Mitochondrial nucleotide translocase from skeletal muscle of halothane sensitive pigs: an electrophoretic study.

ATP translocation into mitochondria isolated from halothane-sensitive pig (HP) muscle was dramatically reduced compared with normal pigs (NP). To determine if this was due to a decreased amount of ATP translocase in the mitochondrial membranes, or a structural modification of this protein, an electrophoretic study was undertaken. Total proteins and purified translocase preparations from (NP) and (HP) mitochondria were analyzed by SDS gel electrophoresis. In the two types of mitochondria no significant differences were observed either in the amount of ATP translocase or in the molecular weight. Also, neither nonequilibrium pH gradient gel electrophoresis nor the analysis of peptides produced by limited proteolysis revealed any structural difference between the two types of protein. On the basis of these results, the depressed translocase activity observed in (HP) mitochondria cannot be explained by a reduced amount of the nucleotide translocase, nor a structural alteration of this protein. Possible inhibition of (HP) translocase activity by Ca2+ accumulation or by other mechanisms is discussed.     

Control of adenine nucleotide metabolism in hepatic mitochondria from rats with ethanol-induced fatty liver

Male rats developed fatty liver after being fed on an ethanol-containing diet for 31 days. Liver mitochondria from these animals catalysed ATP synthesis at a slower rate when compared with mitochondria from pair-fed control rats (control mitochondria), and demonstrated lowered respiratory control with succinate as substrate, owing to a decrease in the State-3 respiratory rate. Respiration in the presence of uncoupler was comparable in mitochondria from both groups of rats. Translocation of both ATP and ADP was decreased in mitochondria from ethanol-fed rats, with ADP uptake being lowered more dramatically by ethanol feeding. Parameters influencing adenine nucleotide translocation were investigated in mitochondria from ethanol-fed rats. Experiments performed suggested that lowered adenine nucleotide translocation in these mitochondria is not the result of inhibition of the translocase by either long-chain acyl-CoA derivatives or unesterified fatty acids. Analysis of endogenous adenine nucleotides in these mitochondria revealed lowered ATP concentrations, but no decrease in total adenine nucleotides. In experiments where the endogenous ATP in these mitochondria was shifted to higher concentrations by incubation with oxidizable substrates or defatted bovine serum albumin, the rate of ADP translocation was increased, with a linear correlation being observed between endogenous ATP concentrations and the rate of ADP translocation. The depressed ATP concentration in mitochondria from ethanol-fed rats suggests that the ATP synthetase complex is replenishing endogenous ATP at a slower rate. The lowered ATPase activity of the ATP synthetase observed in submitochondrial particles from ethanol-fed animals suggests a decrease in the function of the synthetase complex. A decrease in the rate of ATP synthesis in mitochondria from ethanol-fed rats is sufficient to explain the decreased ADP translocation and State-3 respiration.     

Mitochondrial ATP-sensitive potassium channel activity and hypoxic preconditioning are independent of an inwardly rectifying potassium channel subunit in Caenorhabditis elegans

Hypoxic preconditioning (HP) is an evolutionarily-conserved mechanism that protects an organism against stress. The mitochondrial ATP-sensitive K(+) channel (mK(ATP)) plays an essential role in the protective signaling, but remains molecularly undefined. Several lines of evidence suggest that mK(ATP) may arise from an inward rectifying K(+) channel (Kir). The genetic model organism Caenorhabditis elegans exhibits HP and displays mK(ATP) activity. Here, we investigate the tissue expression profile of the three C. elegans Kir genes and demonstrate that mutant strains where the irk genes have been deleted either individually or in combination can be protected by HP and exhibit robust mK(ATP) channel activity in purified mitochondria. These data suggest that the mK(ATP) in C. elegans does not arise from a Kir derived channel.     

