The contribution of uncoupling protein and ATP synthase to state 3 respiration in Acanthamoeba castellanii mitochondria

Mitochondria of the amoeba Acanthamoeba castellanii possess a free fatty acid-activated uncoupling protein (AcUCP) that mediates proton re-uptake driven by the mitochondrial proton electrochemical gradient. We show that AcUCP activity diverts energy from ATP synthesis during state 3 mitochondrial respiration in a fatty acid-dependent way. The efficiency of AcUCP in mitochondrial uncoupling increases when the state 3 respiratory rate decreases as the AcUCP contribution is constant at a given linoleic acid concentration while the ATP synthase contribution decreases with respiratory rate. Respiration sustained by this energy-dissipating process remains constant at a given linoleic acid concentration until more than 60% inhibition of state 3 respiration by n-butyl malonate is achieved. The present study supports the validity of the ADP/O method to determine the actual contributions of AcUCP (activated with various linoleic acid concentrations) and ATP synthase in state 3 respiration of A.castellanii mitochondria fully depleted of free fatty acid-activated and describes how the two contributions vary when the rate of succinate dehydrogenase is decreased by succinate uptake limitation.     

Involvement of the energy-dissipating systems in modulating the energetic efficiency of respiration in mitochondria from etiolated winter wheat seedlings

Effects of cyanide-resistant alternative oxidase (AOX) and modulators of plant uncoupling mitochondrial proteins (PUMP) on respiration rate and generation of transmembrane electric potential (ΔΨ) were investigated during oxidation of various substrates by isolated mitochondria from etiolated coleoptiles of winter wheat (Triticum aestivum L.). Oxidative phosphorylation in wheat mitochondria during malate and succinate oxidation was quite effective (it was characterized by high respiratory control ratio as defined by Chance, high ADP/O ratio, and rapid ATP synthesis). Nevertheless, the effectiveness of oxidative phosphorylation was substantially modulated by operation of energy-dissipating systems. The application of safranin dye revealed the partial dissipation of ΔΨ during inhibition of cytochrome-mediated malate oxidation by cyanide and antimycin A and demonstrated the operation of AOX-dependent compensatory mechanism for ΔΨ generation. The complex I of mitochondrial electron transport chain was shown to play the dominant role in ΔΨ generation and ATP synthesis during AOX functioning upon inhibition of electron transport through the cytochrome pathway. Effects of linoleic acid (PUMP activator) at physiologically low concentrations (4–10 μM) on respiration and ΔΨ generation in mitochondria were examined. The uncoupling effect of linoleic acid was shown in activation of the State 4 respiration, as well as in ΔΨ dissipation; this effect was eliminated in the presence of BSA but was insensitive to purine nucleotides. The uncoupling effect of linoleic acid was accompanied by reversible inhibition of AOX activity. The results are discussed with regard to possible physiological role of mitochondrial energy-dissipating systems in regulation of energy transduction in plant cells under stress conditions.     

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.     

Involvement of oxyleghaemoglobin and cytochrome P-450 in an efficient oxidative phosphorylation pathway which supports nitrogen fixation in Rhizobium.

Cellular ATP level, ATP/ADP ratio and nitrogenase activity rise when oxyleghaemoglobin is added to respiring suspensions of Rhizobium japonicum bacteroids from soybean root nodules. Increased gaseous O2 tension is much less efficient than oxyleghaemoglobin in stimulation of bacteroid ATP production. Studies with the inhibitor carbonyl cyanide m-chlorophenylhydrazone show this ATP to be generated as a consequence of oxidative phosphorylation. N-Phenylimidazole, a specific cytochrome P-450 inhibitor, also lowers the efficiency of bacteroid oxidative phosphorylation. An approximately linear relationship is observed between ATP/ADP ratio and nitrogenase activity as N-phenylimidazole concentration is lowered. It is suggested that cytochrome P-450 is a component of the leghaemoglobin-facilitated respiration pathway and that it may act as intracellular O2 carrier rather than terminal oxidase. A less efficient oxidase appears to function when cytochrome P-450 is inhibited.     

