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The Biochemical Journal May 1999The tetraspans are molecules with four transmembrane domains which are engaged in multimolecular complexes (the tetraspan web) containing a subset of beta1 integrins (in...
The tetraspans are molecules with four transmembrane domains which are engaged in multimolecular complexes (the tetraspan web) containing a subset of beta1 integrins (in particular alpha3beta1, alpha4beta1 and alpha6beta1), MHC antigens and several unidentified molecules. The molecules associated with tetraspans are readily detected after immunoprecipitation performed in mild detergents such as Brij 97 or CHAPS. In this study we show that another classical mild detergent, digitonin, dissociated most of these associated molecules, including integrins, from the tetraspans CD9, CD37, CD53, CD63, CD82, Co-029, Talla-1 and NAG-2. In contrast, reciprocal immunoprecipitations from various cell lines demonstrated that two other tetraspans, CD81 and CD151, formed complexes with integrins not disrupted by digitonin. These complexes were CD81/alpha4beta1, CD151/alpha3beta1 and CD151/alpha6beta1. Furthermore, a new anti-CD151 monoclonal antibody (mAb), TS151r, was shown to have a restricted pattern of expression, inversely related to the sum of the levels of expression of alpha6beta1 and alpha3beta1. This mAb was unable to co-precipitate integrins in digitonin, suggesting that its epitope is blocked by the association with integrins. Indeed, the binding of TS151r to the cell surface was quantitatively diminished following alpha3beta1 overexpression. Altogether, these data suggest that, among tetraspans, CD81 interacts directly with the integrin alpha4beta1, and CD151 interacts directly with integrins alpha3beta1 and alpha6beta1. Because all tetraspan-tetraspan associations are disrupted by digitonin, it is likely that the other tetraspans interact indirectly with integrins, through interactions with CD81 or CD151.
Topics: Animals; Antibodies, Monoclonal; Antigens, CD; Blood Cells; Cell Adhesion; Cell Line; Digitonin; Epithelial Cells; Epitopes; Humans; Integrin alpha3beta1; Integrin alpha4beta1; Integrin alpha6beta1; Integrins; Membrane Proteins; Mice; Mice, Inbred BALB C; Molecular Weight; Precipitin Tests; Protein Binding; Receptors, Lymphocyte Homing; Tetraspanin 24; Tetraspanin 28
PubMed: 10229664
DOI: No ID Found -
The Journal of Biological Chemistry Apr 1988Livers of starved rats refed for 2 h were perfused in situ by a modification of the dual digitonin pulse technique of Quistorff and Grunnet (Quistorff, B., and Grunnet,...
Livers of starved rats refed for 2 h were perfused in situ by a modification of the dual digitonin pulse technique of Quistorff and Grunnet (Quistorff, B., and Grunnet, N. (1987) Biochem. J. 243, 87-95). A pulse of digitonin (2 mg/ml) was infused first antegrade through the portal vein followed retrograde through the vena cava, or in reverse order, 13 mg of digitonin per zone. Microscopic examination showed that this procedure permeabilized the periportal and perivenous zones of the liver without overlap, with a narrow unaffected band of hepatocytes between the zones. The distribution pattern between periportal and perivenous zones ratio for alanine transaminase, lactate hydrogenase, fructose-1,6-bisphosphatase, and phosphoenolpyruvate carboxykinase ranged from 1.5 to 3. Glucokinase activity was higher in the perivenous zone (periportal/perivenous ratio of 0.7) and glutamine synthetase was exclusively present in that zone. Fructose 2,6-bisphosphate concentration was nearly equal in the two zones.
Topics: Alanine Transaminase; Animals; Digitonin; Fructose-Bisphosphatase; Glutamate-Ammonia Ligase; Histocytochemistry; L-Lactate Dehydrogenase; Liver; Male; Rats
PubMed: 2895107
DOI: No ID Found -
Journal of Neurochemistry Dec 2011Mitochondrial outer membrane Bax oligomers are critical for cytochrome c release, but the role of resident mitochondrial proteins in this process remains unclear.... (Comparative Study)
Comparative Study
Mitochondrial outer membrane Bax oligomers are critical for cytochrome c release, but the role of resident mitochondrial proteins in this process remains unclear. Membrane-associated Bax has primarily been studied using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) as the solubilizing agent, as it does not induce conformational artifacts, although recent evidence indicates it may have other artifactual effects. The objective of this study was to investigate digitonin as an alternative detergent to assess Bax oligomeric state, and possible interaction with voltage-dependent anion channel (VDAC)1 in cerebellar granule neurons. VDAC1 co-immunoprecipitated with Bax in digitonin extracts from healthy and apoptotic neurons. Two-dimensional blue native-SDS-PAGE revealed five Bax and VDAC1 oligomers having similar masses from 120 to 500 kDa. The levels of two VDAC1 oligomers in Bax 1D1 immunodepleted extracts negatively correlated with levels of co-precipitated VDAC1, indicating the co-precipitated VDAC1 was derived from these oligomers. Immunodepletion with the 6A7 antibody modestly reduced the levels of Bax oligomers from apoptotic but not healthy neurons. A sixth 170 kDa oligomer containing exclusively 6A7 Bax and no VDAC1 was identified after apoptosis induction. CHAPS failed to solubilize VDAC1, and additionally yielded no distinct oligomers. We conclude that digitonin is a potentially useful detergent preserving Bax-VDAC1 interactions that may be disrupted with CHAPS.
