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The Journal of Biological Chemistry Dec 1986The effects of phorbol esters, dioctanoylglycerol (DiC8), and micromolar Ca2+ on protein phosphorylation and catecholamine secretion in digitonin-treated chromaffin...
The effects of phorbol esters, dioctanoylglycerol (DiC8), and micromolar Ca2+ on protein phosphorylation and catecholamine secretion in digitonin-treated chromaffin cells were investigated. [gamma-32P]ATP was used as a substrate for phosphorylation in the permeabilized cells. 12-O-Tetradecanoylphorbol-13-acetate (TPA) enhanced Ca2+-dependent catecholamine secretion from digitonin-permeabilized cells. The enhancement required MgATP. Only those phorbol esters which activate protein kinase C in vitro enhanced both catecholamine secretion and protein phosphorylation. DiC8, which activates protein kinase C in vitro and mimics phorbol ester effects in situ, also enhanced both catecholamine secretion and protein phosphorylation. Preincubation of intact cells with TPA or DiC8 was necessary for maximal effects on both catecholamine secretion and protein phosphorylation in subsequently digitonin-treated chromaffin cells. The TPA-induced enhancement of protein phosphorylation was almost entirely Ca2+-independent, whereas DiC8-induced enhancement of protein phosphorylation was mainly Ca2+-dependent. Micromolar Ca2+ alone also enhanced the phosphorylation of a large number of proteins. Most of the proteins phosphorylated in response to TPA or potentiated by DiC8 in combination with Ca2+ were also phosphorylated by micromolar Ca2+ in the absence of exogenous protein kinase C activators. In intact cells, 1,1-dimethyl-4-phenylpiperazinium (DMPP) induced Ca2+-dependent phosphorylation of at least 17 proteins which were detected by two-dimensional gel electrophoresis. All of the proteins phosphorylated upon incubation with 1,1-dimethyl-4-phenylpiperazinium were phosphorylated upon incubation with micromolar Ca2+ in digitonin-treated cells. These results demonstrate that TPA- or DiC8-enhanced Ca2+-dependent catecholamine secretion is associated with enhanced protein phosphorylation which is probably mediated by protein kinase C and that activation of protein kinase C modulates catecholamine secretion from digitonin-treated chromaffin cells.
Topics: Adenosine Triphosphate; Adrenal Medulla; Animals; Calcium; Cattle; Cell Membrane Permeability; Cells, Cultured; Digitonin; Diglycerides; Glycerides; Kinetics; Norepinephrine; Phosphoproteins; Phosphorylation; Proteins; Tetradecanoylphorbol Acetate
PubMed: 3491075
DOI: No ID Found -
Journal of Neurochemistry Jan 1985The relationship between catecholamine secretion and arachidonic acid release from digitonin-treated chromaffin cells was investigated. Digitonin renders permeable the...
The relationship between catecholamine secretion and arachidonic acid release from digitonin-treated chromaffin cells was investigated. Digitonin renders permeable the plasma membranes of bovine adrenal chromaffin cells to Ca2+, ATP, and proteins. Digitonin-treated cells undergo exocytosis of catecholamine in response to micromolar Ca2+ in the medium. The addition of micromolar Ca2+ to digitonin-treated chromaffin cells that had been prelabeled with [3H]arachidonic acid caused a marked increase in the release of [3H]arachidonic acid. The time course of [3H]arachidonic acid release paralleled catecholamine secretion. Although [3H]arachidonic acid release and exocytosis were both activated by free Ca2+ in the micromolar range, the activation of [3H]arachidonic acid release occurred at Ca2+ concentrations slightly lower than those required to activate exocytosis. Pretreatment of the chromaffin cells with N-ethylmaleimide (NEM) or p-bromophenacyl bromide (BPB) resulted in dose-dependent inhibition of 10 microM Ca2+-stimulated [3H]arachidonic acid release and exocytosis. The IC50 of NEM for both [3H]arachidonic acid release and exocytosis was 40 microM. The IC50 of BPB for both events was 25 microM. High concentrations (5-20 mM) of Mg2+ caused inhibition of catecholamine secretion without altering [3H]arachidonic acid release. A phorbol ester that activates protein kinase C, 12-O-tetradecanoylphorbol-13-acetate (TPA), caused enhancement of both [3H]arachidonic acid release and exocytosis. The findings demonstrate that [3H]arachidonic acid release is stimulated during catecholamine secretion from digitonin-treated chromaffin cells and they are consistent with a role for phospholipase A2 in exocytosis from chromaffin cells. Furthermore the data suggest that protein kinase C can modulate both arachidonic acid release and exocytosis.
