Conditions Of Oxidative Phosphorylation Research Articles

Permeabilised skeletal muscle reveals mitochondrial deficiency in malignant hyperthermia-susceptible individuals

BackgroundIndividuals genetically susceptible to malignant hyperthermia (MH) exhibit hypermetabolic reactions when exposed to volatile anaesthetics. Mitochondrial dysfunction has previously been associated with the MH-susceptible (MHS) phenotype in animal models, but evidence of this in human MH is limited. MethodsWe used high resolution respirometry to compare oxygen consumption rates (oxygen flux) between permeabilised human MHS and MH-negative (MHN) skeletal muscle fibres with or without prior exposure to halothane. A substrate-uncoupler-inhibitor titration protocol was used to measure the following components of the electron transport chain under conditions of oxidative phosphorylation (OXPHOS) or after uncoupling the electron transport system (ETS): complex I (CI), complex II (CII), CI+CII and, as a measure of mitochondrial mass, complex IV (CIV). ResultsBaseline comparisons without halothane exposure showed significantly increased mitochondrial mass (CIV, P=0.021) but lower flux control ratios in CI+CII(OXPHOS) and CII(ETS) of MHS mitochondria compared with MHN (P=0.033 and 0.005, respectively) showing that human MHS mitochondria have a functional deficiency. Exposure to halothane triggered a hypermetabolic response in MHS mitochondria, significantly increasing mass-specific oxygen flux in CI(OXPHOS), CI+CII(OXPHOS), CI+CII(ETS), and CII(ETS) (P=0.001–0.012), while the rates in MHN samples were unaltered by halothane exposure. ConclusionsWe present evidence of mitochondrial dysfunction in human MHS skeletal muscle both at baseline and after halothane exposure.

Mitochondrial energetics of benthic and pelagic Antarctic teleosts

Antarctic fauna are highly adapted to the frigid waters of the Southern Ocean. This study describes the in vitro temperature sensitivity of oxygen consumption rates measured in liver mitochondria from the pelagic notothenioid Pleuragramma antarcticum between 5 and 35 °C. Oxygen fluxes were measured after the addition of millimolar levels of pyruvate, malate, succinate and glutamate (state II, LEAK) and saturating levels of ADP [state III, oxidative phosphorylation (OXPHOS)]. State III respiration significantly decreased above 18.7 °C. A comparison of the oxidative capacities among P. antarcticum and other notothenioids showed significant differences in state III respiration, where benthic species exhibited about 50 % lower rates than P. antarcticum. In addition, state III respiration rates normalized per milligram of mitochondrial protein of P. antarcticum were up to eight times higher than state III rates reported in the literature for other notothenioids. The comparatively high respiration rates measured in this study may be explained by our approach, which engaged both complexes I and II under conditions of oxidative phosphorylation. State III rates of independently activated complexes I and II were found to range from 42 to 100 % of rates obtained when both complexes were activated simultaneously in the same species. The remarkable tolerance of P. antarcticum OXPHOS toward warmer temperatures was unexpected for an Antarctic stenotherm and may indicate that thermal sensitivity of their mitochondria is not the driving force behind their stenothermy.

The mitochondrial ADP/ATP carrier: Structural, physiological and pathological aspects

Under the conditions of oxidative phosphorylation, the mitochondrial ADP/ATP carrier catalyses the one to one exchange of cytosolic ADP against matrix ATP across the inner mitochondrial membrane. The ADP/ATP transport system can be blocked very specifically by two families of inhibitors: atractyloside (ATR) and carboxyatractyloside (CATR) on one hand, and bongkrekie acid (BA) and isbongkrekic acid (isoBA) on the other hand. It is well established that these inhibitors recognise two different conformations of the carrier protein, the CATR- and BA-conformations, which exhibit different chemical, immunochemical and enzymatic reactivities. The reversible transition of the ADP/ATP carrier between the two conformations was studied by fluorometric techniques. This transconversion, which is only triggered by transportable nucleotides, is probably the same as that which occurs during the functioning ADP/ATP transport system. The fluorometric approach, using the tryptophanyl residues of the yeast carrier as intrinsic fluorescence probes, was combined to a mutagenesis approach to elucidate the ADP/ATP transport mechanism at the molecular level. Finally, recent reports that myopathies might result from defect in ADP/ATP transport led us to develop a method to quantify the carrier protein in muscular biopsies.

