Inhibition of oxidative phosphorylation by Ca2+ or Sr2+: a competition with Mg2+ for the formation of adenine nucleotide complexes

Intramitochondrial Sr2+, similar to Ca2+, inhibits oxidative phosphorylation in intact rat-liver mitochondria. Both Ca2+ and Sr2+ also inhibit the hydrolytic activity of the ATPase in submitochondrial particles. Half-maximal inhibition of ATPase activity was attained at a concentration of 2.5 mM Ca2+ or 5.0 mM Sr2+ when the concentration of Mg2+ in the medium was 1.0 mM. The inhibition of ATPase activity by both cations was strongly decreased by increasing the Mg2+ concentration in the reaction medium. In addition, kinetical data and the determination of the concentration of MgATP, the substrate of the ATPase, in the presence of different concentrations of Ca2+ or Sr2+ strongly indicate that these cations inhibit ATP hydrolysis by competing with Mg2+ for the formation of MgATP. On the basis of a good agreement between these results with submitochondrial particles and the results of titrations of oxidative phosphorylation with carboxyatractyloside or oligomycin in mitochondria loaded with Sr2+ it can be concluded that intramitochondrial Ca2+ or Sr2+ inhibits oxidative phosphorylation in intact mitochondria by decreasing the availability of adenine nucleotides to both the ADP/ATP carrier and the ATP synthase.     

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.     

Inhibition of oxidative phosphorylation by Ca or Sr: A competition with Mg for the formation of adenine nucleotide complexes

Intramitochondrial Sr²⁺, similar to Ca²⁺, inhibits oxidative phosphorylation in intact rat-liver mitochondria. Both Ca²⁺ and Sr²⁺ also inhibit the hydrolytic activity of the ATPase in submitochondrial particles. Half-maximal inhibition of ATPase activity was attained at a concentration of 2.5 mM Ca²⁺ or 5.0 mM Sr²⁺ when the concentration of Mg²⁺ in the medium was 1.0 mM. The inhibition of ATPase activity by both cations was strongly decreased by increasing the Mg²⁺ concentration in the reaction medium. In addition, kinetical data and the determination of the concentration of MgATP, the substrate of the ATPase, in the presence of different concentrations of Ca²⁺ or Sr²⁺ strongly indicate that these cations inhibit ATP hydrolysis by competing with Mg²⁺ for the formation of MgATP. On the basis of a good agreement between these results with submitochondrial particles and the results of titrations of oxidative phosphorylation with carboxyatractyloside or oligomycin in mitochondria loaded with Sr²⁺ it can be concluded that intramitochondrial Ca²⁺ or Sr²⁺ inhibits oxidative phosphorylation in intact mitochondria by decreasing the availability of adenine nucleotides to both the ADP/ATP carrier and the ATP synthase.     

Interaction of a synthetic polyanion with rat liver mitochondria

The effect of a polyanion (a copolymer of methacrylate, malaete and styrene in a 1:2:3 proportion with an average molecular weight of 10 000) on respiration, ATPase activity and ADP/ATP exchange activity of rat liver mitochondria and submitochondrial particles has been studied.The polyanion (at 17–150 μg/ml concentration, 100 μg polyanion corresponding to 0.83 μequiv. of carboxylic groups) inhibits the oxidation of succinate and NAD-linked substrates in state 3 in a concentration-dependent manner. The extent of this inhibition can be decreased by elevating the concentration of ADP. State 4 respiration is not affected by the polyanion. It has also a slight inhibitory effect on the oxidation of the above mentioned substrates in the uncoupled state (a maximum inhibition of 37% at 166 μg/ml polyanion concentration), which is unaffected by ADP. The strong inhibition of state 3 respiration can be relieved by 2,4-dinitrophenol to the low level observed in the uncoupled state. Ascorbate+TMPD oxidation is slightly inhibited in state 3, while it is not inhibited at all in the uncoupled state.The polyanion, depending on its concentration, strongly inhibits also the DNP-activated ATPase activity of mitochondria (50% inhibition at 40 μg/ml polyanion concentration).The ATPase activity of sonic submitochondrial particles is also inhibited. However, this inhibition is incomplete (reaching a maximum of 65%) and higher concentrations of the polyanion are required than to inhibit the ATPase activity of intact mitochondria.The polyanion inhibits the ADP/ATP translocator activity of mitochondria, measured by the “back exchange” of [2-³H]ADP. After a short preincubation of the mitochondria with the polyanion, the concentration dependence of the inhibition by the polyanion corresponds to that of the DNP-activated ATPase activity of intact mitochondria.It is concluded that, in intact mitochondria, the polyanion has at least a dual effect, i.e. it partially inhibits the respiratory chain between cytochrome b and cytochrome c, and strongly oxidative phosphorylation by blocking the ADP/ATP translocator.     