Topics: Acetophenones; Animals; Arachidonic Acid; Arachidonic Acids; Calcium; Catecholamines; Cattle; Chromaffin Granules; Chromaffin System; Digitonin; Ethylmaleimide; Exocytosis; Lipid Metabolism; Magnesium; Norepinephrine; Phorbols; Tetradecanoylphorbol Acetate
PubMed: 3917291
DOI: 10.1111/j.1471-4159.1985.tb07140.x -
Molecular Biology of the Cell Dec 2002The molecular mechanisms of peroxisome biogenesis have begun to emerge; in contrast, relatively little is known about how the organelle functions as cells age. In this...
The molecular mechanisms of peroxisome biogenesis have begun to emerge; in contrast, relatively little is known about how the organelle functions as cells age. In this report, we characterize age-related changes in peroxisomes of human cells. We show that aging compromises peroxisomal targeting signal 1 (PTS1) protein import, affecting in particular the critical antioxidant enzyme catalase. The number and appearance of peroxisomes are altered in these cells, and the organelles accumulate the PTS1-import receptor, Pex5p, on their membranes. Concomitantly, cells produce increasing amounts of the toxic metabolite hydrogen peroxide, and we present evidence that this increased load of reactive oxygen species may further reduce peroxisomal protein import and exacerbate the effects of aging.
Topics: Aging; Animals; Cell Nucleus; Cells, Cultured; Cellular Senescence; Detergents; Digitonin; Dose-Response Relationship, Drug; Endopeptidases; Fibroblasts; Green Fluorescent Proteins; Humans; Hydrogen Peroxide; Immunohistochemistry; Luminescent Proteins; Membrane Proteins; Microscopy, Fluorescence; Octoxynol; Peroxisome-Targeting Signal 1 Receptor; Peroxisomes; Plasmids; Precipitin Tests; Protein Binding; Receptors, Cytoplasmic and Nuclear; Recombinant Fusion Proteins; Time Factors
PubMed: 12475949
DOI: 10.1091/mbc.e02-06-0322 -
Proceedings of the National Academy of... Jun 1980Treatment of isolated rat hepatocptes with low concentrations of digitonin increases the permeability of the plsma membrane to cytosolic proteins without causing release...
Treatment of isolated rat hepatocptes with low concentrations of digitonin increases the permeability of the plsma membrane to cytosolic proteins without causing release of organelles such as mitochondria into the surrounding medium. Electron microscopy showed that treatment of the cells with increasing concentations of digitonin results in a progressive loss in the continuity of the plasma membrane, while most other aspects of cellular morphology remain normal. Depletion of background staining material from the cytosol by digitonin treatment of the cells greatly enhances the visualization of the cytoskeleton. The use of this technique, together with immunofluorescent light microscopy, has verified the presence of an actin-containing filamentous network at the hepatocyte cortex as well as intermediate filaments distributed throughout the cell. Digitonin is thus useful both for selectively permeabilizing the plasma membrane and for intensifying the appearance of intracellular structures such as microfilaments that are normally difficult to observe in cells such as hepatocytes.
Topics: Actins; Animals; Cell Membrane Permeability; Cytoskeleton; Cytosol; Digitonin; Fluorescent Antibody Technique; Immunoelectrophoresis; Liver; Microscopy, Electron; Microscopy, Fluorescence; Rats
PubMed: 6997878
DOI: 10.1073/pnas.77.6.3430 -
European Journal of Immunology Sep 2003By interacting with each others, the tetraspanins are thought to assemble a network of molecular interactions, the tetraspanin web. These tetraspanin/tetraspanin...
By interacting with each others, the tetraspanins are thought to assemble a network of molecular interactions, the tetraspanin web. These tetraspanin/tetraspanin interactions involve in part the palmitoylation of the proteins. We show that tetraspanins interact with cholesterol as indicated by the precipitation of tetraspanin/tetraspanin complexes by digitonin, a cholesterol-precipitating reagent, and the labeling of the tetraspanins CD9, CD81 and CD82 with a photoactivatable cholesterol in vivo. Cholesterol may participate to the interaction of tetraspanins with each other since digitonin-precipitation of tetraspanins was correlated with their mutual interaction, and because these interactions were disrupted following cholesterol depletion by methyl-beta-cyclodextrin (MbetaCD) treatment, or cholesterol sequestration by saponin. A mutant CD9 molecule lacking all palmitoylation sites was not precipitated by digitonin under conditions in which wild-type CD9 was precipitated, indicating a role of palmitoylation for the interaction with cholesterol. Finally, upon ligation of tetraspanins on the surface of a lymphoid B cell line, the tyrosine phosphorylation of several proteins, including the vav nucleotide exchange factor, was inhibited when cells were pretreated with MbetaCD, and increased when they were treated with MbetaCD/cholesterol complexes. Thus, there is a physical and functional link between tetraspanins and cholesterol.
