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The EMBO Journal Apr 2000Around 30-40 years after the first isolation of the five complexes of oxidative phosphorylation from mammalian mitochondria, we present data that fundamentally change...
Around 30-40 years after the first isolation of the five complexes of oxidative phosphorylation from mammalian mitochondria, we present data that fundamentally change the paradigm of how the yeast and mammalian system of oxidative phosphorylation is organized. The complexes are not randomly distributed within the inner mitochondrial membrane, but assemble into supramolecular structures. We show that all cytochrome c oxidase (complex IV) of Saccharomyces cerevisiae is bound to cytochrome c reductase (complex III), which exists in three forms: the free dimer, and two supercomplexes comprising an additional one or two complex IV monomers. The distribution between these forms varies with growth conditions. In mammalian mitochondria, almost all complex I is assembled into supercomplexes comprising complexes I and III and up to four copies of complex IV, which guided us to present a model for a network of respiratory chain complexes: a 'respirasome'. A fraction of total bovine ATP synthase (complex V) was isolated in dimeric form, suggesting that a dimeric state is not limited to S.cerevisiae, but also exists in mammalian mitochondria.
Topics: ATP Synthetase Complexes; Animals; Cattle; Detergents; Digitonin; Electron Transport; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Mitochondria; Mitochondria, Heart; Models, Biological; Multienzyme Complexes; Oxidative Phosphorylation; Phosphotransferases (Phosphate Group Acceptor); Protein Binding; Saccharomyces cerevisiae
PubMed: 10775262
DOI: 10.1093/emboj/19.8.1777 -
Methods (San Diego, Calif.) Jun 2013Rapid analysis of a cell's propensity to undergo apoptosis through the mitochondrial pathway is hindered by the complex network of interactions between more than fifteen...
Rapid analysis of a cell's propensity to undergo apoptosis through the mitochondrial pathway is hindered by the complex network of interactions between more than fifteen known members of the BCL2 family that govern the decision to undergo mitochondrial apoptosis, and measurement of protein levels alone fails to account for critical interactions between the proteins. To address this issue, we have developed two functional assays for same-day analysis of cell lines or primary tissue samples. Using defined inputs in the form of peptides derived primarily from the BH3 domains of pro-apoptotic members of the BCL2 family, we invoke a response in the mitochondria in the form of mitochondrial outer membrane permeabilization measured indirectly using potential sensitive dyes. BH3 profiling can be applied to any viable single cell suspension and provides a response from the sum total of all known and unknown interactions within the BCL2 family for each stimulus, and the pattern of response can provide both a cell's propensity towards mitochondrial apoptosis, or 'priming', as well as indicate dependencies on specific anti-apoptotic proteins. Described here are optimized conditions for both plate-based and FACS-based BH3 profiling for homogeneous and heterogeneous samples.
Topics: Apoptosis; Cell Line, Tumor; Digitonin; Flow Cytometry; Fluorescent Dyes; Fluorometry; Gene Expression Regulation; Humans; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Membranes; Peptide Fragments; Permeability; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2
PubMed: 23607990
DOI: 10.1016/j.ymeth.2013.04.006 -
Journal of Lipid Research Dec 2001The transport of apolipoprotein B (apoB) between the endoplasmic reticulum (ER) and Golgi was studied in puromycin-synchronized HepG2 cells, using an antibody that could...
The transport of apolipoprotein B (apoB) between the endoplasmic reticulum (ER) and Golgi was studied in puromycin-synchronized HepG2 cells, using an antibody that could distinguish between apoB in ER and Golgi compartments. In cells with normal ER-to-Golgi transport, both albumin and apoB colocalized throughout the ER and appeared as intense, compact signals in Golgi. When ER-to-Golgi transport was blocked with brefeldin A, apoB and albumin remained colocalized in the ER network and three-dimensional constructed images showed more intense signals for both proteins in a central, perinuclear region of the ER. When protein synthesis was stopped in cells with brefeldin A-inhibited ER-to-Golgi transport, apoB degradation was visualized as a homogeneous decrease in fluorescence signal intensity throughout the ER that could be slowed with clasto-lactacystin beta-lactone, a proteasome inhibitor. Incubation of cells with CP-10447, an inhibitor of microsomal triglyceride transfer protein, inhibited apoB, but not albumin, transport from ER to Golgi. Nanogold immunoelectron microscopy of digitonin-permeabilized cells showed proteasomes in close proximity to the cytosolic side of the ER membrane. Thus, newly synthesized apoB is localized throughout the entire ER and degraded homogeneously, most likely by neighboring proteasomes located on the cytosolic side of the ER membrane. Although albumin is colocalized with apoB in the ER, as expected, it was not targeted for ER-associated proteasomal degradation.
