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PubMedDate not supplied

Effect of thyroidectomy on oxidative phosphorylation mechanisms in rat liver mitochondria.

(1) In the presence of succinate, 2,4-dinitrophenol raised the oxygen consumption rate of state 3 liver mitochondria, whatever the thyroid state. The rise was clearly greater in thyroidectomized than in normal rats, but the uncoupled state 3 mitochondria of thyroidectomized rats nevertheless consumed oxygen more slowly than normal rat mitochondria. Thyroidectomy therefore weakened respiratory chain activity and phosphorylation reactions. (2) With beta-hydroxybutyrate as substrate, oxygen consumption V of state 3 mitochondria was greatly diminished in thyroidectomized rats, but the KM remained unchanged. In the presence of succinate, state 3 respiration was not affected by thyroidectomy when substrate concentrations were low, but diminished at concentrations above 3 mM. (3) Respiratory chain activity was estimated by determining the succinate cytochrome c reductase activity. After thyroidectomy, catalytic efficiency (V/KM) dropped by 60% in intact mitochondria at high concentrations of the substrate, but only by 30% when concentrations were low. In submitochondrial particles, thyroidectomy reduced V without changing KM. These results suggest that in thyroidectomized rats, succinate penetration is faster when substrate concentrations are low. (4) Mg2+ -stimulated ATPase activity dropped by 20% in thyroidectomized rats. In the presence of increasing concentrations of oligomycin, inhibition of ATPase activity was greater in normal than in thyroidectomized rat mitochondria. Thyroidectomy reduced by over 30% the ATPase activity actually involved in oxidative phosphorylation.

2,4-Dinitrophenol↗
PubMed1992

Mn2+ sequestration by mitochondria and inhibition of oxidative phosphorylation.

Manganese is known to accumulate in mitochondria and in mitochondria-rich tissues in vivo. Although Ca2+ enhances mitochondrial Mn2+ uptake, ATP-bound Mn2+ is not sequestered by suspended rat brain mitochondria, and ATP binds Mn2+ even more tightly than it binds Mg2+. Physiological levels of the polyamine spermine enhanced 54 Mn2+ uptake at the low [Ca2+]s characteristic of unstimulated cells (approximately 100 nM). With succinate as substrate, Mn2+ inhibited oxygen consumption by suspensions of rat liver mitochondria after the addition of ADP but not after the addition of uncoupler. With glutamate/malate as substrate, Mn2+ inhibited ADP-stimulated respiration and also slightly inhibited uncoupler-stimulated respiration. State 4 (resting) respiration was unchanged in all cases, indicating that the inner membrane retained its impermeability to protons. These results suggest that Mn2+ was not oxidized and that it can interfere directly with oxidative phosphorylation, most likely by binding to the F1 ATPase. Mn2+ may also bind to the NADH dehydrogenase complex, but not strongly enough to affect electron transport in vivo. It is suggested that accumulation of manganese within the mitochondria of globus pallidus may help explain the distinctive pathology of manganism.

Adenosine Triphosphate↗
PubMed2004

AIF deficiency compromises oxidative phosphorylation.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that, after apoptosis induction, translocates to the nucleus where it participates in apoptotic chromatinolysis. Here, we show that human or mouse cells lacking AIF as a result of homologous recombination or small interfering RNA exhibit high lactate production and enhanced dependency on glycolytic ATP generation, due to severe reduction of respiratory chain complex I activity. Although AIF itself is not a part of complex I, AIF-deficient cells exhibit a reduced content of complex I and of its components, pointing to a role of AIF in the biogenesis and/or maintenance of this polyprotein complex. Harlequin mice with reduced AIF expression due to a retroviral insertion into the AIF gene also manifest a reduced oxidative phosphorylation (OXPHOS) in the retina and in the brain, correlating with reduced expression of complex I subunits, retinal degeneration, and neuronal defects. Altogether, these data point to a role of AIF in OXPHOS and emphasize the dual role of AIF in life and death.

