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The ADP/ATP carrier (AAC) of mitochondria has been an early example for elucidating the transport mechanism alternating between the external (c-) and internal (m-) states (M. Klingenberg, Biochim. Biophys. Acta 1778 (2008) 1978-2021). An atomic resolution crystal structure of AAC is available only for the c-state featuring a three repeat transmembrane domain structure. Modeling of transport mechanism remained hypothetical for want of an atomic structure of the m-state. Previous molecular dynamics studies simulated the binding of ADP or ATP to the AAC remaining in the c-state. Here, a full description of the AAC switching from the c- to the m-state is reported using well-tempered metadynamics simulations. Free-energy landscapes of the entire translocation from the c- to the m-state, based on the gyration radii of the c- and m-gates and of the center of mass, were generated. The simulations revealed three free-energy basins attributed to the c-, intermediate- and m-states separated by activation barriers. These simulations were performed with the empty and with the ADP- and ATP-loaded AAC as well as with the poorly transported AMP and guanine nucleotides, showing in the free energy landscapes that ADP and ATP lowered the activation free-energy barriers more than the other substrates. Upon binding AMP and guanine nucleotides a deeper free-energy level stabilized the intermediate-state of the AAC2 hampering the transition to the m-state. The structures of the substrate binding sites in the different states are described producing a full picture of the translocation events in the AAC.

International audience; Cardiolipin (CL) is a unique mitochondrial phospholipid potentially affecting many aspects of mitochondrial function/processes, i.e. energy production through oxidative phosphorylation. Most data focusing on implication of CL content and mitochondrial bioenergetics were performed in yeast or in cellular models of Barth syndrome. Previous work reported that increase in CL content leads to decrease in liver mitochondrial ATP synthesis yield. Therefore the aim of this study was to determine the effects of moderate decrease in CL content on mitochondrial bioenergetics in human hepatocytes. For this purpose, we generated a cardiolipin synthase knockdown (shCLS) in HepaRG hepatoma cells showing bioenergetics features similar to primary human hepatocytes. shCLS cells exhibited a 55% reduction in CLS gene and a 40% decrease in protein expression resulting in a 45% lower content in CL compared to control (shCTL) cells. Oxygen consumption was significantly reduced in shCLS cells compared to shCTL regardless of substrate used and energy state analyzed. Mitochondrial low molecular weight supercomplexes content was higher in shCLS cells (+ 60%) compared to shCTL. Significant fragmentation of the mitochondrial network was observed in shCLS cells compared to shCTL cells. Surprisingly, mitochondrial ATP synthesis was unchanged in shCLS compared to shCTL cells but exhibited a higher ATP:O ratio (+ 46%) in shCLS cells. Our results suggest that lowered respiratory chain activity induced by moderate reduction in CL content may be due to both destabilization of supercomplexes and mitochondrial network fragmentation. In addition, CL content may regulate mitochondrial ATP synthesis yield.

Polyethylenimines (PEIs) are highly efficient non-viral transfectants, but can induce cell death through poorly understood necrotic and apoptotic processes as well as autophagy. Through high resolution respirometry studies in H1299 cells we demonstrate that the 25kDa branched polyethylenimine (25k-PEI-B), in a concentration and time-dependent manner, facilitates mitochondrial proton leak and inhibits the electron transport system. These events were associated with gradual reduction of the mitochondrial membrane potential and mitochondrial ATP synthesis. The intracellular ATP levels further declined as a consequence of PEI-mediated plasma membrane damage and subsequent ATP leakage to the extracellular medium. Studies with freshly isolated mouse liver mitochondria corroborated with bioenergetic findings and demonstrated parallel polycation concentration- and time-dependent changes in state 2 and state 4o oxygen flux as well as lowered ADP phosphorylation (state 3) and mitochondrial ATP synthesis. Polycation-mediated reduction of electron transport system activity was further demonstrated in ‘broken mitochondria’ (freeze-thawed mitochondrial preparations). Moreover, by using both high-resolution respirometry and spectrophotometry analysis of cytochrome c oxidase activity we were able to identify complex IV (cytochrome c oxidase) as a likely specific site of PEI mediated inhibition within the electron transport system. Unraveling the mechanisms of PEI-mediated mitochondrial energy crisis is central for combinatorial design of safer polymeric non-viral gene delivery systems.

Mitochondrial carriers link biochemical pathways in the mitochondrial matrix and cytosol by transporting metabolites, inorganic ions, nucleotides and cofactors across the mitochondrial inner membrane. Uncoupling proteins that dissipate the proton electrochemical gradient also belong to this protein family. For almost 35years the general consensus has been that mitochondrial carriers are dimeric in structure and function. This view was based on data from inhibitor binding studies, small-angle neutron scattering, electron microscopy, differential tagging/affinity chromatography, size-exclusion chromatography, analytical ultracentrifugation, native gel electrophoresis, cross-linking experiments, tandem-fusions, negative dominance studies and mutagenesis. However, the structural folds of the ADP/ATP carriers were found to be monomeric, lacking obvious dimerisation interfaces. Subsequently, the yeast ADP/ATP carrier was demonstrated to function as a monomer. Here, we revisit the data that have been published in support of a dimeric state of mitochondrial carriers. Our analysis shows that when critical factors are taken into account, the monomer is the only plausible functional form of mitochondrial carriers. We propose a transport model based on the monomer, in which access to a single substrate binding site is controlled by two flanking salt bridge networks, explaining uniport and strict exchange of substrates.