Accumulation of ATP by plasma membranes of human and rat hepatocytes induced by some growth factors and phosphatidylcholine

Using the method of ATP luminometry it is shown that crude membrane preparations from human and rat hepatocytes accumulate ATP 20–100 nmol/mg protein during a 1-min incubation under conditions of oxidative phosphorylation. Application of appropriate inhibitors shows that a possible contamination of the membrane preparations with mitochondria does not contribute to this ATP accumulation. Phosphatidylcholine, tumor necrosis factor, and cell proliferation factor markedly stimulate the accumulation of ATP by plasma membraneenriched particles isolated from rat and human liver. The hepatocyte plasma membrane is shown to be able to synthesize ATP from inorganic phosphate and ADP using the aerobic mechanism. ATP in the plasma membrane is assumed to participate in the transmembrane signal transduction from growth factors to the cell effector systems.

Complementation of Escherichia coli uncD mutant strains by a chimeric F 1-β subunit constructed from E. coli and spinach chloroplast F 1-β

ATP-synthesizing F 0F 1-ATPases are complex enzymes consisting of at least eight different subunits. These subunits are conserved during evolution to a very variable degree ranging in pairwise comparison between, for example, Escherichia coli and spinach chloroplast from 20% to 66% identical residues. It was surprising to find that some of the less well conserved subunits like δ and ε could replace their E. coli counterparts, whereas the highly conserved β subunit, which carries the active site, in the E. coli enzyme could not be substituted by spinach chloroplast β (Lill et al. (1993) Biochim. Biophys. Acta 1144, 278–284). We constructed a chimeric F 1-β subunit consisting of spinach β in which the 96 N-terminal amino acids were replaced by the respective residue sequence from E. coli β. Whereas spinach β did not complement E. coli uncD mutant strains, the chimeric β subunit restored growth under conditions of oxidative phosphorylation.

Complementation of Escherichia coli unc mutant strains by chloroplast and cyanobacterial F 1-ATPase subunits

The genes encoding the five subunits of the F 1 portion of the ATPases from both spinach chloroplasts and the cyanobacterium Synechocystis sp. PCC 6803 were cloned into expression vectors and expressed in Escherichia coli. The recombinant subunits formed inclusion bodies within the cells. Each particular subunit was expressed in the respective unc mutant, each unable to grow on non-fermentable carbon sources. The following subunits restored growth under conditions of oxidative phosphorylation: α (both sources, cyanobacterial subunit more than spinach subunit), β (cyanobacterial subunit only), δ (both spinach and Synechocystis), and ϵ (both sources), whereas no growth was achieved with the γ subunits from both sources. Despite a high degree of sequence homology the large subunits α and β of spinach and cyanobacterial F 1 were not as effective in the substitution of their E. coli counterparts. On the other hand, the two smallest subunits of the E. coli ATPase could be more effectively replaced by their cyanobacterial or chloroplast counterparts, although the sequence identity or even similarity is very low. We attribute these findings to the different roles of these subunits in F 1: The large α and β subunits contribute to the catalytic centers of the enzyme, a function rendering them very sensitive to even minor changes. For the smaller δ and ϵ subunits it was sufficient to maintain a certain tertiary structure during evolution, with little emphasis on the conservation of particular amino acids.

Energy-dependent dissociation of ATP from high affinity catalytic sites of beef heart mitochondrial adenosine triphosphatase.

