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(1/83) Oligomycin induces a decrease in the cellular content of a pathogenic mutation in the human mitochondrial ATPase 6 gene.

A T --> G mutation at position 8993 in human mitochondrial DNA is associated with the syndrome neuropathy, ataxia, and retinitis pigmentosa and with a maternally inherited form of Leigh's syndrome. The mutation substitutes an arginine for a leucine at amino acid position 156 in ATPase 6, a component of the F0 portion of the mitochondrial ATP synthase complex. Fibroblasts harboring high levels of the T8993G mutation have decreased ATP synthesis activity, but do not display any growth defect under standard culture conditions. Combining the notions that cells with respiratory chain defects grow poorly in medium containing galactose as the major carbon source, and that resistance to oligomycin, a mitochondrial inhibitor, is associated with mutations in the ATPase 6 gene in the same transmembrane domain where the T8993G amino acid substitution is located, we created selective culture conditions using galactose and oligomycin that elicited a pathological phenotype in T8993G cells and that allowed for the rapid selection of wild-type over T8993G mutant cells. We then generated cytoplasmic hybrid clones containing heteroplasmic levels of the T8993G mutation, and showed that selection in galactose-oligomycin caused a significant increase in the fraction of wild-type molecules (from 16 to 28%) in these cells.  (+info)

(2/83) Uncoupling of intestinal mitochondrial oxidative phosphorylation and inhibition of cyclooxygenase are required for the development of NSAID-enteropathy in the rat.

BACKGROUND: The pathogenesis of NSAID-induced gastrointestinal damage is believed to involve a nonprostaglandin dependent effect as well as prostaglandin dependent effects. One suggestion is that the nonprostaglandin mechanism involves uncoupling of mitochondrial oxidative phosphorylation. AIMS: To assess the role of uncoupling of mitochondrial oxidative phosphorylation in the pathogenesis of small intestinal damage in the rat. METHODS: We compared key pathophysiologic events in the small bowel following (i) dinitrophenol, an uncoupling agent (ii) parenteral aspirin, to inhibit cyclooxygenase without causing a 'topical' effect and (iii) the two together, using (iv) indomethacin as a positive control. RESULTS: Dinitrophenol altered intestinal mitochondrial morphology, increased intestinal permeability and caused inflammation without affecting gastric permeability or intestinal prostanoid levels. Parenteral aspirin decreased mucosal prostanoids without affecting intestinal mitochondria in vivo, gastric or intestinal permeability. Aspirin caused no inflammation or ulcers. When dinitrophenol and aspirin were given together the changes in intestinal mitochondrial morphology, permeability, inflammation and prostanoid levels and the macro- and microscopic appearances of intestinal ulcers were similar to indomethacin. CONCLUSIONS: These studies allow dissociation of the contribution and consequences of uncoupling of mitochondrial oxidative phosphorylation and cyclooxygenase inhibition in the pathophysiology of NSAID enteropathy. While uncoupling of enterocyte mitochondrial oxidative phosphorylation leads to increased intestinal permeability and low grade inflammation, concurrent decreases in mucosal prostanoids appear to be important in the development of ulcers.  (+info)

(3/83) Bovine coupling factor 6, with just 14.5% shared identity, replaces subunit h in the yeast ATP synthase.

The mammalian mitochondrial ATP synthase is composed of at least 16 polypeptides. With the exception of coupling factor F(6), there are likely yeast homologs for each of these polypeptides. There are no obvious yeast homologs of F(6), as predicted from primary sequence comparison of the putative peptides encoded by the open reading frames in the yeast genome. In this manuscript, we demonstrate that expression of bovine F(6) complements a null mutant in ATP14 gene in yeast Saccharomyces cerevisiae. Subunit h of the yeast ATP synthase is encoded by ATP14 and is just 14.5% identical to bovine F(6). Expression of bovine F(6) in an atp14 null mutant strain recovers oxidative phosphorylation, and the ATP synthase is active, although functioning with a lower efficiency than the wild type enzyme. Like subunit h, bovine F(6) is shown to interact mainly with subunit 4 (subunit b), a component of the second stalk of the enzyme. These data indicated the subunit h is the yeast homolog of mammalian coupling factor F(6).  (+info)

(4/83) Mitochondrial coupling factor 6 is present on the surface of human vascular endothelial cells and is released by shear stress.

BACKGROUND: We showed that mitochondrial coupling factor 6 (CF6), an endogenous inhibitor of prostacyclin synthesis, is present in the systemic circulation as a pressor substance in rats. We investigated the possibility of vascular endothelial cells as a source of circulating CF6. METHODS AND RESULTS: We used 2 cultured endothelial cell lines, human umbilical vein endothelial cells (HUVECs) and ECV 304 cells (transformed HUVECs), for this study. Immunofluorescence microscopy of both ECV 304 and HUVECs confirmed the surface-associated immunoreactivity of anti-CF6 antibody on the plasma membrane. The concentration of CF6 in the medium increased gradually with time in both ECV 304 and HUVECs in static conditions. Exposure of ECV 304 and HUVECs to a fluid shear stress enhanced the release of CF6: In ECV 304, the concentration of CF6 in the medium (ng. well(-1). 6 hours(-1)) was 2.1+/-0.8 at baseline, 4.3+/-0.8 after shear at 15 dynes/cm(2), and 57.7+/-8.4 after shear at 25 dynes/cm(2). CF6 contents in the cell homogenate and mitochondria were both significantly increased after exposure of ECV 304 to 6-hour shear at 15 dynes/cm(2), whereas they were unchanged after shear stress at 25 dynes/cm(2). The ratio of CF6 to GAPDH mRNA was enhanced significantly, by 1.8+/-0.2-fold, after 6-hour shear stress at 25 dynes/cm(2). Flow cytometry analysis revealed that the surface-associated CF6 was significantly increased in a 3-hour static condition after the previous exposure of the cells to shear stress for 3 hours. CONCLUSIONS: Vascular endothelial cells are a source of CF6, and shear stress regulates the release of the surface-associated CF6.  (+info)