Mitochondria are dynamic organelles that constantly undergo fission, fusion, and movement. Increasing evidence indicates that these dynamic changes are intricately related to mitochondrial function, suggesting that mitochondrial form and function are linked. Calcium (Ca2+) is one signal that has been shown to both regulate mitochondrial fission in various cell types and stimulate mitochondrial enzymes involved in ATP generation. However, although Ca2+ plays an important role in adult cardiac muscle cells for excitation–metabolism coupling, little is known about whether Ca2+ can regulate their mitochondrial morphology. Therefore, we tested the role of Ca2+ in regulating cardiac mitochondrial fission. We found that neonatal and adult cardiomyocyte mitochondria undergo rapid and transient fragmentation upon a thapsigargin (TG)- or KCl-induced cytosolic Ca2+ increase. The mitochondrial fission protein, DLP1, participates in this mitochondrial fragmentation, suggesting that cardiac mitochondrial fission machinery may be regulated by intracellular Ca2+ signaling. Moreover, the TG-induced fragmentation was also associated with an increase in reactive oxygen species (ROS) formation, suggesting that activation of mitochondrial fission machinery is an early event for Ca2+-mediated ROS generation in cardiac myocytes. These results suggest that Ca2+, an important regulator of muscle contraction and energy generation, also dynamically regulates mitochondrial morphology and ROS generation in cardiac myocytes.

Biogenesis of mitochondria depends on the coordinated action of at least six protein translocases present in both mitochondrial membranes. They use different energy sources to drive unidirectional transport of proteins across and into mitochondrial membranes. Here we present an overview on the energetic requirements of different mitochondrial import pathways.

Uncoupling protein-2 (UCP2) is a member of the inner mitochondrial membrane anion-carrier superfamily. Although mRNA for UCP2 is widely expressed, protein expression is detected in only a few cell types, including macrophages. UCP2 functions by an incompletely defined mechanism, to reduce reactive oxygen species production during mitochondrial electron transport. We observed that the abundance of UCP2 in macrophages increased rapidly in response to treatments (rotenone, antimycin A and diethyldithiocarbamate) that increased mitochondrial superoxide production, but not in response to superoxide produced outside the mitochondria or in response to H2O2. Increased UCP2 protein was not accompanied by increases in ucp2 gene expression or mRNA abundance, but was due to enhanced translational efficiency and possibly stabilization of UCP2 protein in the inner mitochondrial membrane. This was not dependent on mitochondrial membrane potential. These findings extend our understanding of the homeostatic function of UCP2 in regulating mitochondrial reactive oxygen production by identifying a feedback loop that senses mitochondrial reactive oxygen production and increases inner mitochondrial membrane UCP2 abundance and activity. Reactive oxygen species-induction of UCP2 may facilitate survival of macrophages and retention of function in widely variable tissue environments.

The MTERF-family is a wide family of proteins identified in Metazoa and plants which includes the known mitochondrial transcription termination factors. With the aim to shed light on the function of MTERF-family members in Drosophila, we performed the cloning and characterization of D-MTERF3, a component of the most conserved group of this family. D-MTERF3 is a mitochondrial protein of 323 amino acids. Sequence analysis in seven different organisms showed that the protein contains five conserved “mTERF-motifs”, three of which include a leucine zipper-like domain. D-MTERF3 knock-down, obtained by RNAi in D.Mel-2 cells, did not affect mitochondrial replication and transcription. On the contrary, it decreased to a variable extent the rate of labelling of about half of the mitochondrial polypeptides, with ND1 being the most affected by D-MTERF3 depletion. These results indicate that D-MTERF3 is involved in mitochondrial translation. This role, likely based on protein–protein interactions, may be exerted either through a direct interaction with the translation machinery or by bridging the mitochondrial transcription and translation apparatus.

In experiments on isolated kidney and liver mitochondria, it is shown that oleate hydroperoxide induces a much smaller increase in the controlled respiration rate and DeltaPsi decrease than the same concentrations of oleate. Palmitate appears to be less efficient than oleate but more efficient than oleate hydroperoxide. In all cases, GDP and CAtr cause some recoupling, CAtr being more effective. Addition of 0.2 mM GDP before CAtr does not prevent further DeltaPsi increase by subsequent CAtr addition. On the other hand, GDP added after CAtr is without any effect. GDP partially prevents the DeltaPsi lowering by ADP at the State 4--State 3 transition if small amounts of CAtr are present. The data are consistent with the suggestion of F. Goglia and V.P. Skulachev (FASEB J. 17, 1585-1591, 2003) that fatty acid anions are translocated by mitochondrial anion carriers much better than their hydroperoxides. As to GDP recoupling, it cannot be regarded as a specific probe for uncoupling by UCPs since it can be mediated by the ATP/ADP antiporter.

To support the rapid growth of tumors, the cell can respond by increasing the number of mitochondria, in a concerted biosynthesis of mitochondrial constituents (nuclear and mitochondria encoded). Increased transcription, availability and stability of oxidative phosphorylation mRNAs, without increasing mitochondria number could also lead to more rapid energy-yielding effects. Mitochondria biogenesis and de novo formation of respiratory chain components imply coordination of nuclear and mt gene transcription. The mitochondrial mass is regulated by a number of physiopathological conditions. In response to external stimuli, mitochondria biogenesis is dependent on an orchestrated crosstalk between the nuclear and the mitochondrial genomes. Based on the higher incidence of glycolysis over oxidative phosphorylation in cancer tissues, we studied by differential proteomics the energy metabolism pathway of matched samples of normal and cancer tissue. Our results indicated that oxidative phosphorylation in cancer cells seemed altered because there is an unbalanced coordination between nuclear- and mitochondria-encoded mitochondrial proteins.

