affected by the digitonin treatment.
Topics: Animals; Cell Membrane Permeability; Digitonin; Liver; Male; Perfusion; Rats; Rats, Inbred Strains
PubMed: 2807995
DOI: 10.1007/BF00524760