Adenosine Triphosphate↗
PubMed1985

Formation of a leakage-type ion pathway in lipid bilayer membranes by divalent cationic cyanine dyes in cooperation with inorganic phosphate. Role of the cyanine dye in uncoupling of oxidative phosphorylation.

Divalent cationic cyanine dyes, such as 2,2'-[3-[2-(3-butyl-4-methyl-2-thiazoline-2-ylidene)-ethylidene] propenylene]bis[3-butyl-4-methylthiazolinium iodide] and platonin induced large membrane current fluctuation when an electrical potential difference was applied across a planar phosphatidylserine bilayer membrane in medium containing inorganic phosphate (Pi). Without Pi, the dyes did not induce current fluctuation at concentrations of less than 30 microM. Noise analysis of current fluctuation indicated formation of a pathway for ion leakage. From measurements of the interfacial tension between oil and aqueous phases, and of water permeability across liposomal membranes, Pi was concluded to relax the phospholipid bilayer structure, resulting in great reduction in the concentration of the cyanine dye necessary for induction of the leakage-type pathway. In the presence of Pi, cationic ions such as tetrabutyl ammonium and tetraphenyl phosphonium did not induce the leakage-type pathway, although they had electrophoretic effects at high concentrations. These facts suggest that the mechanism of the uncoupling of oxidative phosphorylation by dicationic cyanine dyes in mitochondria is different from that of cationic uncouplers such as tetrabutyl ammonium ion.

Cations, Divalent↗
PubMed2004

Differential accumulation of Glut1 in the non-DRM domain of the plasma membrane in response to the inhibition of oxidative phosphorylation.

Approximately 50% of Glut1 in the plasma membrane of Clone 9 cells is localized to the detergent-resistant membrane (DRM) fraction. Acute exposure (90 min) to 5mM azide stimulated glucose transport by approximately 4.7-fold and increased the abundance of Glut1 in the non-DRM fraction of the plasma membrane by approximately 2.9-fold while the abundance of Glut1 in the DRMs was not changed. In parallel experiments, approximately 17 h exposure to azide further increased the rate of glucose transport over that observed at 90 min by approximately 33% and increased plasma membrane Glut1 content by approximately 3.5-fold over control. The increase in total plasma membrane Glut1 reflected a approximately 4.7-fold increase of Glut1 content in the non-DRM fraction and a approximately 2.6-fold increase in the DRMs. We conclude that acute exposure to azide increases Glut1 content in the non-DRM fractions, while prolonged exposure to azide increases the Glut1 content in both non-DRM and DRM fractions. These changes may play an important role in the stimulation of glucose transport in response to the inhibition of oxidative phosphorylation.

Animals↗
PubMed1983

The F1F0-ATPase of Escherichia coli. Substitution of proline by leucine at position 64 in the c-subunit causes loss of oxidative phosphorylation.

The uncE410 allele differs from the normal uncE gene in that C leads to T base changes occur at nucleotides 190 and 191, resulting in proline at position 64 in the c-subunit of the F1F0-ATPase being replaced by leucine. Two partial-revertant strains were isolated in which alanine-20 of the c-subunit was replaced by proline, owing to a G leads to C base change at nucleotide 58. These c-subunits, coded for by the uncE501 and uncE502 alleles, therefore contained two amino acid changes, namely proline-64 leads to leucine, and alanine-20 leads to proline. Membranes prepared from the partial-revertant strains lacked ATP-dependent atebrin-fluorescence-quenching activity but were able to carry out oxidative phosphorylation. The ATPase activity of the F1-ATPase was inhibited when bound to membranes from strains carrying the uncE410, uncE501 and uncE502 alleles. It is concluded that a bend in the helix axis in one of the arms of the c-subunit hairpin structure is required for integration of the c-subunit into a functional F1F0-ATPase.

Adenosine Triphosphatases↗
PubMed1993

Phosphate affects the distribution of flux control among the enzymes of oxidative phosphorylation in rat skeletal muscle mitochondria.