The potential role of dystrophin-mediated control of systems integrating mitochondria with ATPases was assessed in muscle cells. Mitochondrial distribution and function in skinned cardiac and skeletal muscle fibers from dystrophin-deficient (MDX) and wild-type mice were compared. Laser confocal microscopy revealed disorganized mitochondrial arrays in m. gastrocnemius in MDX mice, whereas the other muscles appeared normal in this group. Irrespective of muscle type, the absence of dystrophin had no effect on the maximal capacity of oxidative phosphorylation, nor on coupling between oxidation and phosphorylation. However, in the myocardium and m. soleus, the coupling of mitochondrial creatine kinase to adenine nucleotide translocase was attenuated as evidenced by the decreased effect of creatine on the K-m for ADP in the reactions of oxidative phosphorylation. In m. soleus, a low K-m, for ADP compared to the wild-type counterpart was found, which implies increased permeability for that nucleotide across the mitochondrial outer membrane. In normal cardiac fibers 35% of the ADP flux generated by ATPases was not accessible to the external pyruvate kinase-phosphoenolpyruvate system, which suggests the compartmentalized (direct) channeling of that fraction of ADP to mitochondria. Compared to control, the direct ADP transfer was increased in MDX ventricles. In conclusion. our data indicate that in slow-twitch muscle cells, the absence of dystrophin is associated with the rearrangement of the intracellular energy and feedback signal transfer systems between mitochondria and ATPases. As the mechanisms mediated by creatine kinases become ineffective, the role of diffusion of adenine nucleotides increases due to the higher permeability of the mitochondrial outer membrane for ADP and enhanced compartmentalization of ADP flux. (C) 2001 Elsevier Science B.V. All rights reserved.

Protein sorting into mitochondria is achieved by the concerted action of at least four translocation complexes. Vectorial transport of polypeptide chains by these complexes requires different driving forces. In particular, Δψ, matrix adenosine triphosphate and the free energy of the binding to other protein components are used in series to achieve sorting of proteins to the various mitochondrial subcompartments. The processes providing the translocation energy are presented in this review and their impact for protein sorting into and within mitochondria is discussed.

The expression of a key mitochondrial membrane component, the ADP/ATP carrier, was investigated in two aerobic yeast species, Kluyveromyces lactis and Schizosaccharomyces pombe. Although the two species differ very much in their respiratory capacity, the expression of the carrier in both yeast species was decreased under partially anaerobic conditions and was induced by nonfermentable carbon sources. The single ADP/ATP carrier encoding gene was deleted in S. pombe. The null mutant exhibits impaired growth properties, especially when cultivated at reduced oxygen tension, and is unable to grow on a nonfermentable carbon source. Our results suggest that the inability of K. lactis and S. pombe to grow under anaerobic conditions can be related in part to the absence of a functional ADP/ATP carrier due to repression of the corresponding gene expression.

To characterize the transport mechanism mediated by the mammalian mitochondrial ADP/ATP carrier (AAC), we tried to express bovine heart mitochondrial AAC (bhAAC) in Saccharomyces cerevisiae. The open reading frame of the bhAAC was introduced into the haploid strain WB-12, in which intrinsic AAC genes were disrupted. Growth of the transformant was very low in glycerol medium, and a little amount of bhAAC was detected in the mitochondrial membrane. For improvement of bhAAC expression in WB-12, we introduced DNA fragments encoding chimeric bhAACs, in which the N-terminal region of the bhAAC extending into the cytosol was replaced by the corresponding regions of the type 1 and type 2 yeast AAC isoforms (yAAC1 and yAAC2). These transformants grew well, and the amounts of the chimeric bhAACs in their mitochondria were as high as that of yAAC2. The carriers expressed showed essentially the same ADP transport activities as that of AAC in bovine heart mitochondria.