Topics: Albumins; Apolipoprotein B-100; Apolipoproteins B; Brefeldin A; Cell Membrane Permeability; Cysteine Endopeptidases; Digitonin; Endoplasmic Reticulum; Golgi Apparatus; Humans; Microscopy, Electron; Microscopy, Fluorescence; Multienzyme Complexes; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Protein Transport; Puromycin; Tumor Cells, Cultured
PubMed: 11734567
DOI: No ID Found -
The Biochemical Journal Dec 1980The ratio of ATP content/ADP content in the mitochondrial matrix was found to be 2.07 +/- 0.21 and 2.26 +/- 0.22 as determined with six different preparations of... (Comparative Study)
Comparative Study
The ratio of ATP content/ADP content in the mitochondrial matrix was found to be 2.07 +/- 0.21 and 2.26 +/- 0.22 as determined with six different preparations of isolated hepatocytes subfractionated with the digitonin and non-aqueous-fractionation procedures, respectively. In contrast, the mitochondrial matrix ATP/ADP determined with isolated haemoglobin-free perfused liver by using the non-aqueous-fractionation procedure was about 0.2, whereas the cytosolic values obtained with isolated cells and with the intact organ were similar. It is concluded that the relatively higher ATP/ADP ratio in the mitochondrial matrix of isolated hepatocytes represents a biochemical difference due to properties of the model rather than a methodological artifact.
Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Carbon Tetrachloride; Cell Fractionation; Cytosol; Digitonin; Heptanes; Liver; Male; Mitochondria, Liver; Rats
PubMed: 7236247
DOI: 10.1042/bj1920951 -
European Journal of Biochemistry Jun 1991Digitonin permeabilization of hepatocytes from control and clofibrate-treated (0.5% by mass, 10 days) male C57bl/6 mice was used to study the intracellular distributions...
Studies on the intracellular distributions of soluble epoxide hydrolase and of catalase by digitonin-permeabilization of hepatocytes isolated from control and clofibrate-treated mice.
Digitonin permeabilization of hepatocytes from control and clofibrate-treated (0.5% by mass, 10 days) male C57bl/6 mice was used to study the intracellular distributions of soluble ('cytosolic') epoxide hydrolase and of catalase. The following conclusions were drawn. (1) About 60% of the total soluble epoxide hydrolase activity in control mouse hepatocytes is situated in the cytosol. (2) The rest is not mitochondrial, but probably peroxisomal. (3) Of the total catalase activity in control mouse hepatocytes, 5-10% is found in the cytosol. (4) Treatment of mice with clofibrate increases the total hepatocyte activity of soluble epoxide hydrolase 4-fold, but does not influence the relative distribution of this enzyme between cytosol and peroxisomes. (5) The total catalase activity is increased 3.5-fold by clofibrate treatment and 15-35% of this activity is shifted from the peroxisomes to the cytosol.
Topics: Animals; Catalase; Cell Membrane Permeability; Cells, Cultured; Clofibrate; Digitonin; Epoxide Hydrolases; Kinetics; Liver; Male; Mice; Mice, Inbred C57BL; Reference Values; Subcellular Fractions
PubMed: 2040306
DOI: 10.1111/j.1432-1033.1991.tb16037.x -
The Journal of Biological Chemistry Nov 2000Synaptotagmins (Syt) play important roles in Ca(2+)-induced neuroexocytosis. Insulin secretion of the pancreatic beta-cell is dependent on an increase in intracellular...