Deficiency of uncoupler-stimulated adenosine triphosphatase activity in yeast mitochondria

Oligomycin-sensitive ATPase activity was studied in isolated yeast mitochondria. The protonophore CCCP, at a concentration which completely inhibited ATP synthesis, induced only a low rate of hydrolysis of externally added ATP, and the extent of hydrolysis was dependent upon phosphate (Pi) concentration. CCCP promoted hydrolysis of intramitochondrial ATP. However, hydrolysis of externally added ATP was total in a medium containing potassium phosphate plus valinomycin. Without ionophores, ATPase activity was only observed at high external pH or with detergent-treated mitochondria. Under state 4 conditions, external ATP had access to the catalytic nucleotide site of ATPase as shown by 32Pi-ATP exchange experiments. These results are discussed in terms of a limitation of the translocase-mediated ATP/ADP exchange in uncoupled mitochondria.     

Control of pyruvate dehydrogenase activity in intact cardiac mitochondria. Regulation of the inactivation and activation of the dehydrogenase.

The control of pyruvate dehydrogenase activity by inactivation and activation was studied in intact mitochondria isolated from rabbit heart. Pyruvate dehydrogenase could be completely inactivated by incubating mitochondria with ATP, oligomycin, and NaF. This loss in dehydrogenase activity was correlated with the incorporation of 32P from [gamma-32P]ATP into mitochondrial protein(s) and with a decrease in the mitochondrial oxidation of pyruvate. ATP may be supplied exogenously, generated from endogenous ADP during oxidative phosphorylation, or formed from exogenous ADP in carbonyl cyanid p-trifluoromethoxyphenylhydrazone-uncoupled mitochondria. With coupled mitochondria the concentration of added ATP required to half-inactivate the dehydrogenase was 0.24 mM. With uncoupled mitochondria the apparent Km was decreased to 60 muM ATP. Inactivation of pyruvate dehydrogenase by exogenous ATP was sensitive to atractyloside, suggesting that pyruvate dehydrogenase kinase acts internally to the atractyloside-sensitive barrier. The divalent cation ionophore, A23187, enhanced the loss of dehydrogenase activity. Pyruvate dehydrogenase activity is regulated additionally by pyruvate, inorganic phosphate, and ADP. Pyruvate, in the presence of rotenone, strongly inhibited inactivation. This suggests that pyruvate facilitates its own oxidation and that increases in pyruvate dehydrogenase activity by substrate may provide a modulating influence on the utilization of pyruvate via the tricarboxylate cycle. Inorganic phosphate protected the dehydrogenase from inactivation by ATP. ADP added to the incubation mixture together with ATP inhibited the inactivation of pyruvate dehydrogenase. This protection may result from a direct action on pyruvate dehydrogenase kinase, as ADP competes with ATP, and an indirect action, in that ADP competes with ATP for the translocase. It is suggested that the intramitochondrial [ATP]:[ADP] ratio effects the kinase activity directly, whereas the cytosolic [ATP]:[ADP] ratio acts indirectly. Mg2+ enhances the rate of reactivation of the inactivated pyruvate dehydrogenase presumably by accelerating the rate of dephosphorylation of the enzyme. Maximal activation is obtained with the addition of 0.5 mM Mg2+..     