Topics: Animals; Animals, Newborn; Cerebellar Cortex; Cholic Acids; Cytoplasmic Granules; Digitonin; Neurons; Primary Cell Culture; Rats; Rats, Wistar; Solubility; Voltage-Dependent Anion Channel 1; bcl-2-Associated X Protein
PubMed: 21951169
DOI: 10.1111/j.1471-4159.2011.07499.x -
Journal of Biochemistry Jan 1998Adenylate kinase (AK) is a ubiquitous enzyme that contributes to the homeostasis of the cellular adenine nucleotide composition. Three isozymes, AK1, AK2, and AK3, have...
Adenylate kinase (AK) is a ubiquitous enzyme that contributes to the homeostasis of the cellular adenine nucleotide composition. Three isozymes, AK1, AK2, and AK3, have so far been characterized in vertebrates. They are located in different tissues, while their primary and tertiary structures are similar. Among them, AK2 and AK3 are located in mitochondria, but unlike most mitochondrial proteins, both proteins lack a cleavable presequence. In this study, we first confirmed that AK2 is distributed in liver cells in both the cytosol and the intermembrane space of mitochondria, while AK3 is localized exclusively in the mitochondrial matrix. Next, we analyzed the process of import of AK2 and AK3 by incubating isolated rat mitochondria with proteins that were synthesized in a reticulocyte lysate translation system. The results indicated that both AK2 (an intermembrane-space-targeting protein) and AK3 (a matrix-targeting protein) require an inner membrane electrochemical potential for their import. This finding for AK2 is in contrast with those of other noncleavable intermembrane-space-targeting proteins such as cytochrome c and cytochrome c heme lyase, which do not require the membrane potential for their import. In the transport process, AK2 and AK3 competed with the adrenodoxin precursor, which is imported into the matrix through a mechanism common to other mitochondrial matrix proteins. Thus, AK2 and AK3 were thought to be translocated into mitochondria through the same pathway as that for most mitochondrial protein precursors. Neither AK2, that was previously synthesized in reticulocyte lysates, nor AK2, that was purified from an Escherichia coli overexpression system, was imported into mitochondria in a post-translational import manner. In contrast, AK3 was imported into mitochondria after completion of protein synthesis. Thus, the import of AK2 is likely to be co-translational, and the co-translational import mechanism might contribute to the bi-topological distribution of AK2 in both the cytosol and mitochondria.
Topics: Adenylate Kinase; Adrenodoxin; Animals; Binding, Competitive; Biological Transport; Cycloheximide; Digitonin; In Vitro Techniques; Indicators and Reagents; Intracellular Membranes; Isoenzymes; Membrane Potentials; Mitochondria, Liver; Protein Biosynthesis; Protein Precursors; Protein Synthesis Inhibitors; Rats; Rats, Wistar
PubMed: 9504419
DOI: 10.1093/oxfordjournals.jbchem.a021899 -
The Journal of Biological Chemistry Jul 2004Many proteins of the secretory pathway contain disulfide bonds that are essential for structure and function. In the endoplasmic reticulum (ER), Ero1 alpha and Ero1 beta...
Many proteins of the secretory pathway contain disulfide bonds that are essential for structure and function. In the endoplasmic reticulum (ER), Ero1 alpha and Ero1 beta oxidize protein disulfide isomerase (PDI), which in turn transfers oxidative equivalents to newly synthesized cargo proteins. However, oxidation must be limited, as some reduced PDI is necessary for disulfide isomerization and ER-associated degradation. Here we show that in semipermeable cells, PDI is more oxidized, disulfide bonds are formed faster, and high molecular mass covalent protein aggregates accumulate in the absence of cytosol. Addition of reduced glutathione (GSH) reduces PDI and restores normal disulfide formation rates. A higher GSH concentration is needed to balance oxidative folding in semipermeable cells overexpressing Ero1 alpha, indicating that cytosolic GSH and lumenal Ero1 alpha play antagonistic roles in controlling the ER redox. Moreover, the overexpression of Ero1 alpha significantly increases the GSH content in HeLa cells. Our data demonstrate tight connections between ER and cytosol to guarantee redox exchange across compartments: a reducing cytosol is important to ensure disulfide isomerization in secretory proteins.