The purpose of this work was to investigate the mechanism of regulation of mitochondrial respiration in vivo in different muscles of normal rat and mice, and in transgenic mice deficient in desmin. Skinned fiber technique was used to study the mitochondrial respiration in the cells in vivo in the heart, soleus and white gastrocnemius skeletal muscles of these animals. Also, cardiomyocytes were isolated from the normal rat heart, permeabilized by saponin and the “ghost” (phantom) cardiomyocytes were produced by extraction of myosin with 800 mM KCl. Use of confocal immunofluorescent microscopy and anti-desmin antibodies showed good preservation of mitochondria and cytoskeletal system in these phantom cells. Kinetics of respiration regulation by ADP was also studied in these cells in detail before and after binding of anti-desmine antibodies with intermediate filaments. In skinned cardiac or soleus skeletal muscle fibers but not in fibers from fast twitch skeletal muscle the kinetics of mitochondrial respiration regulation by ADP was characterized by very high apparent K m (low affinity) equal to 300–400 μM, exceeding that for isolated mitochondria by factor of 25. In skinned fibers from m. soleus, partial inhibition of respiration by NaN 3 did not decrease the apparent K m for ADP significantly, this excluding the possible explanation of low apparent affinity of mitochondria to ADP in these cells by its rapid consumption due to high oxidative activity and by intracellular diffusion problems. However, short treatment of fibers with trypsin decreased this constant value to 40–70 μM, confirming the earlier proposition that mitochondrial sensitivity to ADP in vivo is controlled by some cytoplasmic protein. Phantom cardiomyocytes which contain mostly mitochondria and cytoskeleton and retain the normal shape, showed also high apparent K m values for ADP. Therefore, they are probably the most suitable system for studies of cellular factors which control mitochondrial function in the cells in vivo. In these phantom cells anti-desmin antibodies did not change the kinetics of respiration regulation by ADP. However, in skinned fibers from the heart and m. soleus of transgenic desmin-deficient mice some changes in kinetics of respiration regulation by ADP were observed: in these fibers two populations of mitochondria were observed, one with usually high apparent K m for ADP and the second one with very low apparent K m for ADP. Morphological observations by electron microscopy confirmed the existence of two distinct cellular populations in the muscle cells of desmin-deficient mice. The results conform to the conclusion that the reason for observed high apparent K m for ADP in regulation of oxidative phosphorylation in heart and slow twitch skeletal muscle cells in vivo is low permeability of mitochondrial outer membrane porins but not diffusion problems of ADP into and inside the cells. Most probably, in these cells there is a protein associated with cytoskeleton, which controls the permeability of the outer mitochondrial porin pores (VDAC) for ADP. Desmin itself does not display this type of control of mitochondrial porin pores, but its absence results in appearance of cells with disorganised structure and of altered mitochondrial population probably lacking this unknown VDAC controlling protein. Thus, there may be functional connection between mitochondria, cellular structural organisation and cytoskeleton in the cells in vivo due to the existence of still unidentified protein factor(s).

Maintenance of an electrochemical proton gradient across the mitochondrial inner membrane against the significant proton permeability of the membrane accounts for 25–30% of resting oxygen consumption in hepatocytes. It has been proposed that proton leak could be a significant contributor to resting metabolic rate in mammals if it were present in other tissues. Mitochondria were isolated from the major oxygen-consuming tissues (liver, kidney, brain and skeletal muscle) of the rat. In each tissue, the mitochondria showed significant proton leak with the same characteristic non-linear dependence on membrane potential. Liver and kidney mitochondria showed similar membrane proton permeability per mg of mitochondrial protein; brain and muscle permeabilities were greater when expressed in this way. Differences in the kinetic response of the substrate oxidation and phosphorylating systems to membrane potential were observed. The substrate oxidation system was more active in kidney, brain and skeletal muscle mitochondria than in liver mitochondria per mg of mitochondrial protein. Liver and kidney phosphorylating systems were less active than brain and skeletal muscle per mg of mitochondrial protein. The control of oxidative phosphorylation was also assessed. The distribution of control in mitochondria isolated from the four tissue types was found to be similar.

The interaction of several classes of detergents with mitochondrial ATP / ADP carrier (AAC) was studied. The detergents that were best suited for solubilization of active AAC differed in several physico-chemical properties, but contained relatively rigid or planar hydrophobic (sub)moieties. Based on specific binding of AAC to Blue Sepharose, a novel method for the purification of the AAC was developed. The new method gave AAC samples which were devoid of non-essential lipids and allowed to purify AAC isoenzymes from several species and tissues to a significantly higher degree of purity than that achieved up to now. Western blot analysis of purified AACs with an antiserum against chicken heart AAC confirmed that immunological variability is more important between tissues than between species. In contrast to liver and kidney AACs, brain AAC displayed similar antigenic properties to heart AAC.

The conditions of treatment of human skeletal muscle fibers from M. vastus lateralis with saponin were optimized to achieve complete permeabilization of cell membrane at intact mitochondrial oxidative phosphorylation. After 30 min of incubation with saponin all lactate dehydrogenase, 50% of creatine kinase, 30% of adenylate kinase and less than 20% of citrate synthase was released into the permeabilization medium. These skinned fibers behave similar to isolated mitochondria from human skeletal muscle: (i) the respiration with mitochondrial substrates can be stimulated by ADP, (ii) inhibited by carboxyatractyloside and (iii) it is possible to detect fluorescence changes of mitochondrial NAD(P)H on additions of substrates, uncoupler and cyanide. From a comparison of rates of respiration per cytochrome aa3 content of isolated human skeletal muscle mitochondria and saponin-skinned muscle fibers it was possible to calculate that almost 85% of mitochondria in those fibers are accessible for the investigation of oxidative phosphorylation. As shown by the investigation of biopsy samples of two patients with undefined myopathies these fibers are a suitable object for the replacement of isolated mitochondria in the diagnosis of mitochondrial myopathies and encephalomyopathies.

Formation of a transmembrane proton gradient, tight binding of adenine nucleotides and ATPase activity were followed during hydrolysis of a limited amount of ATP by thylakoids preilluminated in the presence of DTT. The following results were obtained: (1) While a micromolar concentration of ADP caused complete deactivation of the ATPase within 15 s, the same concentration of ATP sustained enzyme activity for more than 1 min due to a small proton gradient generated by hydrolysis of the ATP. (2) In the presence of ADP, steady-state ATPase activity and levels of tightly bound nucleotides are inversely influenced by ΔpH. When ADP is replaced by ATP, the activity curve is shifted towards much lower ΔpH values and is not significantly different from the ΔpH curve obtained in the absence of medium nucleotides. (3) At low medium ATP concentration (5 μM), ATP as well as ADP is incorporated into membrane-bound CF1; the ratio of tightly bound ATP ADP increases while the sum of ATP plus ADP decreases with increasing ΔpH. An enzyme with a tightly bound ATP can be active or inactive, depending on the actual ΔpH. (4) Hydrolysis of ATP by thylakoids containing a certain percentage of active ATPases, effects extensive reactivation of inactive enzyme molecules due to coupled ΔpH formation. At exhaustion of the substrate, however, the total enzyme population is inactivated within 1 min. Inactivation is a synergic result of the decay of the proton gradient and tight binding of the hydrolysis product ADP.

