Control of mitochondrial respiration

The control theory of Kacser and Burns [in: Rate Control of Biological Processes (Davies, D.D. ed) pp. 65-104, Cambridge University Press, London, 1973] and Heinrich and Rapoport [Eur. J. Biochem. (1974) 42, 97-105] has been used to quantify the amount of control exerted by different steps on mitochondrial oxidative phosphorylation in rat-liver mitochondria. Inhibitors were used to manipulate the amount of active enzyme. The control strength of the adenine nucleotide translocator was measured by carrying out titrations with carboxyatractyloside. In state 4, the control strength of the translocator was found to be zero. As the rate of respiration was increased by adding hexokinase, the control strength of the translocator increased to a maximum value of approximately 30% at approximately 80% of state 3 respiration. In state 3, control of respiration is distributed between a number of steps, including the adenine nucleotide translocator, the dicarboxylate carrier and cytochrome c oxidase. The measured values for the distribution of control agree very well with those calculated with the aid of a model for mitochondrial oxidative phosphorylation developed by Bohnensack et al. [Biochim. Biophys. Acta (1982) 680, 271-280].     

Hexokinase of rat brain mitochondria: relative importance of adenylate kinase and oxidative phosphorylation as sources of substrate ATP, and interaction with intramitochondrial compartments of ATP and ADP

Interactions between intramitochondrial ATP-generating, ADP-requiring processes and ATP-requiring, ADP-generating phosphorylation of glucose by mitochondrially bound hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) have been investigated using well-coupled mitochondria isolated from rat brain. ADP generated by mitochondrially bound hexokinase was more effective at stimulating respiration than was ADP generated by hexokinase dissociated from the mitochondria, and pyruvate kinase was less effective as a scavenger of ADP generated by the mitochondrially bound hexokinase than was the case with ADP generated by the dissociated enzyme. These results indicate that ADP generated by the mitochondrially bound enzyme is at least partially sequestered and directed toward the mitochondrial oxidative phosphorylation apparatus. Under the conditions of these experiments, the maximum rate of ATP production by oxidative phosphorylation was approximately 10-fold greater than the maximum rate of ATP generation by the adenylate kinase reaction. Moreover, during periods of active oxidative phosphorylation, adenylate kinase made no detectable contribution to ATP production. Thus, adenylate kinase does not represent a major source of ATP for hexokinase bound to actively phosphorylating brain mitochondria. With adenylate kinase as the sole source of ATP, a steady state was attained in which ATP formation was balanced by utilization in the hexokinase reaction. In contrast, when oxidative phosphorylation was the source of ATP, a steady state rate of Glc phosphorylation was attained, but it was equivalent to only about 40-50% of the rate of ATP production and thus there was a continued net increase in ATP concentration in the system. Rates of Glc phosphorylation with ATP generated by oxidative phosphorylation exceeded those seen with equivalent levels of exogenously added ATP. Moreover, at total ATP concentrations greater than approximately 0.2 mM, hexokinase bound to actively phosphorylating mitochondria was unresponsive to continued slow increases in ATP levels; acute increase in ATP (by addition of exogenous nucleotide) did, however, result in increased hexokinase activity. The relative insensitivity of mitochondrially bound hexokinase to extramitochondrial ATP suggested dependence on an intramitochondrial pool (or pools) of ATP during active oxidative phosphorylation. Two intramitochondrial compartments of ATP were identified based on their selective release by inhibitors of electron transport or oxidative phosphorylation. These compartments were distinguished by their sensitivity to inhibitors and the kinetics with which they were filled with ATP generated by oxidative phosphorylation. Exogenous glycerol kinase competed effectively with mitochondrially bound hexokinase for extramitochondrial ATP, with relatively low levels of glycerol kinase completely inhibiting phosphorylation of Glc.(ABSTRACT TRUNCATED AT 400 WORDS)     

Micromolar concentrations of Al3+ induce phase separation, aggregation and dye release in phosphatidylserine-containing lipid vesicles

