Regulation of the reaction rate of ATP synthesis in intact mitochondria

High concentrations of respiration inhibitors are known to sharply decrease the membrane potential in mitochondria. The effect of relatively low concentrations of oxidative phosphorylation inhibitors on the value of membrane potential of intact mitochondria and on the rate of respiration and phosphorylation as well was studied. It has been found that within a certain concentration range the inhibitors of oxidative phsophorylation--malonic acid, sodium cyanide m-chlorophenyl hydrozonecarbonylcyanide, sharply decrease the phosphorylation rate (by 70 divided by 90%) but do not practically a affect the membrane potential value of intact mitochondria in the state 3 according to Chance.     

The role of adenine nucleotide translocator in the regulation of oxidative phosphorylation in heart mitochondria

The regulatory role of adenine nucleotide translocase in oxidative phosphorylation was determined by titration of respiration of isolated rabbit heart mitochondria with carboxyatractyloside in the creatine phosphokinase ADP-regenerating system, which is not rate-limiting. It was found that the respiration rate is not controlled by adenine nucleotide translocase in states 3 and 4. Within the physiological region of respiration (30-70% of the maximal rate), the control coefficient for ADP/ATP translocase is 0.62-0.75. Thus, translocase plays a key role in the regulation of oxidative phosphorylation.     

Top-down control analysis of the effect of temperature on ectotherm oxidative phosphorylation

Top-down control and elasticity analysis was conducted on mitochondria isolated from the midgut of the tobacco hornworm (Manduca sexta) to assess how temperature affects oxidative phosphorylation in a eurythermic ectotherm. Oxygen consumption and protonmotive force (measured as membrane potential in the presence of nigericin) were monitored at 15, 25, and 35 degrees C. State 4 respiration displayed a Q(10) of 2.4-2.7 when measured over two temperature ranges (15-25 degrees C and 25-35 degrees C). In state 3, the Q(10)s for respiration were 2.0 and 1.7 for the lower and higher temperature ranges, respectively. The kinetic responses (oxygen consumption) of the substrate oxidation system, proton leak, and phosphorylation system increased as temperature rose, although the proton leak and substrate oxidation system showed the greatest thermal sensitivity. Whereas there were temperature-induced changes in the activities of the oxidative phosphorylation subsystems, there was no change in the state 4 membrane potential and little change in the state 3 membrane potential. Top-down control analysis revealed that control over respiration did not change with temperature. In state 4, control of respiration was shared nearly equally by the proton leak and the substrate oxidation system, whereas in state 3 the substrate oxidation system exerted over 90% of the control over respiration. The proton leak and phosphorylation system account for <10% of the temperature-induced change in the state 3 respiration rate. Therefore, when the temperature is changed, the state 3 respiration rate is altered primarily because of temperature's effect on the substrate oxidation system.     

The role of state 4 electron transport in the activation of state 3 respiration in potato mitochondria

The initial state 3 respiration rate of potato mitochondria is markedly depressed, or attenuated. With several consecutive state 3/state 4 cycles the state 3 rate rises to a maximum, while the state 4 rate remains essentially unchanged. The development of state 3 respiration has been termed conditioning. An analysis of the process has indicated that state 4 is a better conditioner than state3 per se. Conditioning is also attained by preincubation in state 2, or under conditions designated pseudostate 2, wherein ADP is present, with or without oligomycin, and inorganic phosphate is absent. ADP implements the conditioning process in the absence of oxidative phosphorylation. The action of ADP in its secondary or modulator role appears to be positively cooperative, the kinetics of ADP involvement being second-order. S_0.5 for ADP as a modulator of the conditioning process is approximately 62 M, a value in excess of the K _s for ADP in oxidative phosphorylation. Electron transport is indispensable for conditioning, and it is suggested that conditioning and ATP synthesis represent alternative uses of respiratory energy. It is further suggested that to some extent state 4 underlies state 3.     

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].     

