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The Biochemical Journal May 19651. The excessive accumulation of Ca(2+) by mitochondria suspended in an iso-osmotic buffered potassium chloride medium containing oxidizable substrate and phosphate led...
1. The excessive accumulation of Ca(2+) by mitochondria suspended in an iso-osmotic buffered potassium chloride medium containing oxidizable substrate and phosphate led to extensive swelling and release of accumulated Ca(2+) from the mitochondria. When the Ca(2+) was removed from the medium by chelation with ethylene glycol bis(aminoethyl)tetra-acetate, the swelling was reversed in a respiration-dependent contraction. The contracted mitochondria were shown to have regained some degree of respiratory control. 2. The respiration-dependent contraction could be supported by electron transport through a restricted portion of the respiratory chain, and by substrates donating electrons at different levels in the respiratory chain. 3. Respiratory inhibitors appropriate to the substrate present completely inhibited the contraction. Uncoupling agents, and the inhibitors oligomycin and atractyloside, were without effect. 4. When the reversal of swelling had been prevented by respiratory inhibitors, the addition of ATP induced a contraction of the mitochondria. In the absence of added chelating agent the contraction was very slow. The ATP-induced contraction was completely inhibited by oligomycin and atractyloside, was incomplete in the presence of uncoupling agents and was unaffected by respiratory inhibitors. 5. The relationship between the energy requirements of respiration-dependent contraction and the requirements of ion transport and other contractile systems are discussed.
Topics: Adenosine Triphosphate; Calcium; Cell Respiration; Chelating Agents; Dinitrophenols; Electron Transport; Ions; Liver; Metabolism; Mitochondria; Mitochondria, Liver; Oligomycins; Osmosis; Pharmacology; Phosphates; Research; Uncoupling Agents; Water-Electrolyte Balance
PubMed: 14340089
DOI: 10.1042/bj0950387 -
Proceedings of the National Academy of... Dec 2009The structure of the complex between bovine mitochondrial F(1)-ATPase and a stator subcomplex has been determined at a resolution of 3.2 A. The resolved region of the...
The structure of the complex between bovine mitochondrial F(1)-ATPase and a stator subcomplex has been determined at a resolution of 3.2 A. The resolved region of the stator contains residues 122-207 of subunit b; residues 5-25 and 35-57 of F(6); 3 segments of subunit d from residues 30-40, 65-74, and 85-91; and residues 1-146 and 169-189 of the oligomycin sensitivity conferral protein (OSCP). The stator subcomplex represents its membrane distal part, and its structure has been augmented with an earlier structure of a subcomplex containing residues 79-183, 3-123, and 5-70 of subunits b, d, and F(6), respectively, which extends to the surface of the inner membrane of the mitochondrion. The N-terminal domain of the OSCP links the stator with F(1)-ATPase via alpha-helical interactions with the N-terminal region of subunit alpha(E). Its C-terminal domain makes extensive helix-helix interactions with the C-terminal alpha-helix of subunit b from residues 190-207. Subunit b extends as a continuous 160-A long alpha-helix from residue 188 back to residue 79 near to the surface of the inner mitochondrial membrane. This helix appears to be stiffened by other alpha-helices in subunits d and F(6), but the structure can bend inward toward the F(1) domain around residue 146 of subunit b. The linker region between the 2 domains of the OSCP also appears to be flexible, enabling the stator to adjust its shape as it passes over the changing profile of the F(1) domain during a catalytic cycle. The structure of the membrane extrinsic part of bovine ATP synthase is now complete.
Topics: Animals; Cattle; Mitochondrial Proton-Translocating ATPases; Models, Molecular; Oligomycins; Protein Conformation
PubMed: 19995987
DOI: 10.1073/pnas.0910365106 -
The Biochemical Journal Mar 1977Ligand-binding studies with labelled triethyltin on yeast mitochondrial membranes showed the presence of high-affinity sites (KD = 0.6 micronM; 1.2 +/- 0.3 nmol/mg of...