Incubation of [gamma-32P]ATP with a molar excess of the membrane-bound form of mitochondrial ATPase (F1) results in binding of the bulk of the radioactive nucleotide in high affinity catalytic sites (Ka = 10(12) M-1). Subsequent initiation of respiration by addition of succinate or NADH is accompanied by a profound decrease in the affinity for ATP. About one-third of the bound radioactive ATP appears to dissociate, that is, the [gamma-32P]ATP becomes accessible to hexokinase. The NADH-stimulated dissociation of [gamma-32P]ATP is energy-dependent since the stimulation is inhibited by uncouplers of oxidative phosphorylation and is prevented by respiratory chain inhibitors. The rate of the energy-dependent dissociation of ATP that occurs in the presence of NADH, ADP, and Pi is commensurate with the measured initial rate of ATP synthesis in NADH-supported oxidative phosphorylation catalyzed by the same submitochondrial particles. Thus, the rate of dissociation of ATP from the high affinity catalytic site of submitochondrial particles meets the criterion of kinetic competency under the conditions of oxidative phosphorylation. These experiments provide evidence in support of the argument that energy conserved during the oxidation of substrates by the respiratory chain can be utilized to reduce the very tight binding of product ATP in high affinity catalytic sites and to promote dissociation of the nucleotide.

Role of mitochondrial oxidative phosphorylation in the maintenance of intracellular pH

The oxidative phosphorylation of isolated rabbit heart mitochondria and the accompanying pH changes were simultaneously measured in the same sample. The nearly constant extramitochondrial proton concentration during State 4 respiration decreased considerably under conditions of oxidative phosphorylation. On the other hand, the proton concentration increased when ATP was hydrolysed by the mitochondrial ATPase. The lysis of mitochondria by Triton X-100 equilibrating the pH difference across the inner membrane did not cause significant change in the proton concentration of the sample neither before nor after State 3 respiration. The accumulation of protons during the extramitochondrial hexokinase reaction was reduced by the mitochondrial oxidative phosphorylation. It is suggested that in the normoxic myocardial cell, the protons generated by the extramitochondrial hydrolysis of ATP are utilized during mitochondrial oxidative phosphorylation.

Plastid Development in Etiolated Bean Leaves under Uncoupling Conditions of Oxidative Phosphorylation

Plastid Development in Etiolated Bean Leaves under Uncoupling Conditions of Oxidative Phosphorylation

Adenine nucleotide translocation in yeast mitochondria. Effect of inhibitors of mitochondrial biogenesis on the ADP translocase