Topics: Animals; Antigens, CD; Cholesterol; Digitonin; Humans; Membrane Glycoproteins; Membrane Proteins; Oncogene Proteins; Phosphorylation; Proto-Oncogene Proteins c-vav; Saponins; Tetraspanin 29; Tritium; Tyrosine
PubMed: 12938224
DOI: 10.1002/eji.200323884 -
The Journal of Biological Chemistry Nov 1985Low 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...
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.
Topics: Adenosine Triphosphate; Animals; Caffeine; Calcium; Digitonin; Heart; Microscopy, Electron; Microscopy, Electron, Scanning; Myocardial Contraction; Myocardium; Procaine; Rats; Ruthenium Red; Sarcolemma; Sarcoplasmic Reticulum
PubMed: 2414295
DOI: No ID Found -
Scientific Reports Jul 2021In higher plants, the photosynthetic process is performed and regulated by Photosystem II (PSII). Arabidopsis thaliana was the first higher plant with a fully sequenced...
In higher plants, the photosynthetic process is performed and regulated by Photosystem II (PSII). Arabidopsis thaliana was the first higher plant with a fully sequenced genome, conferring it the status of a model organism; nonetheless, a high-resolution structure of its Photosystem II is missing. We present the first Cryo-EM high-resolution structure of Arabidopsis PSII supercomplex with average resolution of 2.79 Å, an important model for future PSII studies. The digitonin extracted PSII complexes demonstrate the importance of: the LHG2630-lipid-headgroup in the trimerization of the light-harvesting complex II; the stabilization of the PsbJ subunit and the CP43-loop E by DGD520-lipid; the choice of detergent for the integrity of membrane protein complexes. Furthermore, our data shows at the anticipated MnCaO-site a single metal ion density as a reminiscent early stage of Photosystem II photoactivation.
Topics: Arabidopsis; Cryoelectron Microscopy; Digitonin; Photosystem II Protein Complex
PubMed: 34330992
DOI: 10.1038/s41598-021-94914-x -
The Biochemical Journal Oct 1988We have investigated the cause of defective glycogen synthesis in hepatocyte preparations enriched with cells from the periportal or perivenous zones obtained by the...
We have investigated the cause of defective glycogen synthesis in hepatocyte preparations enriched with cells from the periportal or perivenous zones obtained by the methods of Lindros & Penttila [Biochem. J. (1985) 228, 757-760] and of Quistorff [Biochem. J. (1985) 229, 221-226]. A modified procedure which yields hepatocytes capable of consistent rates of glycogen synthesis is described, and the rates of glucose and glycogen syntheses and of glycolysis in hepatocytes from the two zones are compared. Glycogen synthesis in cells was greatly impaired by very low concentrations (0.01-0.05 mg/ml) of digitonin, which had little effect on glucose and protein syntheses and Trypan Blue exclusion. Cells exposed to such low concentrations of digitonin lose all their synthetic capacity and ability to exclude Trypan Blue when incubated with EGTA, which does not affect cells not exposed to digitonin. With a modified procedure based on this phenomenon, our study reveals that hepatocyte preparations enriched with cells from the periportal zone synthesized glucose from lactate and alanine at rates twice those by cells from the perivenous zone, whereas the rate of glycogen synthesis from C3 precursors in periportal cells was 4 times that in the perivenous preparations. With substrates entering the pathway at the triose phosphate level, gluconeogenesis in periportal-cell preparations was 20% higher, and glycogen synthesis was twice that in perivenous preparations. Glycolysis was studied by the formation of 3HOH from [2-3H]glucose, the yield of lactate, and the conversion of [14C]glucose into [14C]lactate. In cell preparations from both zones glycolysis by all criteria was negligible at 10 mM-glucose, but was substantial at higher concentrations. However, there was no difference between the zones. We confirm that the capacities for glucose and glycogen syntheses in periportal cells are higher than in perivenous cells, but that at physiological glucose concentrations there is negligible glycolysis in liver parenchyma in both zones. The metabolic pattern in the perivenous cells is not glycolytic.