Ligand-binding studies with labelled triethyltin on yeast mitochondrial membranes showed the presence of high-affinity sites (KD = 0.6 micronM; 1.2 +/- 0.3 nmol/mg of protein) and low-affinity sites (KD less than 45 micronM; 70 +/- 20 nmol/mg of protein). The dissociation constant of the high-affinity site is in good agreement with the concentration of triethyltin required for inhibition of mitochondrial ATPase (adenosine triphosphatase) and oxidative phosphorylation. The high-affinity site is not competed for by oligomycin or venturicidin, indicating that triethyltin reacts at a different site from these inhibitors of oxidative phosphorylation. Fractionation of the mitochondrial membrane shows a specific association of the high-affinity sites with the ATP synthase complex. During purification of ATP synthase (oligomycin-sensitive ATPase) there is a 5-6-fold purification of oligomycin- and triethyltin-sensitive ATPase activity concomitant with a 7-9-fold increase in high-affinity triethyltin-binding sites. The purified yeast oligomycin-sensitive ATPase complex contains approximately six binding sites for triethyltin/mol of enzyme complex. It is concluded that specific triethyltin-binding sites are components of the ATP synthase complex, which accounts for the specific inhibition of ATPase and oxidative phosphorylation by triethyltin.
Topics: Adenosine Triphosphatases; Binding Sites; Membranes; Mitochondria; Oligomycins; Saccharomyces cerevisiae; Trialkyltin Compounds; Venturicidins
PubMed: 141273
DOI: 10.1042/bj1620575 -
The Journal of Physiology Sep 19661. The ability of the three nitrophenols, m-nitrophenol, p-nitrophenol and 2,4-dinitrophenol, to inhibit acid secretion and stimulate oxygen uptake by gastric mucosa...
1. The ability of the three nitrophenols, m-nitrophenol, p-nitrophenol and 2,4-dinitrophenol, to inhibit acid secretion and stimulate oxygen uptake by gastric mucosa paralleled their extent of dissociation at physiological pH.2. Oligomycin inhibited oxygen uptake by gastric mucosa, and showed a tendency to inhibit acid secretion.3. 2,4-dinitrophenol lowered the value of the ratio between acid secretion and the associated oxygen uptake. Oligomycin did not affect the value of this ratio.4. It would appear that a non-phosphorylated high-energy intermediate of oxidative phosphorylation might be involved in acid secretion by gastric mucosa.
Topics: Animals; Anura; Electrophysiology; Gastric Juice; Gastric Mucosa; Nitrophenols; Oligomycins; Oxidative Phosphorylation; Oximetry; Oxygen Consumption
PubMed: 5914259
DOI: 10.1113/jphysiol.1966.sp008021 -
The Journal of Biological Chemistry Jun 1998Apoptosis is a programmed form of cell death characterized by biochemical and morphological changes affecting the nucleus, cytoplasm, and plasma membrane. These changes...
Apoptosis is a programmed form of cell death characterized by biochemical and morphological changes affecting the nucleus, cytoplasm, and plasma membrane. These changes in various cellular compartments are widely regarded as mechanistically linked events in a single "program" in which activation of caspases and proteolysis of intracellular substrates represent a final common pathway leading to cell death. To date there has been very limited exploration of the linkage of this program to the plasma membrane changes, which bring about swift recognition, uptake, and safe degradation of apoptotic cells by phagocytes. Using the mitochondrial inhibitors antimycin A and oligomycin in human monocytic THP.1 cells triggered into apoptosis, we report the uncoupling of plasma membrane changes from other features of apoptosis. These inhibitors blocked increased plasma membrane permeability, externalization of phosphatidylserine, and recognition by two classes of phagocytes but not activation of caspase-3, cleavage of poly(ADP-ribose) polymerase and DNA fragmentation. Externalization of phosphatidylserine in apoptotic human leukemic U937 cells was also dissociated from caspase activation. Thus changes governing safe clearance of apoptotic cells may be regulated by an independent pathway to those bringing about caspase activation. This finding could have important consequences for attempts to manipulate cell death for therapeutic gain in vivo.
Topics: Amino Acid Chloromethyl Ketones; Antimycin A; Apoptosis; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; DNA Fragmentation; Humans; Intracellular Membranes; Membrane Potentials; Mitochondria; Oligomycins; Phagocytes; Phosphatidylserines; Surface Properties; Tumor Cells, Cultured
PubMed: 9624155
DOI: 10.1074/jbc.273.25.15628 -
The Journal of Biological Chemistry Mar 1976The mechanism by which fatty acid addition leads to the inactivation of pyruvate dehydrogenase in intact rat liver mitochondria was investigated. In all cases the fatty...