Effect of organophosphorus insecticides on the oxidative processes in rat brain synaptosomes

Abstract: This paper describes the effect of four organophosphorus insecticides: Dipterex, DDVP, Ronnel and its oxygen analogue on the respiration of rat brain synaptosomes. Dipterex and DDVP in the concentrations used, 5, 50, or 500 μM, did not change the rate of oxygen uptake and oxidative phosphorylation in rat brain synaptosomes. Ronnel in the highest concentration (500 μM) inhibited respiration in state 3 conditions and abolished respiratory control by ADP. This inhibition was correlated with a change of cytochrome c oxidase activity. The oxygen analogue of Ronnel (OAR) in micromolar concentrations (50 μM) increased the rate of respiration of synaptosomes utilizing glutamate plus malate as substrate. Higher concentrations of OAR produced inhibition of respiration, cytochrome c oxidase and NADH: cytochrome c reductase activities. These observations are typical for uncouplers of oxidative phosphorylation. Noteworthy is the fact that the uncoupling activity of OAR was observed at concentrations which did not inhibit acetylcholinesterase activity. These findings seem to suggest that disturbances in oxidative processes could play an important role in the toxicity of organophosphorus insecticides. The relation between chemical structure and the ability of insecticides to affect oxidative phosphorylation is discussed.     

Elucidating ANTs in worms using genomic and bioinformatic tools — Biotechnological prospects?

Adenine nucleotide translocators (ANTs) belong to the mitochondrial carrier family (MCF) of proteins. ATP production and consumption are tightly linked to ANTs, the kinetics of which have been proposed to play a key regulatory role in mitochondrial oxidative phosphorylation. ANTs are also recognized as a central component of the mitochondrial permeability transition pore associated with apoptosis. Although ANTs have been investigated in a range of vertebrates, including human, mouse and cattle, and invertebrates, such as Drosophila melanogaster (vinegar fly), Saccharomyces cerevisiae (yeast) and Caenorhabditis elegans (free-living nematode), there has been a void of information on these molecules for parasitic nematodes of socio-economic importance. Exploring ANTs in nematodes has the potential lead to a better understanding of their fundamental roles in key biological pathways and might provide an avenue for the identification of targets for the rational design of nematocidal drugs. In the present article, we describe the discovery of an ANT from Haemonchus contortus (one of the most economically important parasitic nematodes of sheep and goats), conduct a comparative analysis of key ANTs and their genes (particularly ant-1.1) in nematodes and other organisms, predict the functional roles utilizing a combined genomic-bioinformatic approach and propose ANTs and associated molecules as possible drug targets, with the potential for biotechnological outcomes.     

Substance P and mitochondrial oxygen consumption: evidence for a direct intracellular role for the peptide

Substance P (SP), a member of the tachykinin group of peptides, has been shown to augment the sensory discharge of the carotid body, an oxygen sensing chemoreceptor. In this study we present evidence that the excitatory effect of SP, in part, could arise from a direct effect of the peptide on mitochondrial oxidative phosphorylation. Measurement of the partition coefficient of SP showed that the peptide has a relatively high apolar partition, which could be consistent with its distribution across lipid bilayers and in intracellular organelles. In addition, the effects of three concentrations of SP were tested on oxygen consumption of mitochondria isolated from rat hearts. The results showed that while the lower concentration of the peptide (0.5 microM) did not affect O2 consumption, higher concentrations, i.e., 1 and 2 microM, enhanced the rate of state 4 respiration by 52 and 64%, respectively. The rate of state 3 respiration, on the other hand, was unaltered with 0.5 and 1 microM, and was only slightly decreased with 2 microM of the peptide. The ADP:O ratio was unaffected by any concentrations of SP tested. The peptide-induced effect on state 4 respiration was even more pronounced with glutamate as a respiratory substrate and in presence of K+ in the medium. These results indicate that SP, in addition to its more accepted role as a neurotransmitter or modulator in the carotid body, may elicit intracellular response by interfering directly with oxidative phosphorylation.     

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.     

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.     