The stimulation of liver mitochondrial metabolism by hormones that raise cytosolic [Ca2+] can be explained, in part, by a 20–40% increase in mitochondrial volume which activates many enzymes associated with the inner membrane. The swelling is caused by a rise in mitochondrial [Ca2+], inhibition of mitochondrial pyrophosphatase and a consequent increase in matrix [PPi]. PPi interaction with the adenine nucleotide carrier stimulates electrogenic transport of K+ into the matrix. At higher [Ca2+] nonspecific increases in permeability occur through a calcium-dependent interaction of peptidyl-prolyl cis-trans isomerase with the carrier. This can be blocked by cyclosporin A.

The respective contribution of exogenous and intramitochondrially formed ATP to D-glucose phosphorylation by mitochondria-bound hexokinase was examined in both rat liver and pancreatic islet mitochondria by comparing the generation of D-glucose 6-[32P]phosphate from exogenous [gamma-32P]ATP to the total rate of D-[U-14C]glucose phosphorylation. In liver mitochondria, the fractional contribution of exogenous ATP to D-glucose phosphorylation ranged from 4 to 74%, depending on the availability of endogenous ATP formed by either oxidative phosphorylation or in the reaction catalyzed by adenylate kinase. Likewise, in islet mitochondria exposed to exogenous ATP but deprived of exogenous nutrient, about 60% of D-glucose phosphorylation was supported by mitochondrial ATP. Such a fractional contribution was further increased in the presence of ADP and succinate, and suppressed by mitochondrial poisons. It is concluded that, in islet like in liver mitochondria, mitochondrial ATP is used preferentially to exogenous ATP as a substrate for D-glucose phosphorylation by mitochondria-bound hexokinase. This may favour the maintenance of a high cytosolic ATP concentration in glucose-stimulated islet cells.

F1ATPase were studied by FT-ESEEM spectroscopy, using Mn(II) as a paramagnetic probe, which replaces the naturally occurring Mg(II) and maintains the enzyme catalytic activity. Purified F1ATPase still containing three endogenous tightly bound nucleotides, named MF1 (1,2), was obtained under mild conditions, whereas a harsher treatment gave a fully nucleotide depleted enzyme, named MF1 (0,0). When MF1(1,2) was loaded with Mn(II) in 1:0.8 ratio, the spectrum showed evidence of a nitrogen interacting with the metal, while this interaction was not present in the spectrum of MF1(0,0) loaded with Mn(II) in the same ratio. However, when MF1(0,0) was loaded with 2.4 Mn(II), the spectrum showed metal-nitrogen interaction resembling that of MF1(1,2) loaded with Mn(II) in 1:0.8 ratio. When MF1(1,2) was loaded with 2.4 Mn(II) the metalnitrogen interaction signal remained and a phosphorous coordination to the metal was also evident, indicating a binding of Mn to a site containing a tightly bound nucleotide but metal free. These results strongly support the role of the metal alone in structuring the catalytic sites of the enzyme while ESEEM technique appears to be a sensitive and suitable spectroscopic method for conformational studies of MF1 with the advantage of using proteins in frozen solution.

To investigate whether or not the mitochondrial intermembrane space together with the extramitochondrial space form a homogeneous pool for adenine nucleotides, rat-heart mitochondria were studied in reconstituted systems with pyruvate kinase and ADP-producing enzymes with varied localization. In the hexokinase system, ADP is produced extramitochondrially by added yeast hexokinase, whereas in the creatine kinase system mitochondrial creatine kinase is responsible for ADP regeneration in the intermembrane space. The dependence of mitochondrial respiration on the extramitochondrial [ ATP ] [ ADP ] ratio in both systems was investigated experimentally and by means of computer simulation. Near the resting state, higher [ ATP ] [ ADP ] ratios were found in the creatine kinase system than in the hexokinase system at the same rate of respiration. This and the maintaining of a substantial creatine kinase-stimulated respiration in the presence of pyruvate kinase in excess is explained by a two-compartment model considering diffusion limitations of adenine nucleotides. A diffusion rate constant of (8.7 ± 4.7) 104μl · mg−1 · min−1 for ADP and ATP was estimated, resulting in rate-dependent concentration differences up to 13.7 μM AdN between the extramitochondrial space and the AdN-translocator at the maximum rate of oxidative phosphorylation of rat-heart mitochondria. The results support the assumption that ADP diffusion towards the AdN-translocator is limited if its extramitochondrial concentration is low, resulting in a dynamic compartmentation of adenine nucleotides in the mitochondrial intermembrane space.