The flux control coefficients of adenine nucleotide translocase, the phosphate transporter, and H(+)-ATPase were determined in rat skeletal muscle mitochondria using glutamate plus malate as substrates and soluble F1-ATPase as load enzyme. It was observed that the flux control coefficients of adenine nucleotide translocase, H(+)-ATPase, and the load enzyme F1-ATPase, at comparable rates of respiration, strongly depend on the phosphate concentration in the incubation medium. So, the flux control exerted by adenine nucleotide translocase, in the intermediate states of mitochondrial respiration (approximately 120 nmol of O2/min/mg) at 10 mM phosphate, was found to be about 0.37. At a phosphate concentration of 1 mM and comparable rates of respiration the flux control coefficient of the translocase decreased to about 0.20. Under these conditions, a sharp increase in the controlling influence of H(+)-ATPase from 0.10 to 0.74 was detected. Furthermore, at this flux rate, the sum of flux control coefficients of adenine nucleotide translocase, H(+)-ATPase, phosphate transporter, and the load enzyme F1-ATPase was noted to be very close to unity. This indicates that under the conditions of intermediate state respiration, all of the other reactions have a negligible controlling influence on oxidative phosphorylation in skeletal muscle mitochondria.

Animals↗
PubMed1992

A procedure for selecting mammalian cells with an impairment in oxidative phosphorylation.

The mitochondrial encephalomyopathies in man are characterized by heterogeneous defects leading to an impairment in the pathway of aerobic energy production. As a means of investigating the molecular and genetic mechanisms underlying these disorders we have developed a procedure for selecting mammalian cell lines with features resembling the human pathological phenotypes. The principle of the selection is the use of a fluorescent amphiphilic dye, 2,4-(dimethylamino)-1-styrylmethylpyridiniumiodine, a cation showing two main features. Firstly, it is accumulated by mitochondria to an extent correlated with the magnitude of the electrochemical gradient of protons across the mitochondrial inner membrane. Secondly, upon irradiation with UV light, it gives rise to formation of free radicals, which inflict damage to the cell. Mutant cells with an impairment in oxidative phosphorylation will have more chance to survive than wild type cells. The selection procedure was applied to a stock of mutagenized Chinese hamster ovary cells. After subcloning of the cells which survived the selection procedure, twenty-six independent clones were isolated. Eighteen of the clones had a partial deficiency of cytochrome c oxidase ranging from 30 to 60% of the activity in control cells. The properties of two of the clones are described. One clone has been cultured under non-selective conditions for at least 12 months with retention of the partial deficiency of cytochrome c oxidase.

Animals↗
PubMed1997

p32 protein, a splicing factor 2-associated protein, is localized in mitochondrial matrix and is functionally important in maintaining oxidative phosphorylation.

Human p32, originally cloned as a splicing factor 2-associated protein, has been reported to interact with a variety of molecules including human immunodeficiency virus Tat and complement 1q (C1q). p32 protein is supposed to be in the nucleus and on the plasma membrane for the association with human immunodeficiency virus Tat and C1q, respectively. None of the interactions, however, is proven to have a physiological role. To investigate the physiological function of p32, we determined the intracellular localization of p32. The fractionation of cells, fluorescent immunocytochemistry, and electron microscopic immunostaining show that p32 is exclusively localized in the mitochondrial matrix. We cloned a Saccharomyces cerevisiae homologue of human p32 gene, referred to yeast p30 gene. The yeast p30 protein is also localized in the mitochondrial matrix. The disruption of the p30 gene caused the growth retardation of yeast cells in a glycerol medium but not in a glucose medium, i.e. the impairment of the mitochondrial ATP synthesis. The growth impairment was restored by the introduction of the human p32 cDNA, indicating that p30 is a functional yeast counterpart of human p32. Taken together, both p32 and p30 reside in mitochondrial matrix and play an important role in maintaining mitochondrial oxidative phosphorylation.

Amino Acid Sequence↗
PubMed2004

A new role for the von Hippel-Lindau tumor suppressor protein: stimulation of mitochondrial oxidative phosphorylation complex biogenesis.