Investigation of the dependence of the intramitochondrial [ATP]/[ADP] ratio on the respiration rate

The relationship between the respiration rate and the intra- and extramito-chondrial adenine nucleotides was investigated in isolated rat liver mitochondria.

For the determination of adenine nucleotide patterns in both compartments a new procedure was developed, based on the evaluation of these metabolites from incubation of various amounts of mitochondria under identical stationary states of oxidative phosphorylation. These identical states were adjusted by addition of appropriate amounts of hexokinase to a glucose-containing incubation mixture.

Adenine nucleotides were measured in aliquots of the total extract of the incubation mixture without any separation. The concentrations of the adenine nucleotides in both compartments were obtained from a plot of the total concentration of these species versus mitochondrial protein. Disturbances of this method by unspecific efflux of adenine nucleotides could be excluded.

The results obtained for the total adenine nucleotide content (12 nmol · mg⁻¹ protein) and the intramitochondrial [ATP]/[ADP] ratio (about 4 in the resting state) are in good agreement with data obtained by other methods.

Strong evidence is provided for a decrease of the intramitochondrial [ATP]/[ADP] ratio with increasing rate of oxygen consumption. Therefore it is not necessary to assume a microcompartmentation of the intramitochondrial adenine nucleotide pool in respect to the ATPase reaction and the adenine nucleotide translocation.     

Mode of inhibition of mitochondrial energy transduction by chlorophenoxyisobutyrate

1. alpha-p-Chlorophenoxyisobutyric acid, the ethyl ester of which is widely used as an antihypercholesterolaemic drug, is an inhibitor of energy-transfer reactions in isolated rat liver mitochondria. 2. The compound at lower concentrations (<4.0mumol/mg of mitochondrial protein) inhibits state 3 oxidation, stimulates state 4 oxidation, abolishes respiratory control and stimulates the latent adenosine triphosphatase activity of mitochondria. The inhibition imposed on state 3 oxidation is relieved by dinitrophenol. 3. At higher concentrations it inhibits coupled phosphorylation as well as dinitrophenol-stimulated adenosine triphosphatase activity. The inhibition of state 3 oxidation under these conditions is not reversed by uncouplers. 4. The three coupling sites of phosphorylation exhibit differential susceptibility to inactivation by this compound. Coupled phosphorylation at the first site is abolished at a drug concentration of 3.0mumol/mg of protein. The third site is inactivated when the concentration of the drug reaches 5.0mumol/mg of protein. The second site is the most refractory and drug concentrations of the order of 10.0mumol/mg of protein are required effectively to inhibit phosphorylation at this site. 5. The compound also inhibits ATP-dependent reversal of electron transport as well as the adenosine triphosphatase activity in submitochondrial particles. 6. The oxidation of NADH and succinate in these particles is not inhibited. 7. These properties indicate that the compound acts as an ;inhibitory uncoupler' of energy-transfer reactions in isolated mitochondria.     

The stimulation of hepatic oxidative phosphorylation following dexamethasone treatment of rats

The effect of short-term treatment of rats with the synthetic glucocorticoid, dexamethasone, on mitochondrial oxidative phosphorylation has been examined. Treatment of rats for 3 h increased the oxidative capacity of the subsequently isolated mitochondria such that they displayed increased uncoupled and State 3 rates of respiration with NAD-linked substrates, succinate or durohydroquinone. The oxidation of ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine was unaffected. No change was apparent in the activity of a variety of dehydrogenase enzymes nor was there any increase in the mitochondrial content of cytochromes a, b, c1 or c. The uncoupler-dependent ATPase activity of the mitochondria was slightly enhanced following hormone treatment, but not the basal or the total ATPase activity measured in the presence of Triton X-100 plus Mg2+. The mitochondria prepared from dexamethasone-treated rats also displayed increased intramitochondrial concentrations of Mg2+, K+ and exchangeable adenine nucleotides but not Ca2+. It is suggested that the effect of glucocorticoids on mitochondrial respiration may be both the result of a direct activation of the respiratory chain within Complex III and an elevated intramitochondrial adenine nucleotide concentration. The evidence for the de novo synthesis of mitochondrial proteins which mediate the response remains inconclusive.     