The activation state of the chloroplast stromal enzymes fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase and ribulose-5-phosphate kinase was assayed by injecting the chloroplasts into an assay medium where the chloroplasts are immediately lysed and the enzymic assay is terminated 15–30 s later. (1) After the onset of illumination the activities of fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase rise 20- and 30-fold, respectively, and rapidly decrease again when the light is turned off. The light activation of both enzymes shows a substrate requirement. The catalytic activity of the activated enzyme is dependent on the pH and the Mg 2+ concentration in the assay medium, with the fructose-1,6-bisphosphatase more markedly affected. (2) The light activation of ribulose-5-phosphate kinase occurs even more rapidly than that of the other two enzymes and leads to about 10-fold activation. Moreover, the inactivated form of the enzyme, as extracted from chloroplasts in the dark, is strongly inhibited when ADP is added to the assay medium. (3) These data clearly indicate that chloroplasts have the ability to inactivate the three mentioned Calvin cycle enzymes virtually completely in the dark phase by a combined effect of changes of the reductive state of the electron-transport carrier on all three enzymes, changes of stromal Mg 2+ and pH on fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase and changes of stromal ATP ADP ratios on ribulose-5-phosphate kinase.

(1) The effect of phospholipids on a preparation containing the ATPase complex and the adenine nucleotide carrier is studied in the presence of ligands known to affect the conformation of these components of the mitochondrial inner membrane. (2) When ATPase activity is abolished by phospholipid depletion, the reactivation induced by phosphatidylcholine is prevented by the simultaneous addition of ATP. ADP partially reproduces the ATP effect. AMP, GTP, UTP, and Pi are ineffective. (3) The influence of ATP is associated with reduced phospholipid binding to the membrane fragments and is reversible. The ATP effect on reconstitution is not manifest when phosphatidylcholine is added together with negatively charged phospholipids. (4) Carboxyatractyloside does not modify the phospholipid-ATPase complex interaction but bongkrekic acid is as effective as ATP. In the presence of ADP, the influence of bongkrekic acid is considerably increased. (5) It is concluded that the binding of ATP to the adenine nucleotide carrier enables the complex to select between the charged and uncharged phospholipids. As a result of the carrier conformational change, the ATPase complex is induced to prefer a negatively charged phospholipid environment.

Endogenous adenine nucleotide contents of isolated intact chloroplasts and activities of the H+-ATPase were measured in parallel under various experimental conditions. In chloroplasts isolated from spinach grown in summer, ATPase activity declines upon light-dark transition in a biphasic manner. Initial rapid inactivation of about 50% of the ATPases corresponds with the rapid increase of the ADP and decrease of the ATP levels. The residual ATPases are slowly deactivated, probably due to the reversal of thiol modulation. In isolated chloroplasts from winter plants the phase of rapid inactivation is largely absent. Those chloroplasts have a low content of endogenous nucleotides. When the ratio of internal ATP ADP is decreased by the addition of 3-phosphoglycerate or the uncoupler FCCP, the activity of the ATPase decreases. The relationship between activity and ATP ADP ratio is nearly linear. Inorganic phosphate, although being ineffective in changing the internal nucleotide levels, significantly raises the ATPase activity. The results suggest that in whole chloroplasts the ATPase not only is regulated by the thioredoxin system but also controlled by the internal ATP ADP ratio. This kind of control is referred to the nucleotide-dependent regulatory mechanism previously demonstrated in isolated thylakoid suspension.

The kinetics in the millisecond range of ADP, ATP counterexchange in rat liver mitochondria were investigated using a quench-flow apparatus. The exchange was stopped with atractylate and the mitochondria were separated by centrifugation. 1. 1. After correcting for leakage due to flow stress, apparent biphasic exchange kinetics were observed, with a more rapid phase within 100–200 ms, which extends to 10% of the total exchange. In general, the extent of the rapid phase increased when the translocation rate was changed under various conditions, in agreement with the model of the quench mechanism by atractylate. 2. 2. The nature of the “rapid” phase was analyzed in the “steady” and “transient” state of the translocation and was shown to be caused by a delayed binding of atractylate due to competition with ADP. This quench delay results in a residual exchange which could explain the rapid part of the kinetics. 3. 3. Deenergization of mitochondria by valinomycin or by uncoupler largely abolishes the rapid kinetic phase. This is explained by an increased availability of carrier sites at the outer face of the membrane to atractylate in the deenergized state, resulting in a more rapid quench. 4. 4. The interpretation of the rapid exchange phase as a function of carrier sites accessible to atractylate quenching at the outer membrane face was simulated by a computer program based on the reorientating carrier model. With a set of rate constants, an approximate fit for the extent of quench delay with the experimental data is obtained.

The relationship between the respiration rate and the intra- and extramito-chondrial adenine nucleotides was investigated in isolated rat liver mitochondria. For the determination of adenine nucleotide patterns in both compartments a new procedure was developed, based on the evaluation of these metabolites from incubation of various amounts of mitochondria under identical stationary states of oxidative phosphorylation. These identical states were adjusted by addition of appropriate amounts of hexokinase to a glucose-containing incubation mixture. Adenine nucleotides were measured in aliquots of the total extract of the incubation mixture without any separation. The concentrations of the adenine nucleotides in both compartments were obtained from a plot of the total concentration of these species versus mitochondrial protein. Disturbances of this method by unspecific efflux of adenine nucleotides could be excluded. The results obtained for the total adenine nucleotide content (12 nmol · mg −1 protein) and the intramitochondrial [ATP]/[ADP] ratio (about 4 in the resting state) are in good agreement with data obtained by other methods. Strong evidence is provided for a decrease of the intramitochondrial [ATP]/[ADP] ratio with increasing rate of oxygen consumption. Therefore it is not necessary to assume a microcompartmentation of the intramitochondrial adenine nucleotide pool in respect to the ATPase reaction and the adenine nucleotide translocation.