Mitochondrial metabolism during daily torpor in the dwarf Siberian hamster: role of active regulated changes and passive thermal effects

During daily torpor in the dwarf Siberian hamster, Phodopus sungorus, metabolic rate is reduced by 65% compared with the basal rate, but the mechanisms involved are contentious. We examined liver mitochondrial respiration to determine the possible role of active regulated changes and passive thermal effects in the reduction of metabolic rate. When assayed at 37 degrees C, state 3 (phosphorylating) respiration, but not state 4 (nonphosphorylating) respiration, was significantly lower during torpor compared with normothermia, suggesting that active regulated changes occur during daily torpor. Using top-down elasticity analysis, we determined that these active changes in torpor included a reduced substrate oxidation capacity and an increased proton conductance of the inner mitochondrial membrane. At 15 degrees C, mitochondrial respiration was at least 75% lower than at 37 degrees C, but there was no difference between normothermia and torpor. This implies that the active regulated changes are likely more important for reducing respiration at high temperatures (i.e., during entrance) and/or have effects other than reducing respiration at low temperatures. The decrease in respiration from 37 degrees C to 15 degrees C resulted predominantly from a considerable reduction of substrate oxidation capacity in both torpid and normothermic animals. Temperature-dependent changes in proton leak and phosphorylation kinetics depended on metabolic state; proton leakiness increased in torpid animals but decreased in normothermic animals, whereas phosphorylation activity decreased in torpid animals but increased in normothermic animals. Overall, we have shown that both active and passive changes to oxidative phosphorylation occur during daily torpor in this species, contributing to reduced metabolic rate.     

Role of oxidative stress in alterations of mitochondrial function in ischemic-reperfused hearts

To study the mechanisms of mitochondrial dysfunction due to ischemia-reperfusion (I/R) injury, rat hearts were subjected to 20 or 30 min of global ischemia followed by 30 min of reperfusion. After recording both left ventricular developed pressure (LVDP) and end-diastolic pressure (LVEDP) to monitor the status of cardiac performance, mitochondria from these hearts were isolated to determine respiratory and oxidative phosphorylation activities. Although hearts subjected to 20 min of ischemia failed to generate LVDP and showed a marked increase in LVEDP, no changes in mitochondrial respiration and phosphorylation were observed. Reperfusion of 20-min ischemic hearts depressed mitochondrial function significantly but recovered LVDP completely and lowered the elevated LVEDP. On the other hand, depressed LVDP and elevated LVEDP in 30-min ischemic hearts were associated with depressions in both mitochondrial respiration and oxidative phosphorylation. Reperfusion of 30-min ischemic hearts elevated LVEDP, attenuated LVDP, and decreased mitochondrial state 3 and uncoupled respiration, respiratory control index, ADP-to-O ratio, as well as oxidative phosphorylation rate. Alterations of cardiac performance and mitochondrial function in I/R hearts were attenuated or prevented by pretreatment with oxyradical scavenging mixture (superoxide dismutase and catalase) or antioxidants [N-acetyl-L-cysteine or N-(2-mercaptopropionyl)-glycine]. Furthermore, alterations in cardiac performance and mitochondrial function due to I/R were simulated by an oxyradical-generating system (xanthine plus xanthine oxidase) and an oxidant (H(2)O(2)) either upon perfusing the heart or upon incubation with mitochondria. These results support the view that oxidative stress plays an important role in inducing changes in cardiac performance and mitochondrial function due to I/R.     

Iron uncouples oxidative phosphorylation in brain mitochondria isolated from vitamin E-deficient rats

Few, if any, studies have examined the effect of vitamin E deficiency on brain mitochondrial oxidative phosphorylation. The latter was studied using brain mitochondria isolated from control and vitamin E-deficient rats (13 months of deficiency) after exposure to iron, an inducer of oxidative stress. Mitochondria were treated with iron (2 to 50 microM) added as ferrous ammonium sulfate. Rates of state 3 and state 4 respiration, respiratory control ratios, and ADP/O ratios were not affected by vitamin E deficiency alone. However, iron uncoupled oxidative phosphorylation in vitamin E-deficient mitochondria, but not in controls. In vitamin E-deficient mitochondria, iron decreased ADP/O ratios and markedly stimulated state 4 respiration; iron had only a modest effect on these parameters in control mitochondria. Thus, vitamin E may have an important role in sustaining oxidative phosphorylation. Low concentrations of iron (2 to 5 microM) oxidized mitochondrial tocopherol that exists in two pools. The release of iron in brain may impair oxidative phosphorylation, which would be exacerbated by vitamin E deficiency. The results are important for understanding the pathogenesis of human brain disorders known to be associated with abnormalities in mitochondrial function as well as iron homeostasis (e.g., Parkinson's disease).     