Although mitochondrial deficiency in cancer has been described by Warburg, many years ago, the mechanisms underlying this impairment remain essentially unknown. Many types of cancer cells are concerned and, in particular, clear cell renal carcinoma (CCRC). In this cancer, the tumor suppressor gene, VHL (von Hippel-Lindau factor) is invalidated. Previous studies have shown that the transfection of the VHL gene in VHL-deficient cells originating from CCRCs could suppress their ability to form tumors when they were injected into nude mice. However, various additional genetic alterations are observed in such cancer cells. In order to investigate whether VHL invalidation was related to the mitochondrial impairment, we have studied the effects of wild-type VHL transfection into VHL-deficient 786-0 or RCC10 cells on their oxidative phosphorylation (OXPHOS) subunit contents and functions. We show that the presence of wild-type VHL protein (pVHL) increased mitochondrial DNA and respiratory chain protein contents and permitted the cells to rely on their mitochondrial ATP production to grow in the absence of glucose. In parallel to mtDNA increase, the presence of pVHL up regulated the mitochondrial transcription factor A, as shown by western blot analysis. In conclusion, in CCRCs, pVHL deficiency is one of the factors responsible for down-regulation of the biogenesis of OXPHOS complexes.

Adenosine Triphosphate↗
PubMedDate not supplied

[A correlation between respiration and synthesis of ATP in mitochondria at different degree of uncoupling of oxidative phosphorylation].

It is known that mitochondrial respiration in state 3 is due to three simultaneous and independent processes: synthesis of ATP (1), endogenous passive proton leakage (2), and proton leakage by protonophoric uncoupler (3). The total rate of processes (2) and (3) is equal to the product of respiration rate in state 4 and coefficient KR, which is defined as the ratio of the deltamuH+ value in state 3 to that in state 4. It is shown that it is possible to calculate both the rates of processes (1), (2) and (3) separately and the protonophoric activity of uncoupler using the coefficient KR and other coefficients, which are determined as the ratio of deltamuH+ values in state 3 or in state 4 to its maximal value. Simple methods of determination of these coefficients were developed, which are based on the study of the dependence of respiration rate in states 3 and 4 on the concentration of protonophoric uncoupler. It was found that the uncoupling action of palmitate, a natural uncoupler of oxidative phosphorylation, unlike classic uncoupler-protonophores DNP and FCCP, depends not only on its protonophoric activity but also on the inhibition of the process (1).

Adenosine Triphosphate↗
PubMed1989

Fluctuations of membrane potential in isolated single ventricular myocytes of guinea-pig upon resumption of oxidative phosphorylation.

Electrical and mechanical activities of guinea-pig single ventricular myocytes were investigated under conditions simulating hypoxia-reoxygenation. The localized movement of sarcomere was recorded simultaneously with membrane potential, and analyzed using microcomputer-based image processing. Exposure to 5 mM CN- caused progressive shortening of action potential duration and attenuation of twitch contraction. The myocytes became inexcitable about 30 to 70 min after the CN- treatment. On removal of CN-, the myocytes exhibited periodic miniature membrane depolarizations from the resting potential level (-95 mV). When depolarizations were smaller than 6 mV in amplitude and longer than 500 ms in duration, they were accompanied by localized sarcomere shortening like a propagating contractile wave (unifocal oscillation). Membrane depolarizations of larger amplitude and shorter duration were associated with a more uniform pattern of localized sarcomere shortening (multifocal oscillation). Trains of electrical stimuli applied during the washing out period caused transient augmentation of potential fluctuation and enhancement of synchronization of sarcomere shortening. These results suggest that non-uniform elevation of intracellular calcium concentration on the resumption of oxidative phosphorylation may initiate oscillatory fluctuations of membrane potential leading to abnormal spontaneous excitation.

Action Potentials↗
PubMed2006

Increase in mitochondrial DNA quantity and impairment of oxidative phosphorylation in bovine fibroblast cells treated with ethidium bromide for 15 passages in culture.