31P-NMR has been used to study the increase of delta pH in mitochondria by externally added ATP. Freshly prepared mitochondria was treated with N-ethylmaleimide to inhibit the exchange between internal and external P(i). Upon addition of ATP, phosphocreatine (30 mM) and creatine kinase to a NMR sample of mitochondria suspension (approx. 120 mg protein/ml) at 0 degrees C, an increase of delta pH by approx. 0.5 pH unit was observed. However the increased delta pH could not be maintained, but slowly decayed along with the increase of external ADP/ATP ratio. Further addition of valinomycin to the suspension induced a larger delta pH (approx. 1) which was maintained by the increased rate of internal ATP hydrolysis as seen in the growth of the internal P(i) peak intensity in NMR spectra and the concomitant decrease of the external phosphocreatine peak. The external P(i) and ATP peaks stayed virtually constant. When carboxyatractyloside was added to inhibit the ATP/ADP translocase, the internal P(i) increase was stopped and the delta pH decayed. These observations in conjunction with those made earlier in respiring mitochondria clearly show the reversible nature of the ATPase function in which the internal ATP hydrolysis is associated with outward pumping of protons.

The decrease in respiration rate following thyroidectomy is preceded by changes in the lipid composition of the mitochondrial membrane (Hoch, F.L., Subramanian, C., Dhopeshwarkar, G.A. and Mead, J.F. (1981) Lipids 16, 328–334) and in concert, changes in the kinetic parameters of the adenine nucleotide translocase (Mak, I.T., Shrago, E. and Elson, C.E. (1981) Fed. Proc. 40, 398). To demonstrate that physiological adaptation also involves this sequence of events, rats were housed at 8°C for 3–4 weeks. Cold adaptation resulted in a modest (5%) increase in the unsaturation index for the mitochondrial fatty acids comprised of a significant increase in arachidonic acid and a reciprocal decrease in linoleic acid. Phospholipid analysis indicated that cold adaptation increased the mitochondrial phosphatidylethanolamine and reciprocally decreased the phosphatidylcholine content. Concomitantly, cold adaptation resulted in 25–30% increases in rat liver mitochondrial respiratory activities without changing the respiratory control or ADP/O ratios. The kinetic parameters of the adenine nucleotide translocase were determined by the back-exchange method (Pfaff, E. and Klingenberg, M. (1968) Eur. J. Biochem. 6, 66–79). At 0–4 and 10°C, the V max and K m of the cold-adapted rat liver adenine nucleotide translocase were not distinguishable from the control values. The K i values determined by Dixon plot studies for atractylate and palmitoyl-CoA were also comparable between the two groups. However, at 25 and 37°C, cold-adapted rat liver adenine nucleotide translocase exhibited a 20% increase in V max and a 20% decrease in K m for external ADP. The results suggest that one adaption to a cold environment involves hormone-mediated changes in the lipid composition in the mitochondrial membranes which in turn modulate the adenine nucleotide translocase and subsequent respiratory activities.

The control of mitochondrial ATP synthesis by the extramitochondrial adenine nucleotide pattern was investigated with rat liver mitochondria. It is demonstrated that any stationary state between the two limit states of maximum activity (state 3) and of resting activity (state 4) can be obtained by a hexokinase-glucose trap as an ADP-regenerating system. These intermediate states are characterized by stationary respiratory rates, stationary redox levels of the cytochromes b and c and stationary levels of extramitochondrial ATP and ADP between the rates and levels of the limit states. At a constant concentration of inorganic phosphate the activity of mitochondria between the limit states is controlled by the extramitochondrial ATP/ADP ratio independent of the total concentration of adenine nucleotides present. The control range was found to be between ratios of about 5 and 100 at 10 mM phosphate. At lower ratios the mitochondria are in their maximum phosphorylating state. With succinate+rotenone and glutamate+malate the same control range was observed, indicating that it is independent of the nature of substrate oxidized. The results suggest that in the control range the mitochondrial activity is limited by the competition of ADP and ATP for the adenine nucleotide translocator.

Chloroplast thylakoids show a light and Mg 2+ -dependent ATP / ADP γ-phosphate-exchange activity in the absence of inorganic phosphate. This exchange activity is inhibited by the energy-transfer inhibitor phlorizin, indicating that it is catalyzed by the chloroplast ATPase. In contrast to ATP / inorganic phosphate exchange no ATP / ADP exchange is observed in the dark after preillumination in presence of dithiothreitol. No net hydrolysis is measured in the light which could account for apparent ATP / ADP exchange. The results suggest that two cooperating catalytic sites approach each other during the catalytic process in a way that a transfer of phosphate from bound ATP on site 1 to bound ADP on site 2 occurs without intermediate inorganic-phosphate release, and might proceed without intermediate ATP hydrolysis and resynthesis. In the present paper evidence is given that the exchange process requires less energy than photophosphorylation.