Topics: Cytoplasm; Cytosol; Digitonin; Disulfides; Electrophoresis, Polyacrylamide Gel; Endoplasmic Reticulum; Genetic Vectors; Glutathione; Glycoproteins; HeLa Cells; Humans; Membrane Glycoproteins; Microscopy, Fluorescence; Oxidation-Reduction; Oxidoreductases; Oxygen; Plasmids; Protein Disulfide-Isomerases; Protein Folding; Protein Isoforms; Temperature; Time Factors; Transfection
PubMed: 15161913
DOI: 10.1074/jbc.M404992200 -
FEBS Letters Sep 1998Apoptosis, a naturally occurring programmed cell death or cell 'suicide', has been paid much attention as one of the critical mechanisms for morphogenesis and tissue...
Apoptosis, a naturally occurring programmed cell death or cell 'suicide', has been paid much attention as one of the critical mechanisms for morphogenesis and tissue remodeling. Activation of cysteine aspartases (caspases) is one of the critical steps leading to apoptosis. Although a mitochondria-mediated pathway has been postulated to be one of the activation mechanism of caspase-3, another subcellular compartment might be involved in the activation of the enzyme. The present study shows that the supernatant fraction of digitonin-treated lysosomes strongly activates Ac-DEVD-CHO inhibitable caspase-3-like protease. Activation of caspase-3-like protease by digitonin-treated lysosomal fractions was specifically suppressed by leupeptin and E-64, inhibitors of cysteine protease. These results indicate that leakage of lysosomal cysteine protease(s) into the cytosolic compartment might be involved in the activation of caspase-3-like protease.
Topics: Animals; Apoptosis; Caspase 3; Caspases; Cysteine Endopeptidases; Digitonin; Enzyme Activation; Enzyme Inhibitors; Enzyme Precursors; Indicators and Reagents; Lysosomes; Rats; Rats, Wistar
PubMed: 9762916
DOI: 10.1016/s0014-5793(98)01080-1 -
Methods in Molecular Biology (Clifton,... 2013Real-time imaging coupled with a permeabilized cell system presents a very versatile platform to visualize the dynamic and intricate nature of nuclear envelope...
Real-time imaging coupled with a permeabilized cell system presents a very versatile platform to visualize the dynamic and intricate nature of nuclear envelope breakdown, one of the major morphological changes of mitosis. Here, we describe such a strategy in which the plasma membrane of cells expressing fluorescently tagged nucleoporin POM121 and Histone H2B is permeabilized with digitonin. These cells are then incubated with mitotic Xenopus egg extract to create conditions that recapitulate the major events of mitotic nuclear remodeling seen in live-cell imaging, providing the opportunity to probe mechanisms and pathways that coordinate nuclear disassembly.
Topics: Animals; Buffers; Cell Culture Techniques; Cell Extracts; Digitonin; HeLa Cells; Humans; Indicators and Reagents; Nuclear Envelope; Oocytes; Permeability; Single-Cell Analysis; Time-Lapse Imaging; Xenopus
PubMed: 23027000
DOI: 10.1007/978-1-62703-056-4_6 -
Zeitschrift Fur Naturforschung. C,... 2000Contrary to Leishmania spp. and Trypanosoma cruzi, Trypanosoma brucei bloodstream forms do not synthesise their own sterols but take these compounds in the form of...
Contrary to Leishmania spp. and Trypanosoma cruzi, Trypanosoma brucei bloodstream forms do not synthesise their own sterols but take these compounds in the form of cholesterol directly from the mammalian host. However, procyclic insect stages synthesise ergosterol rather than cholesterol. Here the sub-cellular localisation of the first committed enzyme of this pathway of isoprenoid synthesis 3-hydroxy-3-methylglutaryl-coenzyme A reductase in T. brucei procyclics (0.9 nmol x min(-1) x mg(-1) protein) was carried out using both cell-fractionation by isopycnic centrifugation and digitonin-titration experiments. The majority of the NADP+-linked 3-hydroxy-3-methylglutaryl-coenzyme A reductase is a soluble enzyme present in the mitochondrial matrix with some additional membrane-associated activity in glycosomes and possibly in the endoplasmic reticulum. It is suggested that the active metabolism of threonine and/or leucine as preferred 2-carbon source for the incorporation of acetyl units into lipids and/or sterols in the mitochondrion of T. brucei procyclics is the explanation for a high 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in these protozoan organelles.