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.     

Almitrine, a new kind of energy-transduction inhibitor acting on mitochondrial ATP synthase

At low concentrations, almitrine inhibits yeast cell multiplication by acting on oxidative metabolism. Studies on isolated mitochondria display the following features: (i) almitrine inhibits ATPase activity and decreases ATP/O ratio during oxidative phosphorylation; (ii) no direct effect on respiration can be evidenced; (iii) ATP/O value decreases without any change in the magnitude of delta p; (iv) the higher the ATP synthesis and respiratory fluxes, the larger is the decrease in ATP/O ratio induced by almitrine. These results indicate that almitrine does not act as a classical protonophoric uncoupler nor as previously studied non protonophoric uncouplers (e.g., general anesthetics). Our data show a direct inhibitory effect of almitrine on ATPase-ATP synthase complex. But, in contrast to the classical inhibitors of this complex, almitrine decreases the ATP/O ratio in a flux-dependent manner. Thus, almitrine could induce either an intrinsic uncoupling of H+/-ATPase (i.e., slip in this proton pump) or a change in the mechanistic H+/ATP stoichiometry at the ATPase level.     

Nature of enhanced mitochondrial oxidative metabolism by a calf blood extract

The effect of calf blood extract (Solcoseryl, SS) on mitochondrial oxidative function in various states was studied polarographically in vitro. 1) Mitochondrial respiration in all 4 conventional study states (Estabrook, 1967) was enhanced by the addition of SS, including states 1 and 2 (endogenous substrates only). 2) The effect of SS on mitochondrial oxygen consumption was concentration dependent, while ADP/O ratio remained constant. The effect of added respiratory substrates varied with the particular substrate at optimally active concentrations. With suboptimal substrate levels, ADP/O ratios were concentration dependent, in contrast to the SS effect. Under oligomycin ATPase inhibition, SS was no longer active, in contrast to DNP, which remained active. 3) In states 3 (added ADP) and 4 (ADP exhausted), oxygen consumption and oxidative phosphorylation were enhanced by SS in the presence or absence of citrate, glutamate, pyruvate, lactate, or ascorbate. However, in the presence of succinate, SS had no effect. 4) ADP/O ratio was decreased by SS in the presence of added substrate, suggesting that SS activation of H(+)-ATPase enhances ATP hydrolysis as well as oxidative phosphorylation and ATP synthesis. 5) The enhancing effect of SS on mitochondrial function is due to hydrophilic components of SS. The lipidic components obtained by Folch fraction of SS have no effect. It is concluded that the effects of SS respiratory substrates and uncouplers on mitochondrial function are essentially different. SS enhances both ATP synthesis and oxygen consumption by mitochondria.     

Regulation of oxidative phosphorylation in mitochondria by external free Ca2+ concentrations

The rate of oxidative phosphorylation was studied in rat liver mitochondria incubated with free Ca2+ concentrations that range from 10(-9) to 5 X 10(-6) M. The highest rate was observed between 0.5-1.0 microM Ca2+. ATP synthesis was measured by polarographic and spectrophotometric techniques and by uptake of radioactive inorganic phosphate. The concentration of Ca2+ at which maximal rates of ATP synthesis take place is modified by Mg2+ and phosphate. The dependence of oxidative phosphorylation on Ca2+ was observed with alpha-ketoglutarate, glutamate + malate, and succinate, but not with beta-hydroxybutyrate. At 10(-9) M Ca2+ there is a continuous exit of endogenous Ca2+, while with 10(-6) M Ca2+, intramitochondrial Ca2+ levels remained constant throughout time. Apparently the control of the level of internal Ca2+ by external Ca2+ modulates the rate of oxidative phosphorylation. Uncoupler-stimulated respiration also depends on Ca2+ concentration, even though at 10(-9) to 10(-6) M Ca2+ the rate of oxidative phosphorylation is lower than the rate of uncoupled respiration. The contribution of the ADP/ATP carrier and the ATP synthase to the kinetic regulation of ATP synthesis at 10(-9) and 10(-6) M Ca2+ was evaluated by titrations with carboxyatractyloside and oligomycin, respectively. The contribution of the carrier and the synthase to the regulation of the final rate of ATP synthesis was different at the two concentrations of Ca2+; therefore, the concentration of extramitochondrial Ca2+ influences the overall kinetics of oxidative phosphorylation.     