It has been proposed that hexokinase bound to mitochondria occupies a preferred site to which ATP from oxidative phosphorylation is channeled directly (Bessman, S. (1966) Am. J. Medicine 40, 740-749). We have investigated this problem in isolated Zajdela hepatoma mitochondria. Addition of ADP to well-coupled mitochondria in the presence of an oxidizable substrate initiates the synthesis of glucose 6-phosphate via bound hexokinase. This reaction is only partially inhibited by oligomycin, carboxyatractyloside, carbonyl cyanide m-chlorophenylhydrazone (CCCP) or any combination of these, suggesting a source of ATP in addition to oxidative phosPhorylation. This source appears to be adenylate kinase, since Ado2P5, an inhibitor of the enzyme, suppresses hexokinase activity by about 50% when added alone or suppresses activity completely when added together with any of the inhibitors of oxidative phosphorylation. Ado2P5 does not uncouple oxidative phosphorylation nor does it inhibit ADP transport (state 3 respiration) or hexokinase. The relative amount of ATP contributed by adenylate kinase is dependent upon the ADP concentration. At low ADP concentrations, glucose phosphorylation is supported by oxidative phosphorylation, but as the adenine nucleotide translocator becomes saturated the ATP contributed by adenylate kinase increases due to the higher apparent Km of the enzyme. Under conditions of our standard experiment ([ADP] = 0.5 mM), adenylate kinase provides about 50% of the ATP used by hexokinase in well-coupled mitochondria. In spite of this, externally added ATP supported higher initial rates of hexokinase activity than ADP. Our findings demonstrate that oxidative phosphorylation is not a specific or preferential source of ATP for hexokinase bound to hepatoma mitochondria. The apparent lack of a channeling mechanism for ATP to hexokinase in these mitochondria is discussed.     

Characterisation of the control of respiration in potato tuber mitochondria using the top-down approach of metabolic control analysis.

Control over oxidative phosphorylation by purified potato mitochondria was determined using the top-down approach of metabolic control analysis. The control over the respiration rate, phosphorylation rate, proton-leak rate and proton motive force exerted by the respiratory chain, phosphorylation reactions and the proton leak were measured over a range of phosphorylation rates from resting (state 4) to maximal (state 3). These rates were obtained by adding different amounts of hexokinase in the presence of glucose, or different amounts of oligomycin in the presence of ADP. The respiratory substrate was NADH or succinate, both of which feed electrons directly to ubiquinone. The rate of oxygen consumption by the alternative oxidase pathway was negligible with NADH as substrate but was measurable with succinate and was subtracted. Control over the respiration rate in potato mitochondria was predominantly exerted by the respiratory chain at all rates except close to state 4, where control by the proton leak was equally or more important. For oxidation of NADH, the flux control coefficient over the respiration rate exerted by the respiratory chain in state 3 was between 0.8 and 1.0, while in state 4, control over the respiration rate was shared about equally between the chain and the proton leak. The control over the phosphorylation rate was predominantly exerted by the respiratory chain, although at low rates control by the phosphorylation system was also important. For oxidation of NADH, the flux control coefficient over the phosphorylation rate exerted by the respiratory chain in state 3 was 0.8-1.0, while near state 4 the flux control coefficients over the phosphorylation rate were about 0.8 for the phosphorylation system and 0.25 for the chain. Control over the proton leak rate was shared between the respiratory chain and the proton leak; the phosphorylation system had negative control. For oxidation of NADH, the flux control coefficients over the leak rate in state 3 were 1.0 for the leak, 0.4 for the chain and -0.4 for the phosphorylation system, while in state 4 the flux control coefficients over leak rate were about 0.5 for the leak and 0.5 for the chain. Control over the magnitude of the protonmotive force was small, between -0.2 and +0.2, reflecting the way the system operates to keep the protonmotive force fairly constant; the respiratory chain and the phosphorylation system had equal and opposite control and there was very little control by the proton leak except near state 4.     

Rate-controlling steps of oxidative phosphorylation in rat liver mitochondria. A synoptic approach of model and experiment

The contribution of different steps to the control of oxidative phosphorylation in isolated rat liver mitochondria was investigated by a combination of experiments and computer simulations. The parameters of the mathematical model of phosphorylating mitochondria were derived from experimental data. The model correctly describes the competition between ATP utilization inside and outside mitochondria for the ATP generated in mitochondria. On the basis of the good agreement between experiments and simulations, the contribution of different steps to the control of respiration was estimated by computing their control strengths, i.e., the influence of their activities on the rate of respiration. The rate-controlling influences vary depending on the load of oxidative phosphorylation. The predominant steps are: in the fully active state (State 3) — the hydrogen supply to the respiratory chain; in the resting state (State 4) — the proton leak of the mitochondrial inner membrane; in states of non-maximum ATP export — the adenine nucleotide translocator. Titrations of respiration with phenylsuccinate, antimycin, oligomycin and carboxyatractyloside completely support these conclusions.     