1. Optimal test conditions for adenine nucleotide translocation in Candida utilis mitochondria are a standard medium, consisting of 0.63 M mannitol, 2 mM EDTA (or ethylene glycol tetraacetic acid, EGTA), 10 mM morpholinopropane sulfonic acid (pH 6.8), and a temperature of 0 °C. 2. Adenine nucleotide translocation in C. utilis mitochondria is an exchange-diffusion process. The whole pool of internal adenine nucleotides is exchangeable, ADP being the most readily exchangeable nucleotide. The rate of mitochondrial ADP exchange, but not the K m value, depends on growth conditions. At 0 °C, the rate is about 3 to 4 nmoles ADP/min per mg protein for mitochondria obtained from yeast grown in the presence of 1.5% glucose; it rises to 11.5 nmoles when glucose is replaced by 3% ethanol in the growth medium. The K m value for ADP is 2 μM. The Q 10 is about 2 between 0 and 20 °C. Among other exchangeable adenine nucleotides are ATP, dADP and the methylene and the hypophosphate analogues of ADP. Unlike mammalian mitochondria, C. utilis mitochondria are able to transport external UDP by a carboxyatractyloside-sensitive process. 3. Under conditions of oxidative phosphorylation (phosphate and substrate present in an aerated medium), added ADP is exchanged with internal ATP. A higher ATP/ADP ratio was found in the extramitochondrial space than in the intramito-chondrial space. The difference between the calculated phosphate potentials in the two spaces was 0.9–1.7 kcal/mole. 4. Atractyloside, carboxyatractyloside, bongkrekic acid and palmityl-CoA inhibit mitochondrial adenine nucleotide translocation in C. utilis as they do in mammalian mitochondria, but 2 to 4 times less efficiently. The inhibition due to atractyloside or palmityl-CoA is competitive with respect to ADP whereas that due to bongkrekic acid and carboxyatractyloside is non-competitive. Carboxyatractyloside and atractyloside inhibitions are additive. The apparent K d for the binding of [ 35S]-carboxyatractyloside and [ 14C]bongkrekic acid is 10–15 nM and the concentration of sites 0.4–0.6 nmole/mg protein in both cases. [ 35S]Carboxyatractyloside binding is competitively displaced by atractyloside and vice versa. 5. Binding of [ 14C]ADP has been carried out with mitochondria depleted of their endogenous adenine nucleotides by incubation with phosphate and Mg 2+ at 20 °C. The amount of bound [ 14C]ADP which is atractyloside removable is 0.08–0.16 nmole/mg protein. 6. The rate of ADP transport is quite different in mitochondria isolated from C. utilis, according to whether it is grown on glucose, or on ethanol or in the presence of chloramphenicol; for instance, it decreases by 10 times when 3% ethanol in the growth medium is replaced by 10% glucose and by 5 times when chloramphenicol is added to the medium. These variations are accompanied by parallel variations in cytochrome aa 3. The number of atractyloside-sensitive ADP binding sites is not modified by the above conditions of culture, nor the number of [ 35S]carboxyatractyloside binding sites. The affinity for ADP is apparently not significantly modified, nor the size of the endogenous adenine nucleotide pool. In contrast to glucose repression or chloramphenicol inhibition, semi-anaerobiosis in C. utilis lowers significantly the mitochondrial binding capacity for carboxyatractyloside. Strict anaerobiosis in S. cerevisiae results in a practical loss of the cytochrome oxidase activity, and also of the carboxyatractyloside and ADP binding capacity. Transition from anaerobiosis to aerobiosis restores the cytochrome oxidase activity and the ADP and carboxyatractyloside binding capacities.

Kinetics of Phosphorylation of Intramitochondrial and Extramitochondrial Adenine Nucleotides As Related to Nucleotide Translocation

The kinetics of oxidative phosphorylation of extramitochondrial (external) ADP and intramitochondrial (internal) ADP and AMP has been studied under conditions required for the exchange-diffusion of adenine nucleotides through mitochondrial membranes (ethylenediaminetetraacetate-supplemented medium at 0°) and under conditions favoring the leakage of internal adenine nucleotides (Mg++-supplemented medium). By means of atractyloside, it is possible to show that under conditions of exchange-diffusion the kinetics of phosphorylation of internal ADP and AMP is not altered by the addition of ADP to mitochondria and is independent of the phosphorylation of external ADP. Internal ADP and AMP are converted into ATP through a three-step process which successively involves first a rapid oxidative phosphorylation of the internal ADP and ATP, second a transient plateau phase, and finally a conversion of AMP into ATP. The oxidative phosphorylation of external ADP starts after a lag period which corresponds to the time interval required for the oxidative phosphorylation of the internal ADP into ATP. The conversion of the internal AMP into ATP proceeds by two coupled reactions, namely an ATP-dependent transphosphorylation of AMP into ADP and the oxidative phosphorylation of ADP into ATP. As suggested by the data of 32P-labeling experiments, the enzymatic systems catalyzing the two above mentioned coupled reactions are structurally and functionally linked to form a complex which catalyzes the over-all conversion of AMP into ATP in a cyclic manner. The ATP which is the phosphate donor for the transphosphorylation reaction is not the free ATP pre-existing in mitochondria, but the ATP produced by the complex itself; on the other hand, the ADP formed as a transient intermediate in the AMP-ATP conversion is immediately phosphorylated to give ATP without mixing with the free internal ADP. These data point to a compartmentalization of the enzymatic complex involved in the AMP-ATP conversion. Addition of magnesium ions to mitochondria incubated in a standard medium for oxidative phosphorylation (supplemented with O2, phosphate, and oxidizable substrate) results in a leakage of internal adenine nucleotides. This leakage is slowed by inhibitors or uncouplers of oxidative phosphorylation or by a low temperature, but not by atractyloside; ATP is preferentially released compared to ADP and AMP. The adenine nucleotides released outside mitochondria are rapidly exchanged with those remaining inside mitochondria. Since the exchange is much faster than the release, the distribution patterns of the adenine nucleotides remaining inside mitochondria or released outside mitochondria are similar; for instance under the conditions of oxidative phosphorylation, the same high level of ATP is found inside and outside mitochondria. The effect of atractyloside on the apparent efficiency of the oxidative phosphorylation of internal adenine nucleotides is discussed with respect to the above data.