The energy-conserving and energy-dissipating processes in mitochondria isolated from wild type and nonripening tomato fruits during development on the plant

Bioenergetics of tomato (Lycopersicon esculentum) development on the plant was followed from the early growing stage to senescence in wild type (climacteric) and nonripening mutant (nor, nonclimacteric) fruits. Fruit development was expressed in terms of evolution of chlorophyll a content allowing the assessment of a continuous time-course in both cultivars. Measured parameters: the cytochrome pathway-dependent respiration, i.e., the ATP synthesis-sustained respiration (energy-conserving), the uncoupling protein (UCP) activity-sustained respiration (energy-dissipating), the alternative oxidase(AOX)-mediated respiration (energy-dissipating), as well as the protein expression of UCP and AOX, and free fatty acid content exhibited different evolution patterns in the wild type and nor mutant that can be attributed to their climacteric/nonclimacteric properties, respectively. In the wild type, the climacteric respiratory burst observed in vitro depended totally on an increse in the cytochrome pathway activity sustained by ATP synthesis, while the second respiratory rise during the ripening stage was linked to a strong increase in AOX activity accompanied by an overexpression of AOX protein. In wild type mitochondria, the 10-M linoleic acid-stimulated UCP-activity-dependent respiration remained constant during the whole fruit development except in senescence where general respiratory decay was observed.     

Free fatty acids as inducers and regulators of uncoupling of oxidative phosphorylation in liver mitochondria with participation of ADP/ATP- and aspartate/glutamate-antiporter

In liver mitochondria fatty acids act as protonophoric uncouplers mainly with participation of internal membrane protein carriers — ADP/ATP and aspartate/glutamate antiporters. In this study the values of recoupling effects of carboxyatractylate and glutamate (or aspartate) were used to assess the degree of participation of ADP/ATP and aspartate/glutamate antiporters in uncoupling activity of fatty acids. These values were determined from the ability of these recoupling agents to suppress the respiration stimulated by fatty acids and to raise the membrane potential reduced by fatty acids. Increase in palmitic and lauric acid concentration was shown to increase the degree of participation of ADP/ATP antiporter and to decrease the degree of participation of aspartate/glutamate antiporter in uncoupling to the same extent. These data suggest that fatty acids are not only inducers of uncoupling of oxidative phosphorylation, but that they also act the regulators of this process. The linear dependence of carboxyatractylate and glutamate recoupling effects ratio on palmitic and lauric acids concentration was established. Comparison of the effects of fatty acids (palmitic, myristic, lauric, capric, and caprylic having 16, 14, 12, 10, and 8 carbon atoms, respectively) has shown that, as the hydrophobicity of fatty acids decreases, the effectiveness decreases to a greater degree than the respective values of their specific uncoupling activity. The action of fatty acids as regulators of uncoupling is supposed to consist of activation of transport of their anions from the internal to the external monolayer of the internal membrane with participation of ADP/ATP antiporter and, at the same time, in inhibition of this process with the participation of aspartate/glutamate antiporter.     

Control of mitochondrial oxidative phosphorylation

The objective of this investigation is to analyze the two following problems of the regulation of mitochondrial oxidative phosphorylation: what is the extramitochondrial parameter that controls ATP production according to the cytoplasmic demands and how the control is distributed between various mitochondrial enzymes. On the basis of the data of Groen et al. (1982) it is shown that as the respiration rates ranged over 30-50% of the maximum (i.e. within the physiological region) the contribution of the adenine nucleotide translocator to the control of the ATP flux is no less than 90%, referring to the total contribution of all mitochondrial enzymes as 100%. Founding on the key role of the adenine nucleotide translocator it has been concluded that besides the extramitochondrial [ATP]/[ADP] ratio the absolute ADP concentration is another extramitochondrial signal controlling significantly the rate of oxidative phosphorylation.     