Synaptotagmins (Syt) play important roles in Ca(2+)-induced neuroexocytosis. Insulin secretion of the pancreatic beta-cell is dependent on an increase in intracellular Ca(2+); however, Syt involvement in insulin exocytosis is poorly understood. Reverse transcriptase-polymerase chain reaction studies showed the presence of Syt isoforms III, IV, V, and VII in rat pancreatic islets, whereas Syt isoforms I, II, III, IV, V, VII, and VIII were present in insulin-secreting betaTC3 cell. Syt III and VII proteins were identified in rat islets and betaTC3 and RINm5F beta-cells by immunoblotting. Confocal microscopy showed that Syt III and VII co-localized with insulin-containing secretory granules. Two-fold overexpression of Syt III in RINm5F beta-cell (Syt III cell) was achieved by stable transfection, which conferred greater Ca(2+) sensitivity for exocytosis, and resulted in increased insulin secretion. Glyceraldehyde + carbachol-induced insulin secretion in Syt III cells was 2.5-fold higher than control empty vector cells, whereas potassium-induced secretion was 6-fold higher. In permeabilized Syt III cells, Ca(2+)-induced and mastoparan-induced insulin secretion was also increased. In Syt VII-overexpressing RINm5F beta-cells, there was amplification of carbachol-induced insulin secretion in intact cells and of Ca(2+)-induced and mastoparan-induced insulin secretion in permeabilized cells. In conclusion, Syt III/VII are located in insulin-containing secretory granules, and we suggest that Syt III/VII may be the Ca(2+) sensor or one of the Ca(2+) sensors for insulin exocytosis of the beta-cell.
Topics: Animals; Bacterial Proteins; Blotting, Western; Calcium; Calcium-Binding Proteins; Carbachol; Cell Line; Cell Membrane Permeability; Digitonin; Exocytosis; Fluorescent Antibody Technique; Glutaral; Insulin; Insulin Secretion; Islets of Langerhans; Membrane Glycoproteins; Nerve Tissue Proteins; Protein Isoforms; Protein Transport; RNA, Messenger; Rats; Streptolysins; Synaptotagmins; Transfection
PubMed: 10938083
DOI: 10.1074/jbc.M004284200 -
The Journal of Biological Chemistry Mar 1999Many substrates for P-glycoprotein, an ABC transporter that mediates multidrug resistance in mammalian cells, have been shown to stimulate its ATPase activity in vitro....
Many substrates for P-glycoprotein, an ABC transporter that mediates multidrug resistance in mammalian cells, have been shown to stimulate its ATPase activity in vitro. In the present study, we used this property as a criterion to search for natural and artificial substrates and/or allosteric regulators of ABCR, the rod photoreceptor-specific ABC transporter responsible for Stargardt disease, an early onset macular degeneration. ABCR was immunoaffinity purified to apparent homogeneity from bovine rod outer segments and reconstituted into liposomes. All-trans-retinal, a candidate ligand, stimulates the ATPase activity of ABCR 3-4-fold, with a half-maximal effect at 10-15 microM. 11-cis- and 13-cis-retinal show similar activity. All-trans-retinal stimulates the ATPase activity of ABCR with Michaelis-Menten behavior indicative of simple noncooperative binding that is associated with a rate-limiting enzyme-substrate intermediate in the pathway of ATP hydrolysis. Among 37 structurally diverse non-retinoid compounds, including nine previously characterized substrates or sensitizers of P-glycoprotein, only four show significant ATPase stimulation when tested at 20 microM. The dose-response curves of these four compounds are indicative of multiple binding sites and/or modes of interaction with ABCR. Two of these compounds, amiodarone and digitonin, can act synergistically with all-trans-retinal, implying that they interact with a site or sites on ABCR different from the one with which all-trans-retinal interacts. Unlike retinal, amiodarone appears to interact with both free and ATP-bound ABCR. Together with clinical observations on Stargardt disease and the localization of ABCR to rod outer segment disc membranes, these data suggest that retinoids, and most likely retinal, are the natural substrates for transport by ABCR in rod outer segments. These observations have significant implications for understanding the visual cycle and the pathogenesis of Stargardt disease and for the identification of compounds that could modify the natural history of Stargardt disease or other retinopathies associated with impaired ABCR function.
Topics: ATP-Binding Cassette Transporters; Adenosine Triphosphatases; Adenosine Triphosphate; Amiodarone; Animals; Carbohydrate Sequence; Cattle; Digitonin; Hydrolysis; In Vitro Techniques; Kinetics; Macular Degeneration; Models, Chemical; Molecular Sequence Data; Molecular Weight; Norisoprenoids; Retinaldehyde; Rod Cell Outer Segment; Terpenes
PubMed: 10075733
DOI: 10.1074/jbc.274.12.8269 -
Journal of Neurochemistry Aug 1997Isolated rat CNS mitochondria and cultured cortical astrocytes were examined for behavior indicative of a mitochondrial permeability transition (mPT). Exposure of...