Hydrogen Ion Concentration Changes in Mitochondrial Membranes

1. Bromthymol blue added to rat liver mitochondria at concentrations in the range 0.4 to 4.0 mµmoles per mg of protein is tightly bound as indicated by centrifugation and washing of the mitochondria. 2. The absorbance of the dye is increased on adding Ca++ or Mn++ to the mitochondria in the absence of a permeant anion. The extent of increase is 0.03 cm-1 for 167 µ m Ca++ at 618 mµ for 0.5 mµmole of bromthymol blue per mg of protein. 3. The spectrum of the indicator bound to the mitochondria has shifted only slightly to longer wave lengths than in the solution. The indicator shows very nearly the same absorbance changes on alkalinization of the mitochondrial suspension as it does upon addition of small amounts of calcium to the mitochondria. 4. The absorbance increase is independent of the pH of the suspension medium (6.5 to 7.4) and of its buffer capacity from 2.0 m m to 0.3 m sodium succinate. 5. At low protein concentrations (1 to 2 mg per ml), the kinetics of the absorbance increase is compared with the glass electrode recordings and indicates an equal and opposite pH change in the suspension medium of a similar time course. 6. At higher protein concentrations (5 to 10 mg per ml), a similar phenomenon is observed, but only after several prior additions of Ca++; endogenous material (termed Hi+) present, in terms of Ca++, at levels of approximately 40 mµmoles per mg of protein counteracts the initial absorbance increase. A small amount of added phosphate readily serves as Hi+, and endogenous phosphate at 8 mµmoles per mg of protein would afford an appropriate source of Hi+. However, the endogenous material may be phospholipid. 7. The absorbance increase on calcium additions with bromthymol blue as an indicator can be counteracted and even reversed with a 30-fold excess of bovine serum albumin, suggesting that the indicator can be removed from the mitochondrial membranes. 8. The Ca++-induced absorbance increase is nullified rapidly by additions of permeant anions such as phosphate or acetate. Ammonium ions at a 3 m m concentration do not prevent the acidification caused by acetate, and 0.1 m concentrations only slightly facilitate the alkalinization on addition of Ca++. 9. It is concluded that rapid accumulation of a cation such as Ca++ in the absence of a permeant anion can result in an intramitochondrial alkalinization and a pH gradient of about 1 pH unit across the mitochondrial membrane. This gradient develops only as a consequence of cation accumulation in the absence of a permeant anion. In the presence of permeant anions, particularly phosphate under the conditions of oxidative phosphorylation, there is no evidence of a measurable pH gradient across the mitochondrial membrane. 10. The intramitochondrial location of the indicator and of the site of calcium accumulation is tentatively assigned to the intracristal space because of the insensitivity of the bromthymol blue response to changes of osmolarity from 0.1 to 0.8 m . 11. The reaction mechanism for cation accumulation based upon the reaction with the X ∼ I intermediate of energy transfer meets the needs of the available experimental data.