Modification of flow-force relationships by external ATP in yeast mitochondria

The purpose of this work is to measure protonmotive force and cytochrome reduction level under different respiratory steady states in isolated yeast mitochondria. The rate of respiration was varied by using three sets of conditions: (a) different external phosphate concentrations with a fixed concentration of ADP (ATP synthesis) and (b) different concentrations of carbonylcyanide m-chlorophenylhydrazone in the presence of oligomycin and carboxyatractylate (uncoupling) either in the absence or (c) in the presence of external ATP. ADP plus phosphate stimulates respiration more than uncoupler at the same protonmotive force value. However, the relationships between respiratory rate and protonmotive force were similar when stimulation was induced either by ADP + Pi or by carbonylcyanide m-chlorophenylhydrazone in the presence of ATP. At the same respiratory rate, cytochrome a + a3 is more reduced by uncoupler than by ADP + Pi additions. However, the relationships between respiratory rate and reduction level of cytochrome-c oxidase are similar both under ATP synthesis and with uncoupling conditions in the presence of external ATP. Control of respiration exerted by cytochrome-c oxidase, and support the view the condition mentioned above. This control was low when the respiratory rate was varied by the ATP synthesis rate; it increased as a function of the respiratory rate with uncoupler in the absence of ATP. ATP decreased this control under uncoupling conditions. These results suggest a regulatory effect of external ATP on cytochrome-c oxidase, and support the view that the relationships between respiratory rate and protonmotive force, on the one hand, and respiratory rate and the reduction level of cytochrome-c oxidase, on the other, depend respectively on the kinetic regulations of the system.     

Adrenal glucocorticoids, adenine nucleotide translocation, and mitochondrial calcium accumulation.

The administration of dexamethasone to rats markedly diminished the initial rate and maximal extent of substrate-dependent calcium uptake in subsequently isolated liver mitochondria, and enhanced the release of calcium. The apparent Km for calcium transport was not altered by dexamethasone treatment and it ranged from 50 to 80 muM when an EDTA/Ca buffer system was used in the presence of magnesium, and 20 muM when an NTA/Ca buffer system without magnesium was employed. In contrast, when ATP was employed as the energy source, there was no significant difference in initial rate, Km, or the extent of calcium accumulation between mitochondria from control and dexamethasone-treated animals. Although mitochondria from dexamethasone-treated animal showed 15% less cytochrome c oxidase activity/mg of protein, overall respiratory capacity and ATP production from ADP were the same as in control mitochondria. However, mitochondria from dexamethasone-treated animals translocated ATP from inside to outside faster than those from control animals. When the ATP in the medium was depleted by glucose and hexokinase, both types of mitochondria retained essentially all the preloaded calcium until total ATP reached a critical level (7 approximately 5 mumol of ATP/mg of protein). When ATP content fell below this critical level, mitochondria released all the calcium quickly. Dexamethasone treatment increased the susceptibility of mitochondria to the depletion of ATP. These data indicate that the dexamethasone-induced decrease in maximal calcium transport and in calcium retention carrier system per se, but o an altered ability of the mitochondria to regulate intramitochondrial ATP content.     

Evaluation of Fe(III) reduction by mitochondria induced with a respiratory substrate NADH or succinate,using a Fe(II)-specific chelator bathophenanthroline disulfonate in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Mitochondria are the centers of the cellular iron metabolism. Iron utilization by mitochondria is deeply related to their respiratory chain activity. We isolated mitochondria from Saccharomyces cerevisiae and examined Fe(III) reduction induced by a respiratory substrate (NADH or succinate), using a Fe(II)-specific chelator (bathophenanthroline disulfonate). In the presence of either 50 μM NADH or 5 mM succinate, the amount of reduced Fe(III) was linearly correlated with the amount of mitochondria. As the concentration of the substrate increased, the rate of the mitochondrial Fe(III) reduction reached a plateau. In the presence of 1 mM ADP or 1 mM ATP, the extramitochondrial Fe(III) reduction was repressed when succinate was used as the substrate, but not when NADH was used. ADP had an inhibitory effect even under low concentration of succinate, suggesting that ADP and ATP acted in a manner of both competitive and uncompetitive inhibition.     