The mechanism by which fatty acid addition leads to the inactivation of pyruvate dehydrogenase in intact rat liver mitochondria was investigated. In all cases the fatty acid octanoate was added to mitochondria oxidizing succinate. Addition of fatty acid caused an inactivation of pyruvate dehydrogenase in mitochondria incubated under State 3 conditions (glucose plus hexokinase), in uncoupled, oligomycin-treated mitochondria, and in rotenone-menadione-treated mitochondria, but not in uncoupled mitochondria or in mitochondria incubated under State 4 conditions. A number of metabolic conditions were found in which pyruvate dehydrogenase was inactivated concomitant with an elevation in the ATP/ADP ratio. This is consistent with the inverse relationship between the ATP/ADP ratio and the pyruvate dehydrogenase activity proposed by various laboratories. However, in several other metabolic conditions pyruvate dehydrogenase was inactivated while the ATP/ADP ratio either was unchanged or even decreased. This observation implies that there are likely other regulatory factors involved in the fatty acid-mediated inactivation of pyruvate dehydrogenase. Incubation conditions in State 3 were found in which the ATP/ADP and the acetyl-CoA/CoASH ratios remained constant and the pyruvate dehydrogenase activity was correlated inversely with the NADH/NAD+ ratio. Other State 3 conditions were found in which the ATP/ADP and the NADH/NAD+ ratios remained constant while the pyruvate dehydrogenase activity was correlated inversely with the acetyl-CoA/CoASH ratio. Further evidence supporting these experiments with intact mitochondria was the observation that the pyruvate dehydrogenase kinase activity of a mitochondrial extract was stimulated strongly by acetyl-CoA and was inhibited by NAD+ and CoASH. In contrast to acetyl-CoA, octanoyl-CoA inhibited the kinase activity. These results indicate that the inactivation of pyruvate dehydrogenase by fatty acid in isolated rat liver mitochondria may be mediated through effects of the NADH/NAD+ ratio and the acetyl-CoA/CoASH ratio on the interconversion of the active and inactive forms of the enzyme complex catalyzed by pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase.
Topics: Acetyl Coenzyme A; Adenosine Diphosphate; Adenosine Triphosphate; Animals; Caprylates; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Carnitine; Coenzyme A; Male; NAD; Oligomycins; Rats; Rotenone
PubMed: 176149
DOI: No ID Found -
European Journal of Biochemistry Feb 1977The relation between the intramitochondrial and extramitochondrial ratio ATP/ADP, the transmembrane potential and pH gradient is investigated in the present...
The relation between the intramitochondrial and extramitochondrial ratio ATP/ADP, the transmembrane potential and pH gradient is investigated in the present communication. For this purpose mitochondria are equilibrated with added [14C]ATP in the presence of substrate and oligomycin for eliminating phosphate transfer by ATPase. The membrane potential was measured by the distribution of 86Rb+ in the presence of valinomycin, the deltapH by the distribution of [14C]acetate. In the energized state by varying deltapsi between 60 and 160 mV, the internal (ATP/ADP)i is decreased 30-fold, the external (ATP/ADP)e remains largely constant. As a result, the deltalog (ATP/ADP)e/(ATP/ADP)i = deltalogphi is increased linerly with deltapsi according to the following relation: deltalogphi = 0.85 deltapsi - 0.35. The deltapH was changed between 0.1 and 0.8 by increasing the Pi concentration causing only a minor decrease of deltalogphi would be expected if the ATP-ADP exchange has a significant electroneutral portion. Also in the uncoupled and respiration-inhibited state the same function between deltalogphi and deltapsi is found as in the energized states. It is concluded that under these conditions the ATP-ADP exchange is largely electrical.
Topics: Adenosine Diphosphate; Adenosine Triphosphatases; Adenosine Triphosphate; Hydrogen-Ion Concentration; Kinetics; Membrane Potentials; Membranes; Mitochondria; Oligomycins; Potassium; Rubidium
PubMed: 14003
DOI: 10.1111/j.1432-1033.1977.tb11298.x -
The Journal of Biological Chemistry Sep 1987Liver mitochondria from rats fed ethanol chronically demonstrated a 35% decrease in mitochondrial ATPase activity. Moreover, the ATPase activity was inhibited only 61%...