Adenylate kinase is a source of ATP for tumor mitochondrial hexokinase

It has proposed that hexokinase bound to mitochondria occupies a preferred site to wich ATP from oxidative phosphorylation is channeled directly (Bessman, S. (1966) Am. J. Medicine 40, 740–749). We have investigated this problem in isolated Zajdela hepatoma mitochondria. Addition of ADP to well-coupled mitochondria in the presence of an oxidizable substrate initiates the synthesis of glucose 6-phosphate via bound hexokinase. This reaction is only partially inhibited by oligomycin, carboxyatractyloside, carbonyl cyanide m-chlorophenylhydrazone (CCCP) ot any combination of these, suggesting a source of ATP in addition to oxidative phosphorylation. This source appears to be adenylate kinase, since Ado₂P₅, an inhibitor of the enzyme, suppresses hexokinase activity by about 50% when added alone or suppresses activity completely when added together with any of the inhibitors of oxidative phosphorylation. Ado₂P₅ does not uncouple oxidative phosphorylation nor does it inhibit ADP transport (state 3 respiration) or hexokinase. The relative amount of ATP contributed by adenylate kinase is dependent upon the ADP concentration. At low ADP concentraions, glucose phosphorylation is supported by oxidative phosphorylation, but as the adenine nucleotide translocator becomes saturated the ATP contributed by adenylate kinase increases due to the higher apparent K_m of the enzyme. Under conditions of our standard experiment ([ADP] = 0.5 mM), adenylate kinase provides about 50% of the ATP used by hexokinase in well-coupled mitochondria. In spite of this, externally added ATP supported higher rates of hexokinase activity than ADP. Our findings demonstrate that oxidative phosphorylation is not a specific or preferential source of ATP for hexokinase bound to hepatoma mitochondria. The apparent lack of a channeling mechanism for ATP to hexokinase in these mitochondria is discussed.     

‘Idealized’ State 4 and State 3 in Mitochondria vs. Rest and Work in Skeletal Muscle

A computer model of oxidative phosphorylation (OXPHOS) in skeletal muscle is used to compare state 3, intermediate state and state 4 in mitochondria with rest and work in skeletal muscle. ‘Idealized’ state 4 and 3 in relation to various ‘experimental’ states 4 and 3 are defined. Theoretical simulations show, in accordance with experimental data, that oxygen consumption (V’O₂), ADP and P_i are higher, while ATP/ADP and Δp are lower in rest than in state 4, because of the presence of basal ATP consuming reactions in the former. It is postulated that moderate and intensive work in skeletal muscle is very different from state 3 in isolated mitochondria. V’O₂, ATP/ADP, Δp and the control of ATP usage over V’O₂ are much higher, while ADP and Pi are much lower in the former. The slope of the phenomenological V’O₂-ADP relationship is much steeper during the rest-work transition than during the state 4-state 3 transition. The work state in intact muscle is much more similar to intermediate state than to state 3 in isolated mitochondria in terms of ADP, ATP/ADP, Δp and metabolic control pattern, but not in terms of V’O₂. The huge differences between intact muscle and isolated mitochondria are proposed to be caused by the presence of the each-step activation (ESA) mechanism of the regulation of OXPHOS in intact skeletal muscle. Generally, the present study suggests that isolated mitochondria (at least in the absence of Ca²⁺) cannot serve as a good model of OXPHOS regulation in intact skeletal muscle.     

Effect of aclarubicin and doxorubicin on rat liver mitochondrial oxidative phosphorylation

The effects of Aclarubicin (aclacinomycin A; ACM) and Doxorubicin (adriamycin; ADM) on oxidative phosphorylation in rat liver mitochondria were studied in vitro. The state 3 oxygen uptake of mitochondria was reduced by only 2% by 20 microM of ADM, while the same concentration of ACM caused a 67% reduction. When 20 microM of ADM acted on state 4a oxygen uptake of mitochondria, only a slight decrease in state 3, state 4b, dinitrophenol-stimulated respiration and the respiratory control index was observed. In contrast 20 microM of ACM caused significant inhibition of all the above factors when compared with the controls. It was concluded that ACM has strong inhibitory action on the mitochondrial electron transfer system in vitro, and that one can expect functional failure of mitochondria to occur clinically during adverse response to the administration of this drug.     