Maintenance of growth rate at low temperature in rice and wheat cultivars with a high degree of respiratory homeostasis is associated with a high efficiency of respiratory ATP production

Some plants have the ability to maintain similar respiratory rates (measured at the growth temperature) when grown at different temperatures. This phenomenon is referred to as respiratory homeostasis. Using wheat and rice cultivars with different degrees of respiratory homeostasis (H), we previously demonstrated that high-H cultivars maintained shoot and root growth at low temperature [Kurimoto et al. (2004) Plant Cell Environ., 27: 853]. Here, we assess the relationship between respiratory homeostasis and the efficiency of respiratory ATP production, by measuring the levels of alternative oxidase (AOX) and uncoupling protein (UCP), which have the potential to decrease respiratory ATP production per unit of oxygen consumed. We also measured SHAM- and CN-resistant respiration of intact roots, and the capacity of the cytochrome pathway (CP) and AOX in isolated mitochondria. Irrespective of H, SHAM-resistant respiration of intact roots and CP capacity of isolated root mitochondria were larger when plants were grown at low temperature, and the maximal activity and relative amounts of cytochrome c oxidase showed a similar trend. In contrast, CN-resistant respiration of intact roots and relative amounts of AOX protein in mitochondria isolated from those roots, were lower in high-H plants grown at low temperature. In the roots of low-H cultivars, relative amounts of AOX protein were higher at low growth temperature. Relative amounts of UCP protein showed similar trends to AOX. We conclude that maintenance of growth rate in high-H plants grown at low temperature is associated with both respiratory homeostasis and a high efficiency of respiratory ATP production.     

Regulation of Malate Oxidation in Isolated Mung Bean Mitochondria: II. Role of Adenylates

Effects of ADP and ATP on products of malate oxidation in the presence or absence of respiratory inhibitors and an uncoupler were investigated in mitochondria isolated from mung bean (Phaseolus aureus var. Jumbo) hypocotyls. Changes in levels of products from malate oxidation generally correlated directly with changes in oxygen uptake. Effects of ADP and ATP were indistinguishable from each other when respiratory chain activity was limited. We concluded that adenylates indirectly act on malate oxidation via the oxidation-reduction status of the pyridine nucleotides which are linked to the respiratory chain. The possibility of allosteric action of ADP and ATP on malate dehydrogenase activity was examined in both intact mitochondria and a partially purified enzyme preparation. Although small inhibition, 16% with 500 μM ATP and 8% with 500 μM ADP, was observed at pH 9.5, this effect was abolished by the addition of magnesium ions or by lowering the pH to 7.2. We concluded that these adenylate effects are probably not a significant factor in regulation under physiological conditions. Furthermore, the equilibrium constant of malate dehydrogenase (to 1.5 × 10⁻⁵) in both mitochondria and the partially purified enzyme calculated from the steady state level of NADH formed suggested that the enzyme functions in an equilibrium manner in intact mitochondria.     

Relationship between ATP level and respiratory rate in sea urchin embryos

In sea urchin embryos, the rate of respiration, as a result of electron transport through the mitochondrial respiratory chain, was enhanced after hatching without any change in the intrinsic capacity of electron transport in mitochondria. The increase in respiratory rate after hatching was accompanied by an evident decrease in intracellular adenosine triphosphate (ATP) concentration without any change in intracellular levels of adenosine diphosphate (ADP) and adenosine monophosphate (AMP). Adenosine triphosphate is proposed to fortify acceptor control of respiration at high concentrations and to reduce the respiratory rate even in the presence of ADP, the acceptor. The relationships between the respiratory rate and intracellular ATP concentration in embryos were the same as those in mitochondria isolated from embryos, obtained in the presence of ADP at the same concentration as in the embryos. Probably, the respiratory rate is enhanced after hatching because of the decrease in the level of ATP. In embryos kept in a medium containing adenosine, intracellular ATP concentration increased especially after hatching, without any change in the ADP level, and the respiratory rate after hatching was made as low as the rate expected, based on the relationships obtained on isolated mitochondria. The respiratory rate in embryos probably depends on intracellular ATP concentration, irrespective of the developmental stage in early development.     

ATP-regulation of cytochrome oxidase in yeast mitochondria: role of subunit VIa.