Topics: Animals; Digitonin; Egtazic Acid; Glucokinase; Gluconeogenesis; Glucose; Glycolysis; In Vitro Techniques; Lactates; Lactic Acid; Liver; Liver Glycogen; Male; Protein Biosynthesis; Rats; Rats, Inbred Strains; Tissue Distribution
PubMed: 3143359
DOI: 10.1042/bj2550099 -
Archives of Biochemistry and Biophysics Sep 2020To conduct biochemical studies in vitro, membrane proteins (MPs) must be solubilized with detergents. While detergents are great tools, they can also inhibit the...
To conduct biochemical studies in vitro, membrane proteins (MPs) must be solubilized with detergents. While detergents are great tools, they can also inhibit the biological activity and/or perturb oligomerization of individual MPs. Nanodisc scaffold peptide (NSP), an amphipathic peptide analog of ApoA1, was recently shown to reconstitute detergent solubilized MPs into peptidiscs in vitro. Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1), also known as sterol O-acyltransferase 1 (SOAT1), plays a key role in cellular cholesterol storage in various cell types and is a drug target to treat multiple human diseases. ACAT1 contains nine transmembrane domains (TMDs) and primarily forms a homotetramer in vitro and in intact cells; deletion of the N-terminal dimerization domain produces a homodimer with full retention in catalytic activity. ACAT1 is prone to inactivation by numerous detergents. Here we pursued the use of NSP to overcome the detergent-induced inactivation of ACAT1 by generating near detergent-free ACAT1 peptidiscs. Based on native-PAGE analysis, we showed that NSP reconstitutes ACAT1 into soluble peptidiscs, in which ACAT1 exists predominantly in oligomeric states greater than a homotetramer. The formation of these higher-order oligomeric states was independent of the N-terminal dimerization domain, suggesting that the oligomerization is mediated through hydrophobic interactions of multiple ACAT1 subunits. ACAT1 peptidiscs were still susceptible to heat-mediated inactivation, presumably due to the residual detergent (CHAPS) bound to ACAT1. We then conditioned ACAT1 with phosphatidylcholine (PC) to replace CHAPS prior to the formation of ACAT1 peptidiscs. The results showed, when PC was included, ACAT1 was present mainly in higher-order oligomeric states with greater enzymatic activity. With PC present, the enzymatic activity of ACAT1 peptidiscs was protected from heat-mediated inactivation. These results support the use of NSP to create a near detergent-free solution of ACAT1 in peptidiscs for various in vitro studies. Our current results also raise the possibility that, under certain conditions, ACAT1 may form higher-order oligomeric states in vivo.
Topics: Amino Acid Sequence; Animals; CHO Cells; Cholic Acids; Cricetulus; Detergents; Digitonin; Humans; Peptides; Protein Domains; Protein Multimerization; Sterol O-Acyltransferase; Surface-Active Agents
PubMed: 32735863
DOI: 10.1016/j.abb.2020.108518 -
Biochimica Et Biophysica Acta Mar 1996The effects of the glycoalkaloids alpha-solanine, alpha-chaconine and alpha-tomatine on different cell types were studied in order to investigate the membrane action of...
The effects of the glycoalkaloids alpha-solanine, alpha-chaconine and alpha-tomatine on different cell types were studied in order to investigate the membrane action of these compounds. Hemolysis of erythrocytes was compared to 6-carboxyfluorescein leakage from both ghosts and erythrocyte lipid vesicles, whereas leakage of enzymes from mitochondria and the apical and baso-lateral side of Caco-2 cells was determined. Furthermore, the effects of glycoalkaloids on the gap-junctional communication between Caco-2 cells was studied. From these experiments, it was found that glycoalkaloids specifically induced membrane disruptive effects of cholesterol containing membranes as was previously reported in model membrane studies. In addition, alpha-chaconine was found to selectively decrease gap-junctional intercellular communication. Furthermore, the glycoalkaloids were more potent in permeabilizing the outer membrane of mitochondria compared to digitonin at the low concentrations used.
Topics: Adenylate Kinase; Animals; Caco-2 Cells; Cell Membrane; Cell Membrane Permeability; Cholesterol; Digitonin; Erythrocyte Membrane; Erythrocytes; Fluoresceins; Fluorescent Dyes; Gap Junctions; Hemolysis; Humans; L-Lactate Dehydrogenase; Male; Mitochondria, Liver; Rats; Rats, Wistar; Solanaceous Alkaloids
PubMed: 8603093
DOI: 10.1016/0005-2736(95)00253-7