Metabolic control over the oxygen consumption flux in intact skeletal muscle: in silico studies

It has been postulated previously that a direct activation of all oxidative phosphorylation complexes in parallel with the activation of ATP usage and substrate dehydrogenation (the so-called each-step activation) is the main mechanism responsible for adjusting the rate of ATP production by mitochondria to the current energy demand during rest-to-work transition in intact skeletal muscle in vivo. The present in silico study, using a computer model of oxidative phosphorylation developed previously, analyzes the impact of the each-step-activation mechanism on the distribution of control (defined within Metabolic Control Analysis) over the oxygen consumption flux among the components of the bioenergetic system in intact oxidative skeletal muscle at different energy demands. It is demonstrated that in the absence of each-step activation, the oxidative phosphorylation complexes take over from ATP usage most of the control over the respiration rate and oxidative ATP production at higher (but still physiological) energy demands. This leads to a saturation of oxidative phosphorylation, impossibility of a further acceleration of oxidative ATP synthesis, and dramatic drop in the phosphorylation potential. On the other hand, the each-step-activation mechanism allows maintenance of a high degree of the control exerted by ATP usage over the ATP turnover and oxygen consumption flux even at high energy demands and thus enables a potentially very large increase in ATP turnover. It is also shown that low oxygen concentration shifts the metabolic control from ATP usage to cytochrome oxidase and thus limits the oxidative ATP production. respiration rate; parallel activation; oxidative phosphorylation; metabolic control analysis; flux control coefficient; muscle contraction     

Simulation of state 4 --> state 3 transition in isolated mitochondria

The mathematical dynamic model of oxidative phosphorylation developed previously and in the accompanying paper was modified to involve isolated mitochondria conditions; it was also used to simulate state 4 --> state 3 transition in rat liver mitochondria incubated with succinate as respiratory substrate and glucose-hexokinase as an ADP-regenerating system. Changes in the respiration rate, protonmotive force and reduction level of ubiquinone and cytochrome c as well as the internal (i) and external (e) ATP/ADP ratio between state 4 and state 3 were calculated and compared with the experimental data. Flux control coefficients with respect to oxygen consumption flux for different reactions and processes of oxidative phosphorylation were simulated for different values of the respiration rate (state 4, state 3 and intermediate states). Flux control coefficients for the oxidation, phosphorylation and proton leak subsystems with respect to the oxidation, phosphorylation and proton leak fluxes for different values of the respiration rate were also calculated. These theoretical data were compared with the experimental results obtained in the frame of metabolic control analysis and the 'top-down' approach to this analysis. A good agreement was obtained. Simulated time courses of the respiration rate, the protonmotive force (Deltap) and other parameters after addition of a small amount of ADP to mitochondria in state 4 mimicked at least semiquantitatively the experimentally measured time courses of these parameters. It was concluded, therefore, that in the present stage, the model is able to reflect different properties of the oxidative phosphorylation system in a broad range of conditions fairly well, allows deeper insight into the mechanisms responsible for control and regulation of this process, and can be used for simulation of new experiments, thus inspiring experimental verification of the theoretical predictions.     

Studies on mitochondria from dystrophic skeletal muscle of mice

Mitochondrial respiration and oxidative phosphorylation were compared in normal and dystrophic mouse skeletal muscles. To obtain the maximum respiration control ratio (RCR) and adenosine diphosphate/oxygen (ADP/O) ratio from isolated muscle mitochondria, it is found that there is an advantage in having a low concentration of proteinase and EGTA present in the medium during preparation of mitochondria by centrifugation fractionation. Using pyruvate, acetylcarnitine, and palmitylcarnitine as substrates for oxidation, a highly significant reduction (40-60%) is shown in oxygen uptake by dystrophic muscle mitochondria as compared to normal muscle mitochondria. Studies of the integrity of the oxidative phosphorylation apparatus in these samples showed that there is a reduction of the RCR and ADP/O ratio in dystrophic muscle mitochondria as compared to normal muscle mitochondria.     