(5/83) Coupling between voltage sensors and activation gate in voltage-gated K+ channels.

Current through voltage-gated K+ channels underlies the action potential encoding the electrical signal in excitable cells. The four subunits of a voltage-gated K+ channel each have six transmembrane segments (S1-S6), whereas some other K+ channels, such as eukaryotic inward rectifier K+ channels and the prokaryotic KcsA channel, have only two transmembrane segments (M1 and M2). A voltage-gated K+ channel is formed by an ion-pore module (S5-S6, equivalent to M1-M2) and the surrounding voltage-sensing modules. The S4 segments are the primary voltage sensors while the intracellular activation gate is located near the COOH-terminal end of S6, although the coupling mechanism between them remains unknown. In the present study, we found that two short, complementary sequences in voltage-gated K+ channels are essential for coupling the voltage sensors to the intracellular activation gate. One sequence is the so called S4-S5 linker distal to the voltage-sensing S4, while the other is around the COOH-terminal end of S6, a region containing the actual gate-forming residues.  (+info)

(6/83) Salmonella typhimurium HfrA, a mutant in which adenosine triphosphate can drive amino acid transport but not energy-dependent nicotinamide nucleotide transhydrogenation.

In contrast with wild-type Salmonella typhimurium LT2, strain HfrA did not have ATP-driven energy-dependent transhydrogenase activity, although ATP-dependent quenching of atebrin fluorescence was normal. Respiration-dependent and energy-independent transhydrogenase, and Ca2+-activated ATPase (adenosine triphosphatase) activities were similar in both strains. Purified ATPases from the two strains had similar specific activities, similar subunit polypeptides, and were equally effective in restoring energy-dependent transhydrogenase activities to membrane particles of strain LT2 from which the ATPase had been stripped. The purified ATPases from both strains could restore respiration-dependent but not ATP-dependent transhydrogenation to stripped particles of strain HfrA. Both strains grew aerobically equally well on salts media containing glucose, malate, succinate, citrate, acetate, pyruvate, fumarate, lactate or aspartate as substrates. Growth on glucose under anaerobic conditions was similar. Strains LT2 and HfrA were equally effective in the accumulation under both aerobic and anaerobic conditions of the amino acids proline, phenylalanine, histidine, lysine, isoleucine and aspartic acid. Inhibition of amino acid accumulation by KCN and dicyclohexylcarbodi-imide occurred to the same extent in both strains. The complete inhibition by dicyclohexylcarbodi-imide of amino acid uptake under anaerobic conditions suggested that ATP could drive amino acid uptake in both strains. The ability of strain HfrA to carry out ATP-dependent transport or quenching of atebrin fluorescence but not ATP-dependent transhydrogenation is different from the wild-type strain and from any previously described energy-coupling mutant. It is difficult to reconcile the properties of this mutant with the chemiosmotic hypothesis.  (+info)

(7/83) Salt inactivation as a mechanistic probe of membrane-bound chloroplast coupling factor 1.

Conditions are reported under which ATP protects membrane-bound coupling factor 1 against sodium bromide inactivation. The presence of Mg2+ was found to be obligatory for this protection. ADP and GTP also protected the enzyme against salt inactivation but to a much smaller extent. Other nucleotides tested were ineffective. At low ATP concentrations ADP prevented the effect of ATP and modified the saturation curve for ATP from hyperbolic to sigmoidal. Treatment of chloroplasts with 0.4 M MgCl2 or 2 M LiCl resulted in inactivation of photophosphorylation. In contrast to NaBr-depleted particles the MgCl2 or LiCl-depleted chloroplasts can be reconstituted by purified coupling factor 1. A binding site for Mg2+ and two different sites for ATP upon the coupling factor 1 are suggested to explain the mechanism of their protection against salt inactivation.  (+info)

(8/83) Studies on the turnovers in vivo of adenosine di- and triphosphates in a coupling factor of Escherichia coli.

The metabolic stabilities of bound adenine nucleotides in a membrane-bound ATPase (EF1) [EC 3.6.1.3] of Escherichia coli were studied by estimating their rates of turnover in vivo. Two-thirds of the bound ATP prelabelled with 32Pi in EF1 molecules was retained after 3 h in a chase medium. The bound ADP was chased rapidly with a half time of decrease of less than 1 h, the rate similar to that of cytoplasmic free nucleotides. These results suggest that bound ATP in the EF1 is not a direct intermediate in oxidative phosphorylation.  (+info)