Inhibition of mitochondrial respiration by phosphoenolpyruvate

The cytosolic factors that influence mitochondrial oxidative phosphorylation rates are relatively unknown. In this report, we examine the effects of phosphoenolpyruvate (PEP), a glycolytic intermediate, on mitochondrial function. It is reported here that in rat heart mitochondria, PEP delays the onset of state 3 respiration in mitochondria supplied with either NADH-linked substrates or succinate. However, the maximal rate of state 3 respiration is only inhibited when oxidative phosphorylation is supported by NADH-linked substrates. The capacity of PEP to delay and/or inhibit state 3 respiration is dependent upon the presence or absence of ATP. Inhibition of state 3 is exacerbated in uncoupled mitochondria, with a 40% decrease in respiration seen with 0.1mM PEP. In contrast, ATP added exogenously or produced by oxidative phosphorylation completely prevents PEP-mediated inhibition. Mechanistically, the results support the conclusion that the main effects of PEP are to impede ADP uptake and inhibit NADH oxidation. By altering the NADH/NAD(+) status of mitochondria, it is demonstrated that PEP enhances succinate dehydrogenase activity and increase free radical production. The results of this study indicate PEP may be an important modulator of mitochondrial function under conditions of decreased ATP.     

Glucose-stimulated insulin secretion of insulinoma INS-1E cells is associated with elevation of both respiration and mitochondrial membrane potential

Increased ATP/ADP ratio resulting from enhanced glycolysis and oxidative phosphorylation represents a plausible mechanism controlling the glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells. Although specific bioenergetics might be involved, parallel studies of cell respiration and mitochondrial membrane potential (ΔΨ_m) during GSIS are lacking. Using high resolution respirometry and parallel ΔΨ_m monitoring by two distinct fluorescence probes we have quantified bioenergetics in rat insulinoma INS-1E cells representing a suitable model to study in vitro insulin secretion. Upon glucose addition to glucose-depleted cells we demonstrated a simultaneous increase in respiration and ΔΨ_m during GSIS and showed that the endogenous state 3/state 4 respiratory ratio hyperbolically increased with glucose, approaching the maximum oxidative phosphorylation rate at maximum GSIS. Attempting to assess the basis of the “toxic” effect of fatty acids on insulin secretion, GSIS was studied after linoleic acid addition, which diminished respiration increase, ΔΨ_m jump, and magnitude of insulin release, and reduced state 3/state 4 dependencies on glucose. Its effects were due to protonophoric function, i.e. uncoupling, since without glucose, linoleic acid accelerated both state 3 and state 4 respiration by similar extent. In turn, state 3 respiration increased marginally with linoleic acid at 10–20 mM glucose. We conclude that upon glucose addition in physiological range, the INS-1E cells are able to regulate the oxidative phosphorylation rate from nearly zero to maximum and that the impairment of GSIS by linoleic acid is caused by mitochondrial uncoupling. These findings may be relevant to the pathogenesis of type 2 diabetes.     

The contribution of adenine nucleotide loss to ischemia-induced impairment of rat kidney cortex mitochondria.