Presence of a unit for actin-myosin interaction during the superprecipitation of actomyosin.

The interaction of actin with myosin was studied in the presence of ATP at low ionic strength by means of measurements of the actin-activated ATPase activity of myosin and superprecipitation of actomyosin. At high ATP concentrations the ATPase activities of myosin, heavy meromyosin (HMM) and myosin subfragment 1 (S-1) were activated by actin in the same extent. At low ATP concentrations the myosin ATPase activity was activated about 30-fold by actin, whereas those of HMM and S-1 were stimulated only several-fold. This high actin activation of myosin ATPase was coupled with the occurrence of superprecipitation. The activation of HMM or S-1 ATPase by actin shows a simple hyperbolic dependence on actin concentration, but the myosin ATPase was maximally activated by actin at a 2:1 molar ratio of actin to myosin, and a further increase in the actin concentration had no effect on the activation. These results suggest the presence of a unit for actin-myosin interaction, composed of two actin monomers and one myosin molecule in the filaments.     

Competition between extramitochondrial and intramitochondrial ATP-consuming processes.

The relationship between intra- and extramitochondrial ATP utilization was investigated in liver mitochondria isolated from normally fed, starved and high-protein fed rats. ATP export was provoked by adding a hexokinase-glucose-trap and intramitochondrial ATP consumption by adding ammonia, bicarbonate and ornithine in order to stimulate citrulline synthesis. Both processes compete for ATP produced via oxidative phosphorylation; the rate of citrulline formation declines as the extramitochondrial [ATP]/[ADP] ratio decreases. It is concluded that ATP for adenine nucleotide translocation and that for carbamoyl phosphate synthesis are delivered from a common intramitochondrial pool of adenine nucleotides. In mitochondria from rats with a high-protein diet, citrulline synthesis greatly stimulates the rate of oxidative phosphorylation (about two thirds of state 3 respiration). Under these conditions the intramitochondrial [ATP]/[ADP] ratio is significantly reduced. The intramitochondrial [ATP]/[ADP] ratio is not in thermodynamic equilibrium with the extramitochondrial one.     

Effect of rapid freezing on the functional state of rat heart mitochondria

As evidenced by respiration, oxidative phosphorylation, ATPase and NADH-oxidase activities, mitochondria composing heart tissue slices are more damaged by freezing-thawing than isolated mitochondria. A change in the functional activity of mitochondria is manifested in an increased respiratory rate in the second metabolic state and decreased respiratory rate in the third metabolic state upon oxidation of succinate and alpha-ketoglutarate; the ability of mitochondria to synthetize ATP (inhibition of the respiratory control) varied and the ATPase and NADH-oxidase activities increased. These changes in the functional state of mitochondria appeared to be due to a rise of the proton conductivity of the inner mitochondrial membrane by freezing-thawing.     

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)     

Respiration-department uncoupler-stimulated ATPase activity in castor bean endosperm mitochondria and submitochondrial particles.