Theoretical studies on the regulation of oxidative phosphorylation in intact tissues

The theoretical studies on the regulation of oxidative phosphorylation that were performed with the aid of kinetic models of this process are overviewed. A definition of the regulation of the flux through a metabolic pathway is proposed and opposed to the control exerted by particular enzymes over this flux. Different kinetic models of oxidative phosphorylation proposed in the literature are presented, of which only the model proposed by myself and co-workers was extensively used in theoretical studies on the regulation and compensation in the oxidative phosphorylation system. These theoretical studies have led to the following conclusions: (1) in isolated mitochondria, an increase in the activity of an artificial ATP-using system stimulates mitochondria mainly via changes in [ADP], while changes in [ATP] and [P(i)] play only a minor role; (2) in non-excitable tissues (e.g. liver), hormones (acting via some cytosolic factor(s)) activate directly both ATP usage and at least some enzymes of the ATP-producing block; (3) in excitable tissues (e.g. skeletal muscle), neural signals stimulate (via some cytosolic factor(s)) in parallel all the steps of oxidative phosphorylation together with ATP usage and substrate dehydrogenation; (4) the decrease in the flux through cytochrome oxidase caused by a decrease in oxygen concentration is, at least partially, compensated by a decrease in Delta p and increase in the reduction level of cytochrome c. A theoretical prediction is formulated that there should exist and be observable a universal cytosolic factor/regulatory mechanism which directly activates (at least in excitable tissues) all complexes of oxidative phosphorylation during an increased energy demand.     

A model of oxidative phosphorylation in mammalian skeletal muscle

A dynamic computer model of oxidative phosphorylation in oxidative mammalian skeletal muscle was developed. The previously published model of oxidative phosphorylation in isolated skeletal muscle mitochondria was extended by incorporation of the creatine kinase system (creatine kinase plus phosphocreatine/creatine pair), cytosolic proton production/consumption system (proton production/consumption by the creatine kinase-catalysed reaction, efflux/influx of protons), physiological size of the adenine nucleotide pool and some additional minor changes. Theoretical studies performed by means of the extended model demonstrated that the CK system, which allows for large changes in P(i) in relation to isolated mitochondria system, has no significant influence on the kinetic properties of oxidative phosphorylation, as inorganic phosphate only slightly modifies the relationship between the respiration rate and [ADP]. Computer simulations also suggested that the second-order dependence of oxidative phosphorylation on [ADP] proposed in the literature refers only to the ATP synthesis flux, but not to the oxygen consumption flux (the difference between these two fluxes being due to the proton leak). Next, time courses of changes in fluxes and metabolite concentrations during transition between different steady-states were simulated. The model suggests, in accordance with previous theoretical predictions, that activation of oxidative phosphorylation by an increase in [ADP] can (roughly) explain the behaviour of the system only at low work intensities, while at higher work intensities parallel activation of different steps of oxidative phosphorylation is involved.     

Control of succinate oxidation by cucumber (Cucumis sativus L.) cotyledon mitochondria

The aim of this work was to assess the extent to which mitochondria control the gluconeogenic flux in cucumber (Cucumis sativus L.) cotyledons, by quantifying the distribution of control of succinate oxidation by cotyledon mitochondria. The methods of metabolic control analysis were applied under state 3 and state 4 conditions and in the presence of cell-free extracts in order to simulate in-vivo conditions. Oxygen uptake by isolated cotyledon mitochondria oxidising succinate under state 3 conditions was examined using inhibitor titrations. During lipid mobilisation in light-grown cotyledons (3-4 d post-imbibition), control was shared between the adenine-nucleotide translocator (flux-control coefficient, C = 0.25–0.28) and the dicarboxylate-uptake system (C = 0.69–0.72). The dicarboxylate-uptake system was also important in dark-grown cotyledons at this stage (C = 0.55–0.57). In the photosynthetic phase of development (more than 5 d post-imbibition) control rested with the respiratory chain. Application of an external ATP demand provided either by cell-free extracts of cucumber cotyledons or a glucose/hexokinase ADP-regenerating system showed that the reactions outside the mitochondria exert control (C = 0.45–0.54 and C = 0.24–0.38, for cytosolic extract and glucose/hexokinase, respectively). The adenine-nucleotide translocator was a controlling step of both oxygen uptake (C = 0.11–0.32) and the flux between succinate and hexose phosphates (C = 0.28). Other mitochondrial steps made a significant contribution to control. Control of oxygen uptake was dependent on both the nature of the external load and on the rate of phosphorylation. A potential role for mitochondrial membrane-transport processes, including the adenine-nucleotide translocator, is proposed for the integration of lipid breakdown and gluconeogenesis in vivo.Abbreviations AdNT adenine-nucleotide translocator - C flux-control coefficient - F1,6BP fructose-1,6-biphosphate - F1,6BPase fructose-1,6-biphosphatase - F2,6BP fructose-2,6-bi-phosphate - F6P fructose-6-phosphate - OAA oxaloacetate - PEP phospho(enol)pyruvate - PFK phosphofructokinase - PFP pyrophosphate fructose-6-phosphate 1-phosphotransferase This work was supported by the Gatsby Charitable Foundation (Sainsbury Research Studentship to S.A.H.) and the Agricultural and Food Research Council (grant No. PG43/516 to C.J.L.).     