The role of the nuclear-encoded subunit VIa in the regulation of cytochrome oxidase by ATP was investigated in isolated yeast mitochondria. As the subunit VIa-null strain possesses a fully active and assembled cytochrome oxidase, multiple ATP-regulating sites were characterized with respect to their location and their kinetic effect: (a) intra-mitochondrial ATP inhibited the complex IV activity of the null strain, whereas the prevailing effect of ATP on the wild-type strain, at low ionic strength, was activation on the cytosolic side of complex IV, mediated by subunit VIa. However, at physiological ionic strength (i.e. approximately 200 mM), activation by ATP was absent but inhibition was not impaired; (b) in ethanol-respiring mitochondria, when the electron flux was modulated using a protonophoric uncoupler, the redox state of aa3 cytochromes varied with respect to activation (wild-type) or inhibition (null-mutant) of the cytochrome oxidase by ATP; (c) consequently, the control coefficient of cytochrome oxidase on respiratory flux, decreased (wild-type) or increased (null-mutant) in the presence of ATP; (d) considering electron transport from cytochrome c to oxygen, the response of cytochrome oxidase to its thermodynamic driving force was increased by ATP for the wild-type but not for the mutant subunit. Taken together, these findings indicate that at physiological concentration, ATP regulates yeast cytochrome oxidase via subunit-mediated interactions on both sides of the inner membrane, thus subtly tuning the thermodynamic and kinetic control of respiration. This study opens up new prospects for understanding the feedback regulation of the respiratory chain by ATP.     

Inhibition of mitochondrial bioenergetics: the effects on structure of mitochondria in the cell and on apoptosis

The effects of specific inhibitors of respiratory chain, F(o)F(1)ATP synthase and uncouplers of oxidative phosphorylation on survival of carcinoma HeLa cells and on the structure of mitochondria in the cells were studied. The inhibitors of respiration (piericidin, antimycin, myxothiazol), the F(1)-component of ATP synthase (aurovertin) and uncouplers (DNP, FCCP) did not affect viability of HeLa cells, apoptosis induced by TNF or staurosporin and the anti-apoptotic action of Bcl-2. Apoptosis was induced by combined action of respiratory inhibitors and uncouplers indicating possible pro-apoptotic action of reactive oxygen species (ROS) generated by mitochondria. Short-term incubation of HeLa cells with the mitochondrial inhibitors and 2-deoxyglucose followed by 24-48 h recovery resulted in massive apoptosis. Apoptosis correlated to transient (3-4 h) and limited (60-70%) depletion of ATP. More prolonged or more complete transient ATP depletion induced pronounced necrosis. The inhibitors of respiration and uncouplers caused fragmentation of tubular mitochondria and formation of small round bodies followed by swelling. These transitions were not accompanied with release of cytochrome c into the cytosol and were fully reversible. The combined effect of respiratory inhibitors and uncouplers developed more rapidly indicating possible involvement of ROS generated by mitochondria. More prolonged (48-72 h) incubation with this combination of inhibitors caused clustering and degradation of mitochondria.     

ATP produced by oxidative phosphorylation is channeled toward hexokinase bound to mitochondrial porin (VDAC) in beetroots (Beta vulgaris)

Mitochondrial porins or voltage-dependent anion channels (VDAC) are the main route for solute transport through outer mitochondrial membranes (OMM). In mammals, hexokinase (HK) binds to VDAC, which allows the channeling of ATP synthesized by oxidative phosphorylation toward HK. In plants, although HK has been found associated with OMM, evidence for an interaction with VDAC is scarce. Thus, in this work, we studied the physical and functional interaction between these proteins in beetroot mitochondria. To observe a physical interaction between HK and VDAC, OMM presenting HK activity were prepared from purified mitochondria. Protein complexes were solubilized from OMM with mild detergents and separated by centrifugation in glycerol gradients. Both HK activity and immunodetected VDAC were found in small (9S–13S) and large (>40S) complexes. OMM proteins were also separated according to their hydropathy by serial phase partitioning with Triton X-114. Most of HK activity was found in hydrophobic fractions where VDAC was also present. These results indicated that HK could be bound to VDAC in beetroot mitochondria. The functional interaction of HK with VDAC was demonstrated by observing the effect of apyrase on HK-catalyzed glucose phosphorylation in intact mitochondria. Apyrase, which hydrolyzes freely soluble ATP, competed efficiently with hexokinase for ATP when it was produced outside mitochondria (with PEP and pyruvate kinase), but not when it was produced inside mitochondria by oxidative phosphorylation. These results suggest that HK closely interacts with VDAC in beetroot mitochondria, and that this interaction allows the channeling of respiratory ATP toward HK through VDAC.     