1. The uncoupler-stimulated ATPase activity of castor bean endosperm mitochondria and submitchondrial particles has been studied. The rate of ATP hydrolysis catalyzed by intact mitochondria was slow and little enhanced by addition of uncouplers at the concentration required for uncoupling the oxidative phosphorylation. ATP-ase activity was stimulated at higher concentrations of uncouplers. 2. 1-Anilinonaphthalene 8-sulfonate fluorescence was decreased when the mitochondria were oxidizing succinate. Carbonylcyanide-p-trifluoromethoxyphenylhydrazone and antimycin reversed the succinate-induced fluorescence diminution. ATP did not induce the fluorescence response. 3. The addition of succinate, NADH or ascorbate/N,N,N'-N'-tetramethyl-p-phenylenediamine as electron donor induced high ATPase activity in the presence of low concentrations of uncouplers. Stimulating effect of uncouplers was completely abolished by further addition of antimycin. 4. Submitochondrial particles were prepared by sonication. The particles catalyzed a rapid hydrolysis of ATP and carbonylcyanide-p-trifluoromethoxyphenylhydrazone at 10-8 M did not stimulate the ATPase activity. Addition of succinate induced uncoupler-stimulated ATPase activity. The effect of succinate was completely abolished by further addition of antimycin. 5. The treatment of submitochondrial particles by trypsin or high pH also induced uncoupler-stimulated ATPase activity. 6. The above results were interpreted to indicate that ATPase inhibitor regulated the back-flow reaction of mitochondrial oxidative phosphorylation.     

Bioenergetic studies of mitochondrial oxidative phosphorylation using 31phosphorus NMR

The relationship between phosphorylation ratio [( ATP])/[ADP][Pi], phosphocreatine (PCr)/Pi, and ATPase activity was determined for isolated rat heart mitochondria, and the use of phosphorylation ratio and/or PCr/Pi as bioenergetic indices (Chance, B., Eleff, S., Leigh, J. S., Sokolow, D., and Sapega, A. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6714-6718) was evaluated. Isolated rat heart mitochondria were suspended at low concentration (0.5-2.0 mg of protein/ ml) in oxygenated KCl/sucrose/4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid medium at 25 degrees C and pyruvate, malate, PCr, ATP, Pi, and Mg2+ were added. Changes in extramitochondrial phosphorus compounds were followed by 31P NMR. The ATPase activity was varied by the addition of potato apyrase. It was found that the logarithm of steady state PCr/Pi decreased linearly with increasing ATPase rate with a PCr/Pi intercept of 32.8 at 0 ATPase rate. The log phosphorylation ratio was also linearly related to the ATPase rate with an extrapolated maximum value of 6.87 at 0 ATPase rate, corresponding to a phosphorylation ratio of 7.41 X 10(6) M(-1) and a delta GATP of -16.3 kcal. The phosphorylation ratio in these experiments (for state 4 respiration) was greater by 1 or 2 orders of magnitude than previously reported for either isolated mitochondria or for whole tissue.     

Interdependence of mitochondrial ATP production and extramitochondrial ATP utilization in intact spermatozoa

The dependence of both respiration and total activity of ATP-consuming reactions on the cellular adenine nucleotide pattern was investigated in intact bovine spermatozoa. ATP consumption was manipulated by inhibition with vanadate and activation with caffeine, leading to a decrease or increase in the rate of respiration up to 70% or 20%, respectively. Oligomycin blocked the respiration to the same extent as did vanadate, suggesting that the total extramitochondrial ATP-consuming activity is vanadate-sensitive. The major part of ATP utilization must be linked to dynein ATPase, since inhibition of (Na⁺, K⁺) ATPase by ouabain showed only a small effect on respiration (−17%). Being a potent inhibitor of dynein ATPase, vanadate drastically reduced the amount of motile cells, whereas caffeine tended to increase the intensity of motion. The effects of vanadate or caffeine on respiration were paralleled by changes in cellular ATP, reflecting the response of mitochondrial respiration on the cellular ATP/ADP ratio. Respiration was found to depend on changes in the ATP/ADP ratio in the range from about 3 (+ caffeine) to 9 (+ vanadate). The range of response of ATP consumption to the ATP/ADP ratio was determined by varying the mitochondrial ATP production via the concentration of lactate which was used as substrate. The measured effects on both respiratory rate and ATP/ADP ratio suggested that ATP consumption was markedly dependent on ATP/ADP ratios below 5. It is concluded that lactate concentrations above 1 mM sufficiently supply bovine spermatozoa with substrate and the energy turnover is mainly limited by the activity of dynein ATPase rather than by the capacity of mitochondrial oxidative phosphorylation.     