Mitochondrial bound hexokinase activity as a preventive antioxidant defense: steady-state ADP formation as a regulatory mechanism of membrane potential and reactive oxygen species generation in mitochondria

Brain hexokinase is associated with the outer membrane of mitochondria, and its activity has been implicated in the regulation of ATP synthesis and apoptosis. Reactive oxygen species (ROS) are by-products of the electron transport chain in mitochondria. Here we show that the ADP produced by hexokinase activity in rat brain mitochondria (mt-hexokinase) controls both membrane potential (Deltapsi(m)) and ROS generation. Exposing control mitochondria to glucose increased the rate of oxygen consumption and reduced the rate of hydrogen peroxide generation. Mitochondrial associated hexokinase activity also regulated Deltapsi(m), because glucose stabilized low Deltapsi(m) values in state 3. Interestingly, the addition of glucose 6-phosphate significantly reduced the time of state 3 persistence, leading to an increase in the Deltapsi(m) and in H(2)O(2) generation. The glucose analogue 2-deoxyglucose completely impaired H(2)O(2) formation in state 3-state 4 transition. In sharp contrast, the mt-hexokinase-depleted mitochondria were, in all the above mentioned experiments, insensitive to glucose addition, indicating that the mt-hexokinase activity is pivotal in the homeostasis of the physiological functions of mitochondria. When mt-hexokinase-depleted mitochondria were incubated with exogenous yeast hexokinase, which is not able to bind to mitochondria, the rate of H(2)O(2) generation reached levels similar to those exhibited by control mitochondria only when an excess of 10-fold more enzyme activity was supplemented. Hyperglycemia induced in embryonic rat brain cortical neurons increased ROS production due to a rise in the intracellular glucose 6-phosphate levels, which were decreased by the inclusion of 2-deoxyglucose, N-acetyl cysteine, or carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Taken together, the results presented here indicate for the first time that mt-hexokinase activity performed a key role as a preventive antioxidant against oxidative stress, reducing mitochondrial ROS generation through an ADP-recycling mechanism.     

Hexose metabolism in pancreatic islets: preferential utilization of mitochondrial ATP for glucose phosphorylation

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.     

Effect of visible light on the ATP synthetase function of mitochondria

Dependence of oxidative phosphorylation in rat liver mitochondria on redox state of endogenous flavoenzymes was studied. Regulation of the redox state of endogenous flavins in mitochondria was performed by visible light illumination of lambda = 380 nm in oxygen free as well as in oxygenated media. A decrease of oxidative phosphorylation was obtained at full reduction of flavins (respiratory control and ATP synthesis were measured); this effect was partially recovered when flavin was reoxidized by oxygen. Results of ESR measurements of the signal gl = 2.00 associated with semireduced flavins were in accordance with the appearance of diamagnetically completely reduced form of flavin. These facts may be considered as evidence of the regulative role of flavins in oxidative phosphorylation.     

Functional Coupling between Nucleoside Diphosphate Kinase of the Outer Mitochondrial Compartment and Oxidative Phosphorylation