Bovine fetal fibroblast cells were treated with ethidium bromide at a low concentration for 15 passages in culture to determine its effect on mitochondrial DNA copy number and on cell metabolism. Mitochondrial membrane potential and lactate production were estimated in order to characterize cell metabolism. In addition, mitochondrial DNA ND5 in proportion to a nuclear gene (luteinizing hormone receptor) was determined at the 1st, 2nd, 3rd, 10th, and 15th passages using semi-quantitative PCR amplification. Treated cells showed a lower mitochondrial membrane potential and higher levels of lactate production compared with control cells. However, the mitochondrial DNA/nuclear DNA ratio was higher in treated cells compared with control cells at the 10th and 15th passages. This ratio changed between the 3rd and 10th passages. Despite a clear impairment in mitochondrial function, ethidium bromide treatment did not lead to mitochondrial DNA depletion. It is possible that in response to a lower synthesis of ATP, due to an impairment in oxidative phosphorylation, treated cells develop a mechanism to resist the ethidium bromide effect on mtDNA replication, resulting in an increase in mitochondrial DNA copy number.

Animals↗
PubMed1996

Expression of oxidative phosphorylation genes in renal tumors and tumoral cell lines.

To investigate the regulation of genes encoding the proteins involved in energy metabolism in cancer cells, we studied the expression of several mitochondrial and nuclear genes involved in ATP production. Northern blot analysis was performed on renal tumors of different types: a clear cell carcinoma, an oncocytoma, and urothelial tumors at two different stages. The steady-state transcript patterns were compared with those observed in cell lines derived from renal tumors and in transformed cell lines. Striking differences were revealed among the three types of tumors, their respective controls, and the cultured renal cells. The levels of all mitochondrial transcripts were lower in tumor biopsies and tumoral cell lines than in the normal cell types. Moreover, a higher transcript level of nuclear genes involved in oxidative phosphorylation was observed in the oncocytomas and in the more malignant urothelial tumor. Different transcript patterns were observed in each of the tumoral and transformed cell lines, explaining the difference in metabolism between the different tumors and the tumoral or transformed cell lines. In particular, a high transcript level for the adenine nucleotide translocator isoform 2(ANT2) gene, which is usually not expressed in differentiated cells, was observed in oncocytoma and malignant urothelial renal tumor. This phenomenon was also observed in renal carcinoma cell lines and transformed cells. These data provide the first argument for the involvement of the ANT2 protein in glycolytic ATP uptake in cancer cell mitochondria and suggest a possible ANT2 antisense strategy for cancer therapy.

Blotting, Northern↗
PubMed2000

Oxidative phosphorylation defect in the brains of carriers of the tRNAleu(UUR) A3243G mutation in a MELAS pedigree.

MELAS is a mitochondrial encephalomyopathy characterized clinically by recurrent stroke-like episodes, seizures, sensorineural deafness, dementia, hypertrophic cardiomyopathy, and short stature. The majority of patients are heteroplasmic for a mutation (A3243G) in the tRNAleu(UUR) gene in mitochondrial DNA (mtDNA). In cells cultured in vitro, the mutation produces a severe mitochondrial translation defect only when the proportion of mutant mtDNAs exceeds 95% of total mtDNAs. However, most patients are symptomatic well below this threshold, a paradox that remains unexplained. We studied the relationship between the level of heteroplasmy for the mutant mtDNA and the clinical and biochemical abnormalities in a large pedigree that included 8 individuals carrying the A3243G mutation, 4 of whom were asymptomatic. Unexpectedly, we found that brain lactate, a sensitive indicator of oxidative phosphorylation dysfunction, was linearly related to the proportion of mutant mtDNAs in all individuals carrying the mutation, whether they were symptomatic or not. There was no evidence for threshold expression of the metabolic defect. These results suggest that marked tissue-specific differences may exist in the pathogenic expression of the A3243G mutation and explain why a neurological phenotype can be observed at relatively low levels of heteroplasmy.

Adult↗
PubMed1991

2-Ethylhexanol uncouples oxidative phosphorylation in rat liver mitochondria.