Relation between extra- and intramitochondrial ATP/ADP ratios in rat liver mitochondria

Changes of the extra- and intramitochondrial ATP/ADP ratios as a function of the respiratory state were measured in incubations with rat liver mitochondria. ATPase or creatine/creatine kinase was used to change the extramitochondrial ATP/ADP ratio; the separation of the mitochondrial pellet was performed by a Millipore filtration technique. Under all conditions tested, the intramitochondrial ratio changed in the same direction as the extramitochondrial one, except in the presence of atractylate where this correlation was not observed. Furthermore, it could be shown that the oxygen uptake and pyruvate carboxylase activity correlated with the intramitochondrial ATP/ADP ratio and not with the extramitochondrial one. These results do not support the proposal that the adenine nucleotide translocase is rate limiting for respiration.     

Relation between extra- and intramitochondrial ATP/ADP ratios in rat liver mitochondria

Changes of the extra- and intramitochondrial ATP/ADP ratios as a function of the respiratory state were measured in incubations with rat liver mitochondria. ATPase or creatine/creatine kinase was used to change the extramitochondrial ATP/ADP ratio; the separation of the mitochondrial pellet was performed by a Millipore filtration technique. Under all conditions tested, the intramitochondrial ratio changed in the same direction as the extramitochondrial one, except in the presence of atractylate where this correlation was not observed. Furthermore, it could be shown that the oxygen uptake and pyruvate carboxylase activity correlated with the intramitochondrial ATP/ADP ratio and not with the extramitochondrial one. These results do not support the proposal that the adenine nucleotide translocase is rate limiting for respiration.     

Regulation of pyruvate oxidation in blowfly flight muscle mitochondria: Requirement for ADP

Blowfly (Phormia regina) flight muscle mitochondria oxidized pyruvate (+ proline) in the presence of either ADP (coupled respiration) or carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP-uncoupled respiration). There was an absolute requirement for ADP (K_m = 8.0 μm) when pyruvate oxidation was stimulated by FCCP in the presence of oligomycin. This requirement for ADP was limited to the oxidation of pyruvate; uncoupled α-glycerolphosphate oxidation proceeded maximally even in the absence of added ADP. Atractylate inhibited uncoupled pyruvate oxidation whether added before (>99%) or after (95%) initiation of respiration with FCCP. In the presence of FCCP, oligomycin, and limiting concentrations of ADP (less than 110 μm), there was a shutoff in the uptake of oxygen. This inhibition of respiration was completely reversed by the addition of more ADP. Plots of net oxygen uptake as a function of the limiting ADP concentration were linear; the observed ADP/O ratio was 0.22 ± 0.025. An ADP/O ratio of 0.2 was predicted if phosphorylation occurred only at the succinyl-CoA synthetase step of the tricarboxylate cycle. Experiments performed in the presence of limiting concentrations of ADP, and designed to monitor changes in the mitochondrial content of ADP and ATP, demonstrated that the shutoff in oxygen uptake was not due to the presence of a high intramitochondrial concentration of ATP. Indeed, ATP, added to the medium prior to the addition of FCCP, inhibited uncoupled pyruvate oxidation; the apparent K_I was 0.8 mm. These results are consistent with the hypothesis that it is the intramitochondrial ATP/ADP ratio that is one of the controlling factors in determining the rate of flux through the tricarboxylate cycle. Changes in the mitochondrial content of citrate, isocitrate, α-ketoglutarate, and malate during uncoupled pyruvate oxidation in the presence of a limiting concentration of ADP were consistent with the hypothesis that the mitochondrial NAD⁺-linked isocitric dehydrogenase is a major site for such control through the tricarboxylate cycle.