HP30‐2, a mitochondrial PRAT protein for import of signal sequence‐less precursor proteins in Arabidopsis thaliana

Chloroplasts and mitochondria contain a family of putative preprotein and amino acid transporters designated PRAT. Here,we analyzed the role of two previously characterized PRAT protein family members,encoded by At3g49560(HP30) and At5g24650(HP30-2),in planta using a combination of genetic,cell biological and biochemical approaches. Expression studies and green fluorescent protein tagging identified HP30-2 both in chloroplasts and mitochondria,whereas HP30 was located exclusively in chloroplasts. Biochemical evidence was obtained for an association of mitochondrial HP30-2 with two distinct protein complexes,one containing the inner membrane translocase TIM22 and the other containing an alternative NAD(P)H dehydrogenase subunit(NDC_1)implicated in a respiratory complex 1-like electron transport chain. Through its association with TIM22,HP30-2 is involved in the uptake of carrier proteins and other,hydrophobic membrane proteins lacking cleavable NH2-terminal presequences,whereas HP30-2's interaction with NDC1 may permit controlling mitochondrial biogenesis and activity.     

Influence of different energy drains on the interrelationship between the rate of respiration,proton-motive force and adenine nucleotide patterns in isolated mitochondria

The respiration of rat liver mitochondria was stimulated by three different ways of energy drain: (a) partial uncoupling (equivalent to direct collapse of the proton-motive force), (b) intramitochondrial utilization of ATP for citrulline synthesis, and (c) extramitochondrial utilization of ATP for glucose phosphorylation. At identical rates of respiration, the intramitochondrial ATP: ADP ratios were the same in all three systems. Furthermore, the proton-motive force was the same in partially uncoupled mitochondria and in the presence of hexokinase plus glucose up to a respiration rate amounting to about 60% of that of the fully active state. However, external ATP: ADP ratios were considerably different in various systems at comparable rates of oxygen uptake, being the lowest under conditions when ATP was being utilized externally. On this basis, it is concluded that the respiratory rate is controlled directly by the proton-motive force and the mitochondrial ATP-synthesizing system operates under near-equilibrium conditions with respect to the membrane energy state parameters. However, a disequilibrium exists at the step of the transport of ATP from mitochondria to the external (cytoplasmic) compartment.     

Changes in respiratory mitochondrial machinery and cytochrome and alternative pathway activities in response to energy demand underlie the acclimation of respiration to elevated CO2 in the invasive Opuntia ficus-indica

Studies on long-term effects of plants grown at elevated CO(2) are scarce and mechanisms of such responses are largely unknown. To gain mechanistic understanding on respiratory acclimation to elevated CO(2), the Crassulacean acid metabolism Mediterranean invasive Opuntia ficus-indica Miller was grown at various CO(2) concentrations. Respiration rates, maximum activity of cytochrome c oxidase, and active mitochondrial number consistently decreased in plants grown at elevated CO(2) during the 9 months of the study when compared to ambient plants. Plant growth at elevated CO(2) also reduced cytochrome pathway activity, but increased the activity of the alternative pathway. Despite all these effects seen in plants grown at high CO(2), the specific oxygen uptake rate per unit of active mitochondria was the same for plants grown at ambient and elevated CO(2). Although decreases in photorespiration activity have been pointed out as a factor contributing to the long-term acclimation of plant respiration to growth at elevated CO(2), the homeostatic maintenance of specific respiratory rate per unit of mitochondria in response to high CO(2) suggests that photorespiratory activity may play a small role on the long-term acclimation of respiration to elevated CO(2). However, despite growth enhancement and as a result of the inhibition in cytochrome pathway activity by elevated CO(2), total mitochondrial ATP production was decreased by plant growth at elevated CO(2) when compared to ambient-grown plants. Because plant growth at elevated CO(2) increased biomass but reduced respiratory machinery, activity, and ATP yields while maintaining O(2) consumption rates per unit of mitochondria, we suggest that acclimation to elevated CO(2) results from physiological adjustment of respiration to tissue ATP demand, which may not be entirely driven by nitrogen metabolism as previously suggested.     