Adenine nucleotides and respiration were assayed with rat kidney mitochondria depleted of adenine nucleotides by pyrophosphate treatment and by normothermic ischemia, respectively, with the aim of identifying net uptake of ATP as well as elucidating the contribution of adenine nucleotide loss to the ischemic impairment of oxidative phosphorylation. Treatment of rat kidney mitochondria with pyrophosphate caused a loss of adenine nucleotides as well as a decrease of state 3 respiration. After incubation of pyrophosphate-treated mitochondria with ATP, Mg2+ and phosphate, the content of adenine nucleotides increased. We propose that kidney mitochondria possess a mechanism for net uptake of ATP. Restoration of a normal content of matrix adenine nucleotides was related to full recovery of the rate of state 3 respiration. A hyperbolic relationship between the matrix content of adenine nucleotides and the rate of state 3 respiration was observed. Mitochondria isolated from kidneys exposed to normothermic ischemia were characterized by a decrease in the content of adenine nucleotides as well as in state 3 respiration. Incubation of ischemic mitochondria with ATP, Mg2+ and phosphate restored the content of adenine nucleotides to values measured in freshly-isolated mitochondria. State 3 respiration of ischemic mitochondria reloaded with ATP recovered only partially. The rate of state 3 respiration increased by ATP-reloading approached that of uncoupler-stimulated respiration measured with ischemic mitochondria. These findings suggest that the decrease of matrix adenine nucleotides contributes to the impairment of ischemic mitochondria as well as underlining the occurrence of additional molecular changes of respiratory chain limiting the oxidative phosphorylation.     

Effects of ATP on various steps controlling the rate of oxidative phosphorylation in newborn rat liver mitochondria

Preincubation of newborn rat liver mitochondria with ATP increases their state 3 respiration rate [J. K. Pollak (1975) Biochem. J. 150, 477-488; J. R. Aprille, and G. K. Asimakis (1980) Arch. Biochem. Biophys. 201, 564-575]. To determine which reactions contribute to control the rate of succinate oxidation with and without prior exposure to ATP, the effects of inhibitors specific for various reactions were studied. The adenine nucleotide translocator does not control the respiration in newborn more than in the adult mitochondria. The supply of reducing equivalents to the respiratory chain is an important step controlling the rate of oxidative phosphorylation by mitochondria from newborn rat liver, especially after preincubation with ATP. On the contrary, titrations with oligomycin show that the preincubation with ATP markedly decreases the control exerted by the ATPase-ATP synthase complex. That the rate of ATP synthesis is one of the steps controlling the rate of oxidative phosphorylation in newborn rat liver mitochondria is in striking contrast to the behavior of adult rat liver mitochondria. Other differences include a greater permeability to protons and a marked increase in sensitivity to mersalyl, indicating an easier accessibility of the proteins involved in oxidative phosphorylation to the thiol reagent.     

The effect of ATP on the malate regulation of oxidative phosphorylation in brain mitochondria

Malate was studied for its effect on the oxidative phosphorylation rate in the rat brain mitochondria in the presence and absence of ATP, succinate being used as a substrate of the respiration. It has been found that malate in the 0.05-0.4 mM concentration range increases the oxidation phosphorylation rate. ATP inhibiting oxidative phosphorylation intensifies the malate stimulation. The malate 0.8 mM concentration removes the inhibiting action of ATP. The regulatory effects of malate and ATP are supposed to be realized at the adenine nucleotide translocator step.     

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.     

Uncoupling proteins in mitochondria of plants and some microorganisms.

Uncoupling proteins, members of the mitochondrial carrier family, are present in mitochondrial inner membrane and mediate free fatty acid-activated, purine-nucleotide-inhibited H+ re-uptake. Since 1995, it has been shown that the uncoupling protein is present in many higher plants and some microorganisms like non-photosynthetic amoeboid protozoon, Acanthamoeba castellanii and non-fermentative yeast Candida parapsilosis. In mitochondria of these organisms, uncoupling protein activity is revealed not only by stimulation of state 4 respiration by free fatty acids accompanied by decrease in membrane potential (these effects being partially released by ATP and GTP) but mainly by lowering ADP/O ratio during state 3 respiration. Plant and microorganism uncoupling proteins are able to divert very efficiently energy from oxidative phosphorylation, competing for deltamicroH+ with ATP synthase. Functional connection and physiological role of uncoupling protein and alternative oxidase, two main energy-dissipating systems in plant-type mitochondria, are discussed.