2-Ethylhexanol (70 microM), a non-genotoxic carcinogen and peroxisome proliferator, stimulated oxygen uptake in the perfused rat liver by about 10% during the first 10 min of infusion. Perfusion with a higher, hepatotoxic dose of ethylhexanol (3 mM) led to a transient increase in oxygen uptake followed by a rapid inhibition of respiration of over 50% in 10 min. Lactate dehydrogenase (LDH) release, indicative of irreversible cell death, was detected in the effluent perfusate after 20 min. After 10 min of perfusion with ethylhexanol, livers were freeze-clamped, acid extracts were prepared and adenine nucleotides were measured by high-pressure liquid chromatography. Ethylhexanol decreased the ATP/ADP ratio from 2.5 to 0.9. Thus, marked decreases in hepatic energy state due to inhibition of respiration preceded cell death. To attempt to understand this phenomenon, the effect of ethylhexanol on isolated mitochondria was studied. Similar to classical uncoupling agents, ethylhexanol stimulated state-4 rates of respiration, diminished coupled rates of respiration, and decreased the P/O ratio in a dose-dependent manner in isolated mitochondria. Ethylhexanol also decreased uptake of radiolabeled 45CaCl2 by isolated mitochondria 4- to 5-fold. Therefore, we hypothesize that ethylhexanol initially uncouples oxidative phosphorylation leading to diminished ATP synthesis and collapse of ion gradients across the mitochondrial membrane.

Adenosine Diphosphate↗
PubMed1983

[Comparative study of the effects of alpha-tocopherol and a synthetic antioxidant on respiration and oxidative phosphorylation in rat liver mitochondria].

In order to decide whether vitamin E action on ubiquinone-dependent enzymatic systems of mitochondria and on ubiquinone metabolism consisting in its antioxidative function, the effects of alpha-tocopherol and the most effective synthetic antioxidant, chlorohydrate 2-ethyl-6-methyl-3-hydroxypyridine, were compared. It was shown that the contents of vitamin E and ubiquinone as well as the activities of succinate- and NADH-ubiquinone reductase, succinate- and NADH-dehydrogenases in liver mitochondria are increased and the levels of ATP, adenine nucleotides and phosphate potential in the livers of vitamin E-deficient rats are elevated 3 hours after alpha-tocopherol injection (5 micrograms per 1 g of body weight). The synthetic antioxidant injected under identical conditions at a dose of 50 micrograms per 1 g of body weight did not change the ubiquinone content or the enzymatic activities, but considerably enhanced the ATP level. The same antioxidant when injected at a dose of 105 micrograms per 1 g of body weight did not significantly affect the parameters under study but decreased the activity of succinate-ubiquinone reductase. Thus, the role of vitamin E in oxidative phosphorylation may not only consist in its antioxidant action and is largely due to the regulation of metabolism and functioning of ubiquinone.

Adenine Nucleotides↗
PubMed2006

Sustained inhibition of oxidative phosphorylation impairs cell proliferation and induces premature senescence in human fibroblasts.

The mitochondrial theory of aging predicts that functional alterations in mitochondria contribute to the aging process. Whereas this hypothesis implicates increased production of reactive oxygen species (ROS) as a driving force of the aging process, little is known about molecular mechanisms by which mitochondrial impairment might contribute to aging. Using cellular senescence as a model for human aging, we have recently reported partial uncoupling of the respiratory chain in senescent human fibroblasts. In the present communication, we address a potential cause-effect relationship between mitochondrial impairment and the appearance of a senescence-like phenotype in young cells. We found that treatment by antimycin A delays proliferation and induces premature senescence in a subset of the cells, associated with increased reactive oxygen species (ROS) production. Quenching of ROS by antioxidants did however not restore proliferation capacity nor prevent premature senescence. Premature senescence is also induced upon chronic exposure to oligomycin, irrespective of ROS production, and oligomycin treatment induced the up-regulation of the cdk inhibitors p16, p21 and p27, which are also up-regulated in replicative senescence. Thus, besides the well-established influence of ROS on proliferation and senescence, a reduction in the level of oxidative phosphorylation is causally related to reduced cell proliferation and the induction of premature senescence.

Acetylcysteine↗