Uncoupling proteins outside the animal and plant kingdoms: functional and evolutionary aspects

The appearance of intracellular oxidative phosphorylation at the time of acquisition of mitochondria in Eukarya was very soon accompanied by the emergence of uncoupling protein, a carrier specialized in free fatty acid-mediated H⁺ recycling that can modulate the tightness of coupling between mitochondrial respiration and ATP synthesis, thereby maintaining a balance between energy supply and demand in the cell and defending cells against damaging reactive oxygen species production when electron carriers of the respiratory chain become overreduced. The simultaneous occurrence of redox free energy-dissipating oxidase, which has the same final effect, could be related to the functional interactions between both dissipative systems.     

Profound effects of the general anesthetic etomidate on oxidative phosphorylation without effects on their yield

We investigated the effects of the general anesthetic Etomidate on oxidative phosphorylation in isolated rat liver mitochondria. The study of each electron transfer site shows that there is an inhibition: mainly at complex I but also, to a lesser extent, at complex III. Moreover, with succinate as substrate, the increase in non-phosphorylating respiration is accompanied by a decrease in ΔΨ. However, this effect is not due to classical uncoupling of oxidative phosphorylation, since ADP addition at high Etomidate concentrations restores the transmembrane difference of electrical potential. Also, in the same range of Etomidate concentration, the ATP/O ratio is not significantly affected. In conclusion, the main effect of Etomidate is to decrease the oxidative phosphorylation rate without changing yield. The H⁺ leak which appears under non-phosphorylating conditions becomes negligible in physiological conditions.     

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)     

Cyanide-resistant, ATP-synthesis-sustained, and uncoupling-protein-sustained respiration during postharvest ripening of tomato fruit

Tomato (Lycopersicon esculentum) mitochondria contain both alternative oxidase (AOX) and uncoupling protein as energy-dissipating systems that can decrease the efficiency of oxidative phosphorylation. We followed the cyanide (CN)-resistant, ATP-synthesis-sustained, and uncoupling-protein-sustained respiration of isolated mitochondria, as well as the immunologically detectable levels of uncoupling protein and AOX, during tomato fruit ripening from the mature green stage to the red stage. The AOX protein level and CN-resistant respiration of isolated mitochondria decreased with ripening from the green to the red stage. The ATP-synthesis-sustained respiration followed the same behavior. In contrast, the level of uncoupling protein and the total uncoupling-protein-sustained respiration of isolated mitochondria decreased from only the yellow stage on. We observed an acute inhibition of the CN-resistant respiration by linoleic acid in the micromolar range. These results suggest that the two energy-dissipating systems could have different roles during the ripening process.     

Uncoupling of mitochondrial respiration by ADP

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.     

The alternative oxidase of Yarrowia lipolytica mitochondria is unable to compete with the cytochrome pathway for electrons

The activity of the cyanide-resistant alternative oxidase (pathway) of Y. lipolytica mitochondria was studied as a function of the activity of the major, cyanide-sensitive, cytochrome pathway. The contribution of the alternative oxidase to the total respiration of mitochondria was evaluated by measuring the rate of oxygen consumption in the presence of cyanide (an inhibitor of the cytochrome pathway). The potential activity of the cytochrome pathway was evaluated spectrophotometrically, by measuring the oxidation rate of cytochrome c by ferricyanide, which accepts electrons from complex III (cytochrome c) of this pathway. The oxidation of succinate by mitochondria in the presence of ferricyanide and cyanide was accompanied by oxygen consumption due to the transfer of electrons through the alternative pathway. The subsequent addition of ADP or FCCP (an uncoupler of oxidative phosphorylation in the cytochrome pathway) completely inhibited the consumption of oxygen by the mitochondria. Under these conditions, the inhibition of the alternative pathway by benzohydroxamic acid failed to affect the transfer of electrons from cytochrome c to ferricyanide. Benzohydroxamic acid did not influence the rate of ferricyanide reduction by the cytochrome pathway occurring in controlled state 4, nor could it change the phosphorylation quotient ATP/O upon the oxidation of various substrates. These findings indicate that the alternative pathway is unable to compete with the cytochrome respiratory chain for electrons. The alternative pathway transfers only electrons that are superfluous for the cytochrome chain.     