The effect of osmotic `shock'' on the swelling pattern and respiratory control of rat-liver mitochondria

1. Rat-liver mitochondria suspended in 0.25m-sucrose were exposed for a few seconds to strongly hypo-osmotic conditions, and then the osmolarity of the medium was raised again to 0.25 with the aid of tris chloride (osmotic ;shock'). 2. Mitochondria after hypo-osmotic pretreatment lost their capacity for slow energy-dependent swelling in iso-osmotic tris buffer and showed no respiratory control. 3. Swelling could be induced in the ;shocked' mitochondria by ATP but not by addition of respiratory substrates. 4. It was shown that cytochrome c is lost from ;shocked' mitochondria when they come into contact with the tris buffer present in the assay medium, and that the changes observed in the pattern of swelling, as well as in respiratory control, are directly connected with this loss of cytochrome c. 5. The results of the investigation are discussed with regard to the role of cytochrome c in swelling and respiratory control.     

Adenine nucleotides, glutamate and respiratory function of heart mitochondria during acute hypoxia

The effect of acute hypoxia on adenine nucleotides, glutamate, aspartate, alanine and respiration of heart mitochondria was studied in rats. The losses of intramitochondrial adenine nucleotides (ATP+ADP+AMP) during hypoxia were related to depression of state 3 respiration supported by glutamate and malate, as well as decrease in uncoupled respiration. Hypoxia had less prominent effect on succinate-dependent state 3 respiration. Non-phosphorylating (state 4) respiratory rates and ADP/O ratios were slightly affected by oxygen deprivation. Glutamate fall in tissue and mitochondria of hypoxic hearts was concomitant with significant increase in tissue alanine and mitochondrial aspartate. The losses of intramitochondrial ATP and respiratory activity with NAD-dependent substrates during hypoxia were related to a decrease in mitochondrial glutamate. The results suggest that hypoxia-induced impairment of complex I of respiratory chain and a loss of glutamate from the matrix may limit energy-producing capacity of heart mitochondria.     

In vitro 14C-leucine incorporation into nonsynaptic and synaptic rat brain mitochondria isolated by Ficoll gradients

The in vitro incorporation of 14C-leucine by nonsynaptic and synaptic rat brain mitochondria purified by means of discontinuous Ficoll gradients has been characterised. The incorporation was linear for the first 45 min for both populations. Synaptic mitochondria showed a higher rate of incorporation than the nonsynaptic mitochondria at high concentrations of leucine. The incorporation was more effective in the presence of Mg2+ and inhibited by dinitrophenol. The incorporation was sensitive to chloramphenicol and insensitive to cycloheximide. Bacterial contamination was in any case lower than 1,000 colonies per ml after the incubation period. The incorporation was carried out in the presence of either an external ATP-generating system consisting of ATP, phosphoenolpyruvate and pyruvate kinase or with mitochondria respiring with oxidisable substrates plus ADP (state III). The rates obtained for incorporation in this state III were higher for all the substrates assayed (succinate, pyruvate and glutamate) than in the presence of exogenous ATP. The highest rate obtained was found when glutamate was the respiratory substrate. No significant metabolic oxidation of leucine occurs in either synaptic or nonsynaptic mitochondria in the presence of exogenous ATP. Glutamate did not increase leucine uptake in any mitochondrial populations.     

Valproic acid metabolites inhibit dihydrolipoyl dehydrogenase activity leading to impaired 2-oxoglutarate-driven oxidative phosphorylation

The effect of the antiepileptic drug valproic acid (VPA) on mitochondrial oxidative phosphorylation (OXPHOS) was investigated in vitro. Two experimental approaches were used, in the presence of selected respiratory-chain substrates: (1) formation of ATP in digitonin permeabilized rat hepatocytes and (2) measurement of the rate of oxygen consumption by polarography in rat liver mitochondria. VPA (0.1-1.0 mM) was found to inhibit oxygen consumption and ATP synthesis under state 3 conditions with glutamate and 2-oxoglutarate as respiratory substrates. No inhibitory effect on OXPHOS was observed when succinate (plus rotenone) was used as substrate. We tested the hypothesis that dihydrolipoyl dehydrogenase (DLDH) might be a direct target of VPA, especially its acyl-CoA intermediates. Valproyl-CoA (0.5-1.0 mM) and valproyl-dephosphoCoA (0.5-1.0 mM) both inhibited the DLDH activity, acting apparently by different mechanisms. The decreased activity of DLDH induced by VPA metabolites may, at least in part, account for the impaired rate of oxygen consumption and ATP synthesis in mitochondria if 2-oxoglutarate or glutamate were used as respiratory substrates, thus limiting the flux of these substrates through the citric acid cycle.