Topics: Animals; Cell Fractionation; Centrifugation, Density Gradient; Digitonin; Hydroxymethylglutaryl CoA Reductases; Kinetics; Mitochondria; Trypanosoma brucei brucei
PubMed: 10928562
DOI: 10.1515/znc-2000-5-626 -
Biochimica Et Biophysica Acta Oct 2015Recently we found that cytoplasm of permeabilized mammalian cells behaves as a hydrogel displaying intrinsic osmosensitivity. This study examined the role of...
Recently we found that cytoplasm of permeabilized mammalian cells behaves as a hydrogel displaying intrinsic osmosensitivity. This study examined the role of microfilaments and microtubules in the regulation of hydrogel osmosensitivity, volume-sensitive ion transporters, and their contribution to volume modulation of intact cells. We found that intact and digitonin-permeabilized A549 cells displayed similar rate of shrinkage triggered by hyperosmotic medium. It was significantly slowed-down in both cell preparations after disruption of actin microfilaments by cytochalasin B, suggesting that rapid water release by intact cytoplasmic hydrogel contributes to hyperosmotic shrinkage. In hyposmotic swelling experiments, disruption of microtubules by vinblastine attenuated the maximal amplitude of swelling in intact cells and completely abolished it in permeabilized cells. The swelling of intact cells also triggered ~10-fold elevation of furosemide-resistant (86)Rb+ (K+) permeability and the regulatory volume decrease (RVD), both of which were abolished by Ba2+. Interestingly, RVD and K+ permeability remained unaffected in cytocholasin/vinblastine treated cells demonstrating that cytoskeleton disruption has no direct impact on Ba2+-sensitive K+-channels involved in RVD. Our results show, for the first time, that the cytoskeleton network contributes directly to passive cell volume adjustments in anisosmotic media via the modulation of the water retained by the cytoplasmic hydrogel.
Topics: Cell Line, Tumor; Cell Membrane Permeability; Cell Size; Cytoskeleton; Digitonin; Humans; Lung Neoplasms; Osmotic Pressure
PubMed: 26171817
DOI: 10.1016/j.bbamem.2015.07.005 -
The Journal of Biological Chemistry Jan 2004Mobile light-harvesting complex II (LHCII) is implicated in the regulation of excitation energy distribution between Photosystem I (PSI) and Photosystem II (PSII) during...
Mobile light-harvesting complex II (LHCII) is implicated in the regulation of excitation energy distribution between Photosystem I (PSI) and Photosystem II (PSII) during state transitions. To investigate how LHCII interacts with PSI during state transitions, PSI was isolated from Arabidopsis thaliana plants treated with PSII or PSI light. The PSI preparations were made using digitonin. Chemical cross-linking using dithio-bis(succinimidylpropionate) followed by diagonal electrophoresis and immunoblotting showed that the docking site of LHCII (Lhcb1) on PSI is comprised of the PSI-H, -L, and -I subunits. This was confirmed by the lack of energy transfer from LHCII to PSI in the digitonin-PSI isolated from plants lacking PSI-H and -L. Digitonin-PSI was purified further to obtain an LHCII.PSI complex, and two to three times more LHCII was associated with PSI in the wild type in State 2 than in State 1. Lhcb1 was also associated with PSI from plants lacking PSI-K, but PSI from PSI-H, -L, or -O mutants contained only about 30% of Lhcb1 compared with the wild type. Surprisingly, a significant fraction of the LHCII bound to PSI in State 2 was not phosphorylated. Cross-linking prior to sucrose gradient purification resulted in copurification of phosphorylated LHCII in the wild type, but not with PSI from the PSI-H, -L, and -O mutants. The data suggest that migration of LHCII during state transitions cannot be explained sufficiently by different affinity of phosphorylated and unphosphorylated LHCII for PSI but is likely to involve structural changes in thylakoid organization.
Topics: Arabidopsis; Binding Sites; Centrifugation, Density Gradient; Cross-Linking Reagents; Detergents; Digitonin; Immunoblotting; Light; Light-Harvesting Protein Complexes; Mutation; Peptides; Phosphates; Phosphorylation; Phosphothreonine; Photosystem I Protein Complex; Plant Leaves; Protein Binding; Sucrose; Thylakoids; Time Factors
PubMed: 14617624
DOI: 10.1074/jbc.M311640200