Isolated rat CNS mitochondria and cultured cortical astrocytes were examined for behavior indicative of a mitochondrial permeability transition (mPT). Exposure of isolated CNS mitochondria to elevated calcium or phosphate or both produced loss of absorbance indicative of mitochondrial swelling. The absorbance decreases were prevented by ADP and Mg2+ and reduced by cyclosporin A, dithiothreitol, and N-ethylmaleimide. Ruthenium red prevented calcium cycling-induced, but only attenuated phosphate-induced losses of absorbance. In cultured astrocytes permeabilized with digitonin or treated with the calcium ionophore, 4-bromo-A23187, elevations of external calcium altered mitochondrial morphology visualized with the dye, JC-1, from rod-like to rounded, swollen structures. Similar changes were observed in digitonin-permeabilized astrocytes exposed to phosphate. The incidence of calcium-induced changes in astrocyte mitochondria was prevented by Mg2+ and pretreatment with dithiothreitol and N-ethylmaleimide, and was reduced by cyclosporin A, ADP, and butacaine alone or in combinations. Ruthenium red and the Na+/Ca2+ exchange inhibitor CGP 37157 blocked calcium cycling and prevented mitochondrial shape changes in digitonin-treated, but not ionophore-treated astrocytes. Thus, the demonstrated induction conditions and pharmacological profile indicated the existence of an mPT in brain mitochondria. The mPT occurred consequent to activation of calcium cycling-dependent and -independent pathways. Induction of an mPT could contribute to neuronal injury following ischemia and reperfusion.
Topics: Adenosine Diphosphate; Animals; Astrocytes; Brain; Calcimycin; Calcium; Cells, Cultured; Cyclosporine; Digitonin; Ionophores; Magnesium; Male; Mitochondria; Mitochondrial Swelling; Permeability; Phosphates; Rats; Rats, Inbred F344; Spectrophotometry
PubMed: 9231710
DOI: 10.1046/j.1471-4159.1997.69020524.x -
The Journal of Biological Chemistry Jun 1982An 18O exchange method has been used to determine the location of carbonic anhydrase in mitochondria from rat liver and to examine the role of this enzyme in the...
An 18O exchange method has been used to determine the location of carbonic anhydrase in mitochondria from rat liver and to examine the role of this enzyme in the kinetics of CO2 in resting and respiring mitochondria. Using digitonin subfractionation, we have determined that a substantial fraction, 40 to 60%, of the carbonic anhydrase activity in the mitochondrion from rat liver is located in the space between the inner and outer membranes; the remaining activity was found in the matrix with no detectable activity in the sedimented membranes. The total catalytic CO2 hydration activity measured in intact mitochondria from rat liver was about 1% of that found in an equal volume of rat erythrocytes. The apparent permeability constant representing the barrier for the diffusion of HCO3(-) from external solution to intramitochondrial carbonic anhydrase, 9 X 10(-5) cm s-1, is near in magnitude to the permeability constant for the diffusion of HCO3(-) across the rat erythrocyte membrane, 4 X 10(-4) cm s-2. Calcium-induced respiratory jumps were shown to cause changes in the rate of 18O exchange between CO2 and H2O that were consistent with a net uptake of CO2 by the mitochondria.
Topics: Animals; Carbonic Anhydrases; Cell Fractionation; Digitonin; Kinetics; Male; Mitochondria, Liver; Oxygen Isotopes; Rats; Rats, Inbred Strains
PubMed: 6806256
DOI: No ID Found -
Acta Veterinaria Scandinavica 1973A survey is given of presently known mycoplasmas of bovine source. Media and methods of cultivation are described. Cholesterol dependence, being the basis of division of...
A survey is given of presently known mycoplasmas of bovine source. Media and methods of cultivation are described. Cholesterol dependence, being the basis of division of Mycoplasmatales into Mycoplasmataceae and Acholeplasmataceae, was examined directly and also indirectly employing sensitivity tests to digitonin and sodium polyanethole sulphonate (SPS). All by now recognized species were found to be correctly classified in Mycoplasma and Acholeplasma, respectively. Of the unnamed “serogroups” 2 should be classified in the latter genus, while 6 serogroups were members of the genus Mycoplasma. Correlation was found between the digitonin test and the direct determination of cholesterol requirement, whereas this was not the case with the SPS test.
Topics: Animals; Anisoles; Atmospheric Pressure; Bacteriological Techniques; Cattle; Cholesterol; Culture Media; Digitonin; Drug Resistance, Microbial; Filtration; Methods; Mycoplasma; Polymers; Sulfonic Acids
PubMed: 4586124
DOI: 10.1186/BF03547431