1. 1. Beef heart submitochondrial particles prepared by sonication loaded with ADP and MgCl2 are competent in ADP/ATP transport. The Δψ and ΔpH developed by these particles have been determined, and the electrogenic nature of the ADP/ATP transport that they catalyze has been assessed. 2. 2. MgCl2 particles, prepared by sonication, oxidizing succinate in the presence of oligomycin develop a Δψ of 95 mV (positive inside) and a ΔpH of 1.5 pH units (acidic inside), as measured by the distribution of S14CN− (Δψ) and [14C]methylamine (ΔpH) using the flow dialysis technique. The Δψ values determined with the thiocyanate electrode are somewhat higher (110–130 mV). Δψ and ΔpH are partly interchangeable. 3. 3. The fluorescent dye, dipropylthiodicarbocyanine, and the absorbing dye, neutral red, are suitable probes for continuous, qualitative monitoring of Δψ and ΔpH respectively. The Δψ and ΔpH responses to respiratory activity in particles prepared by sonication are opposite to those found with intact mitochondria, in agreement with the reverse polarity of the membrane in particles prepared by sonication. 4. 4. ATP added to oligomycin-pretreated particles prepared by sonication is taken up in exchange for internal ADP. Under these conditions, the fluorescence intensity of dipropylthiodicarbocyanine is transitorily quenched, which corresponds to the net influx of an excess of negative charges inside the particles. A subsequent addition of ADP results in a transient rise in fluorescence. The ATP-induced fluorescence quenching of dipropylthiocarbocyanine is abolished by addition of SCN−, a strong permeant anion which collapses the Δψ. It is inhibited by ADP in a competitive manner. This fluorescence quenching is specific for ATP among a number of nucleotides tested, is inhibited by bongkrekic acid and is highly dependent on temperature. All these features are typical properties of the ADP/ATP carrier. 5. 5. Assays with neutral red as a pH indicator show that the internal phase of particles prepared by sonication treated by oligomycin becomes more acidic upon addition of ATP. However the pH changes are markedly diminished at high concentration of K+ and totally abolished by K+ plus valinomycin and, on the contrary, enhanced by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. This suggests that the ATP-dependent changes of pH are not characteristic of the mechanism of the ADP/ATP carrier, but correspond to a passive transport of H+. 6. 6. In respiring particles prepared by sonication, addition of SCN− at a concentration of 5 mM, sufficient to collapse the Δψ, strongly modifies the kinetics of ATP4−ex/ADP3−in exchange, but not that of ADP3−ex/ADP3−in. It increases the Km value for ATPex and decreases the maximal velocity of transport. Nigericin has no effect. 7. 7. In non-respiring particles prepared by sonication, addition of a sufficiently high concentration of SCN− (30 mM) induces a transient diffusion potential which results in the inhibition of ATP4−ex/ADP3−in exchange. When the excess of negative charges carried inside the particles by SCN− are neutralized by the simultaneous entry of K+, the ATP4−ex/ADP3−in exchange remains unaltered. 8. 8. The above data indicate that ADP/ATP transport across the mitochondrial membrane is primarily electrogenic.

Even when oxidative phosphorylation is blocked completely by addition of high concentrations of oligomycin plus aurovertin, the addition of ADP to a suspension of mitochondria containing a high concentration of ATP inside the mitochondria induces a stimulation of respiration and oxidation of nicotinamide nucleotide. It is concluded that transport of ADP into mitochondria with a high endogenous ATP/ADP ratio requires energy.

Purified pea mitochondrial F1-ATPase reconstituted oxidative phosphorylation in both partially and completely F1-depleted bovine heart mitochondrial membranes. The isolated plant enzyme exhibited high rates of ATP synthesis when combined with bovine heart membranes, suggesting great evolutionary conservation of the ATP synthase complex in mitochondria.

Na+-dependent ADP/ATP exchange activity, of a (Na+ + K+)-dependent ATPase preparation from eel electric organ, was measured in terms of the incorporation of 14C into ATP during incubations with unlabeled ATP and [14C]ADP. Estimates of initial rates of exchange were possible by keeping changes in nucleotide concentrations, from both exchange and extraneous hydrolytic processes, to less than 10%. Under these conditions, increases in MgCl2 concentration, from 0.2 to 3 mM, generally inhibited this exchange activity. The concentrations of free Mg2+, Mg · ATP, and Mg · ADP present, with a range of MgCl2, ATP, and ADP concentrations, were calculated from measured dissociation constants. Inhibition was associated with Mg · ATP as well as with Mg2+, at concentrations from 0.4 to 1 mM (Mg · ADP, in the same concentration range, probably inhibited also). The affinity of the enzyme for these inhibitors is in fair correspondence with demonstrated affinities for Mg2+, Mg · ATP, and Mg · ADP at low affinity substrate sites, measured kinetically. These observations are considered in terms of a dimeric enzyme with high and low affinity substrates sites: ADP/ATP exchange being catalyzed at the high affinity sites, with inhibition occurring through occupancy by Mg2+, Mg · ATP, or Mg · ADP, of the low affinity sites, thereby pulling the reaction process away from those steps involved in exchange.

1. 1. Oxygen evolution by reconstituted chloroplasts with 3-phosphoglycerate as substrate was inhibited by ADP. This inhibition was overcome by increased concentrations of 3-phosphoglycerate or by the addition of excess ATP. 2. 2. The initial rate of 3-phosphoglycerate reduction by chloroplast stromal extracts, measured as 3-phosphoglycerate-dependent oxidation of NADPH, was also dependent on the concentrations of 3-phosphoglycerate, ADP and ATP. 3. 3. Within the range of concentrations of 3-phosphoglycerate, ADP and ATP expected to occur within intact chloroplasts, the rate of 3-phosphoglycerate (PGA) reduction by stromal extract was proportional to the ratio [ATP] [PGA]/[ADP]. 4. 4. These results are consistent with the notion that the substrates and products of 3-phosphoglycerate kinase control the rate of 3-phosphoglycerate reduction via a mass action effect.