Observations on the control of chloroplast structure by adenosine triphosphate

Membranes of mitochondria (1–2), chloroplasts (3–4), and photosynthetic bacteria (5), manifest mechanochemical changes that are coupled to energy transfer reactions. These changes in membrane structure or membrane deformations, conveniently measured by the recording of a physical parameter such as light-scattering, can be closely correlated to conditions favorable for oxidative or photophosphorylation. Since all of the reactants required for phosphorylation coupled to electron transport in both photosynthetic and non-photosynthetic systems are required for the change in structure, and since inhibitors of these functions abolish the change, it has been suggested (1–2, 4–5) that the structural parameter is under the control of energy-containing intermediates. Recent experiments with mitochondria (6) have demonstrated that swelling induced by electron transport is oligomycin sensitive, whereas reversal of swelling by ADP (under conditions of oxidative phosphorylation) is specifically blocked by oligomycin. These observations implicate the ATPase site in the control of mechanochemical changes. Ohnishi and Ohnishi (7) have isolated a protein from mitochondria which undergoes conformational changes with ATP (light-scattering, viscosity) and manifests ATPase activity. It is an attractive hypothesis that in mitochondria, a contractile-like substance may be involved in the changes in macromolecular structure coupled to energy transduction. In view of this, it seemed worthwhile to examine the role of ATP and ATPase activity in the control of chloroplast structure in more detail. In brief, effects of ATP on structural changes in spinach chloroplasts have been demonstrated. Furthermore, a protein fraction has been isolated from chloroplast membranes which undergoes light-scattering changes with ATP and which hydrolyzes this substance.

Structural changes correlated with photochemical phosphorylation in chloroplast membranes

Mitochondria manifest structural changes in their membranes under conditions of oxidative phosphorylation. It was postulated and confirmed here that similar changes occur under conditions of photophosphorylation in chloroplasts. Spinach chloroplasts isolated in buffered 0.5 M sucrose or 0.35 M NaCl and incubated under conditions of non-cyclic and cyclic phosphorylation at 25 ± 0.1°, on illumination with red light, show a rapid increase in 90° scattering of low-intensity 546-mμ light, which is reversed by turning the red light off. To detect such light-scattering changes, a low chlorophyll concentration is required so that pigments do not mask the scattering response, and also the scattering can be measured at the minimum of the photochemical reaction spectrum. The light-scattering responses are precisely correlated with conditions for non-cyclic and cyclic photophosphorylation, when tested in 0.035 M NaCl media in the presence of phosphate, Mg 2+, ADP and appropriate electron acceptors. Sonic treatment caused a decline in the ability to manifest light-scattering responses, and this decline parallels the decline in photophosphorylation. The light-scattering responses are abolished by NH 4 + and 3-(3,4-dichlorophenyl)-1,1-dimethylurea which inhibit photophosphorylation and photosynthetic electron transport, respectively. The magnitude of the scattering response is dependent on red-light intensity. The angle of dependence of the scattering increase rules out a fluorescence change. Aging of chloroplasts results in a loss of the light-scattering response, but this can be restored by ATP. It is concluded that some structural parameter of the chloroplast membrane changes in response to photophosphorylation. A hypothesis for the coupling of the energy-transducing systems to membrane structure in mitochondria and in chloroplasts is presented.

Fat metabolism in higher plants: XIII. phosphatidic acid synthesis and diglyceride phosphokinase activity in mitochondria from peanut cotyledons

A phosphatidic acid is the major lipid to become labeled when Pi32 (inorganic orthophosphate labeled with P32), under conditions of oxidative phosphorylation, or ATP32 is fed to mito-choline and phosphatidylethanolamine in the mitochondria obtained a much higher level of P32, the only cofactor required was Mg++. The stimulation of phosphatidic acid synthesis by an α,β-diglyceride provided support for the view that this synthesis is due to diglyceride phosphokinase activity in the mitochondria. Evidence is also presented that this enzyme preparation is capable of phosphorylating α-monoglycerides with the formation of monoacyl phosphatidic acids. When Pi32 was added to slices of peanut cotyledons, the phosphatidyl-choline and phosphatidylethanolamine in the mitochondria obtained a much higher level of radioactivity than that observed in experiments with the isolated mitochondria.