Liver mitochondria from rats fed ethanol chronically demonstrated a 35% decrease in mitochondrial ATPase activity. Moreover, the ATPase activity was inhibited only 61% by addition of oligomycin. Treatment of mitochondria from ethanol-fed rats with the detergent, Lubrol-WX, caused the release of 36% of the F1 from the resulting inner membrane particles. In comparison, only 5% of the F1 was dissociated when control mitochondria were subjected to the Lubrol treatment. However, when the units of ATPase activity from the supernatant and particles obtained after Lubrol treatment were added together, their sums were equivalent in preparations from control and ethanol-fed animals. Moreover, polyacrylamide gel electrophoresis analyses indicated equal amounts of the alpha + beta subunits of F1 in mitochondria from control and ethanol-fed rats. Reconstitution experiments with urea particles and F1 prepared from both control and ethanol mitochondria revealed a decrease in oligomycin sensitivity which could be attributed to an alteration in the functioning of either the oligomycin sensitivity conferring protein or a membrane sector subunit that interacts with oligomycin. Analysis by reconstitution also demonstrated that there were no ethanol-elicited alterations in the properties of the F1 portion of the ATP synthase complex. These observations indicate that the activity of the ATP synthase complex is altered significantly by ethanol-elicited changes in the functioning of those polypeptides involved in modulating both oligomycin sensitivity and the association of F1 with membrane sector subunits.
Topics: Animals; Enzyme Stability; Ethanol; Intracellular Membranes; Male; Mitochondria, Liver; Oligomycins; Proton-Translocating ATPases; Rats; Rats, Inbred Strains; Submitochondrial Particles
PubMed: 2888757
DOI: No ID Found -
FEBS Letters Jul 1983Lipid peroxidation in mitochondria induced by Fe2+ in the presence of ascorbate or by cumene hydroperoxide in the presence of phosphate results in a drop of the membrane...
Lipid peroxidation in mitochondria induced by Fe2+ in the presence of ascorbate or by cumene hydroperoxide in the presence of phosphate results in a drop of the membrane potential and in K+ efflux. The inhibitors of ATP-synthetase (oligomycin and dicyclohexylcarbodiimide (DCCD)) are capable of preventing lipid peroxidation, stabilizing the membrane potential and inhibiting potassium efflux. The same effects are observed in the presence of ionol or alpha-tocopherol. In contrast to antioxidant protection the effects of oligomycin and DCCD are reversed by the uncoupler (FCCP). The functional link between non-enzymatic lipid peroxidation, proton conduction through Fo component of ATP-synthetase and induced cation transport is suggested.
Topics: ATP Synthetase Complexes; Animals; Biological Transport; Carbodiimides; Cations; Dicyclohexylcarbodiimide; In Vitro Techniques; Lipid Peroxides; Membrane Potentials; Mitochondria, Liver; Multienzyme Complexes; Oligomycins; Oxidation-Reduction; Phosphotransferases; Rats
PubMed: 6305725
DOI: 10.1016/0014-5793(83)80669-3 -
Journal of Bacteriology Feb 1976The cytochrome spectra of two extranuclear mutants of Aspergillus nidulans and the double-mutant recombinant formed from them have been examined both at room temperature...
The cytochrome spectra of two extranuclear mutants of Aspergillus nidulans and the double-mutant recombinant formed from them have been examined both at room temperature and at the temperature of liquid N2 and compared with those of the wild-type strain. The oligomycin-resistant, slow growing mutant contained an increased amount of cytochrome c without any loss of cytochromes b and a,a3. The cold-sensitive mutant, apparently normal when grown at 37 C, showed an increased amount of cytochrome c and a partial loss of cytochromes b and a,a3 when grown at 20 C. A combination of these effects was observed in the double-mutant recombinant. Cyanide-resistant respiration was present in both mutant strains and in the recombinant at much higher levels than in the wild-type strain. In the oligomycin-resistant mutant, this was usually present together with cyanide-sensitive respiration, whereas in the cold-sensitive mutant and recombinant grown at 20 C cyanide-resistant approached 100%. Inhibitor and growth yield studies indicated that the cyanide-resistant pathway was not used by the cold-sensitive mutant during growth at 20 C.
Topics: Antimycin A; Aspergillus nidulans; Cyanides; Cytochromes; Drug Resistance, Microbial; Extrachromosomal Inheritance; Hydroxamic Acids; Mitochondria; Mutation; Oligomycins; Oxygen Consumption; Recombination, Genetic; Temperature
PubMed: 1107321
DOI: 10.1128/jb.125.2.389-397.1976