The inhibition of isocitrate oxidation by palmitoyl-l-carnitine and palmitoyl-C0 A in rat liver mitochondria.

Palmitoyl-L carnitine decreases the oxidation of isocitrate in rat liver mitochondria in state 3 by 25-30%. Palmitoyl-L-carnitine acts as an additional substrate raising the rate of oxidative phosphorylation, NAD reduction and ATP/ADP ratio in mitochondria. Palmitoyl-CoA added to mitochondria oxidizing isocitrate in state 3 causes a strong inhibition of isocitrate oxidation and of oxidative phosphorylation and a considerable elevation of intramitochondrial NADH/NAD and ATP/ADP ratios. The effect of palmitoyl-CoA is dependent on its concentration and is competitive with ADP. Carnitine restores only oxidative phosphorylation, but the oxidation of isocitrate remains inhibited. Evidence is presented that the transport of isocitrate is not affected by palmitoyl-CoA is due to the inhibition of adenine nucleotide translocation. The kinetic studies of NAD-dependent isocitrate dehydrogenase in the soluble fraction of sonicated mitochondria revealed that the enzyme is very sensitive towards the inhibition by NADH and only very slightly affected by ATP (Ki for NADH and ATP are 0.017 and 3.6 mM respectively). On the basis of the kinetic data the relative contribution of NADH and ATP in the inhibition of isocitrate oxidation by fatty acids was calculated. It is concluded that the inhibition of isocitrate oxidation caused by palmitoyl-L-carnitine and palmitoyl-CoA is primarily due to the increased reduction of NAD, whereas the increase of ATP/ADP ratio is much less important.     

Involvement of oxyleghaemoglobin and cytochrome P-450 in an efficient oxidative phosphorylation pathway which supports nitrogen fixation in Rhizobium

Cellular ATP level, ATP/ADP ratio and nitrogenase activity rise when oxyleghaemoglobin is added to respiring suspensions of Rhizobium japonicum bacteroids from soybean root nodules. Increased gaseous O₂ tension is much less efficient than oxyleghaemoglobin in stimulation of bacteroid ATP production. Studies with the inhibitor carbonyl cyanide m-chlorophenylhydrazone show this ATP to be generated as a consequence of oxidative phosphorylation. N-Phenylimidazole, a specific cytochrome P-450 inhibitor, also lowers the efficiency of bacteroid oxidative phosphorylation. An approximately linear relationship is observed between ATP/ADP ratio and nitrogenase activity as N-phenylimidazole concentration is lowered. It is suggested that cytochrome P-450 is a component of the leghaemoglobin-facilitated respiration pathway and that it may act as intracellular O₂ carrier rather than terminal oxidase. A less efficient oxidase appears to function when cytochrome P-450 is inhibited.     

A correlation between respiration and synthesis of ATP in mitochondria at different degree of uncoupling of oxidative phosphorylation

It is known that mitochondrial respiration in state 3 is due to three simultaneous and independent processes: synthesis of ATP (1), endogenous passive proton leakage (2), and proton leakage by protonophoric uncoupler (3). The total rate of processes (2) and (3) is equal to the product of respiration rate in state 4 and coefficient KR, which is defined as the ratio of the deltamuH+ value in state 3 to that in state 4. It is shown that it is possible to calculate both the rates of processes (1), (2) and (3) separately and the protonophoric activity of uncoupler using the coefficient KR and other coefficients, which are determined as the ratio of deltamuH+ values in state 3 or in state 4 to its maximal value. Simple methods of determination of these coefficients were developed, which are based on the study of the dependence of respiration rate in states 3 and 4 on the concentration of protonophoric uncoupler. It was found that the uncoupling action of palmitate, a natural uncoupler of oxidative phosphorylation, unlike classic uncoupler-protonophores DNP and FCCP, depends not only on its protonophoric activity but also on the inhibition of the process (1).