In rat liver mitochondria all nucleoside diphosphate kinase of the outer compartment is associated with the outer surface of the outer membrane (Lipskaya, T. Yu., and Plakida, K. N. (2003) Biochemistry (Moscow), 68, 1136-1144). In the present study, three systems operating as ADP donors for oxidative phosphorylation have been investigated. The outer membrane bound nucleoside diphosphate kinase was the first system tested. Two others employed yeast hexokinase and yeast nucleoside diphosphate kinase. The two enzymes exhibited the same activity but could not bind to mitochondrial membranes. In all three systems, muscle creatine phosphokinase was the external agent competing with the oxidative phosphorylation system for ADP. Determination of mitochondrial respiration rate in the presence of increasing quantities of creatine phosphokinase revealed that at large excess of creatine phosphokinase activity over other kinase activities (of the three systems tested) and oxidative phosphorylation the creatine phosphokinase reaction reached a quasi-equilibrium state. Under these conditions equilibrium concentrations of all creatine phosphokinase substrates were determined and K(eq)app of this reaction was calculated for the system with yeast hexokinase. In samples containing active mitochondrial nucleoside diphosphate kinase the concentrations of ATP, creatine, and phosphocreatine were determined and the quasi-equilibrium concentration of ADP was calculated using the K(eq)app value. At balance of quasi-equilibrium concentrations of ADP and ATP/ADP ratio the mitochondrial respiration rate in the system containing nucleoside diphosphate kinase was 21% of the respiration rate assayed in the absence of creatine phosphokinase; in the system containing yeast hexokinase this parameter was only 7% of the respiration rate assayed in the absence of creatine phosphokinase. Substitution of mitochondrial nucleoside diphosphate kinase with yeast nucleoside diphosphate kinase abolished this difference. It is concluded that oxidative phosphorylation is accompanied by appearance of functional coupling between mitochondrial nucleoside diphosphate kinase and the oxidative phosphorylation system. Possible mechanisms of this coupling are discussed.     

Calcium regulation of oxidative phosphorylation in rat skeletal muscle mitochondria

Activation of oxidative phosphorylation by physiological levels of calcium in mitochondria from rat skeletal muscle was analysed using top-down elasticity and regulation analysis. Oxidative phosphorylation was conceptually divided into three subsystems (substrate oxidation, proton leak and phosphorylation) connected by the membrane potential or the protonmotive force. Calcium directly activated the phosphorylation subsystem and (with sub-saturating 2-oxoglutarate) the substrate oxidation subsystem but had no effect on the proton leak kinetics. The response of mitochondria respiring on 2-oxoglutarate at two physiological concentrations of free calcium was quantified using control and regulation analysis. The partial integrated response coefficients showed that direct stimulation of substrate oxidation contributed 86% of the effect of calcium on state 3 oxygen consumption, and direct activation of the phosphorylation reactions caused 37% of the increase in phosphorylation flux. Calcium directly activated phosphorylation more strongly than substrate oxidation (78% compared to 45%) to achieve homeostasis of mitochondrial membrane potential during large increases in flux.     

Rapid isolation of muscle and heart mitochondria, the lability of oxidative phosphorylation and attempts to stabilize the process in vitro by taurine, carnitine and other compounds

We modified the isolation procedure of muscle and heart mitochondria. In human muscle, this resulted in a 3.4 fold higher yield of better coupled mitochondria in half the isolation time. In a preparation from rat muscle we studied factors that affected the stability of oxidative phosphorylation (oxphos) and found that it decreased by shaking the preparation on a Vortex machine, by exposure to light and by an increase in storage temperature. The decay was found to be different for each substrate tested. The oxidation of ascorbate was most stable and less sensitive to the treatments.When mitochondria were stored in the dark and the cold, the decrease in oxidative phosphorylation followed first order kinetics. In individual preparations of muscle and heart mitochondria, protection of oxidative phosphorylation was found by adding candidate stabilizers, such as desferrioxamine, lazaroids, taurine, carnitine, phosphocreatine, N-acetylcysteine, Trolox-C and ruthenium red, implying a role for reactive oxygen species and calcium-ions in the in vitro damage at low temperature to oxidative phosphorylation.In heart mitochondria oxphos with pyruvate and palmitoylcarnitine was most labile followed by glutamate, succinate and ascorbate.We studied the effect of taurine, hypotaurine, carnitine, and desferrioxamine on the decay of oxphos with these substrates. 1 mM taurine (n = 6) caused a significant protection of oxphos with pyruvate, glutamate and palmitoylcarnitine, but not with the other substrates. 5 mM L-carnitine (n = 6), 1 mM hypotaurine (n = 3) and 0.1 mM desferrioxamine (n = 3) did not protect oxphos with any of the substrates at a significant level.These experiments were undertaken in the hope that the in vitro stabilizers can be used in future treatment of patients with defects in oxidative phosphorylation. (Mol Cell Biochem 174: 61–66, 1997)