Uptake Of Adenine Nucleotides Research Articles

Glutamate excitotoxicity and Ca2+-regulation of respiration: Role of the Ca2+ activated mitochondrial transporters (CaMCs)

Glutamate elicits Ca2+ signals and workloads that regulate neuronal fate both in physiological and pathological circumstances. Oxidative phosphorylation is required in order to respond to the metabolic challenge caused by glutamate. In response to physiological glutamate signals, cytosolic Ca2+ activates respiration by stimulation of the NADH malate–aspartate shuttle through Ca2+-binding to the mitochondrial aspartate/glutamate carrier (Aralar/AGC1/Slc25a12), and by stimulation of adenine nucleotide uptake through Ca2+ binding to the mitochondrial ATP-Mg/Pi carrier (SCaMC-3/Slc25a23). In addition, after Ca2+ entry into the matrix through the mitochondrial Ca2+ uniporter (MCU), it activates mitochondrial dehydrogenases. In response to pathological glutamate stimulation during excitotoxicity, Ca2+ overload, reactive oxygen species (ROS), mitochondrial dysfunction and delayed Ca2+ deregulation (DCD) lead to neuronal death. Glutamate-induced respiratory stimulation is rapidly inactivated through a mechanism involving Poly (ADP-ribose) Polymerase-1 (PARP-1) activation, consumption of cytosolic NAD+, a decrease in matrix ATP and restricted substrate supply. Glutamate-induced Ca2+-activation of SCaMC-3 imports adenine nucleotides into mitochondria, counteracting the depletion of matrix ATP and the impaired respiration, while Aralar-dependent lactate metabolism prevents substrate exhaustion. A second mechanism induced by excitotoxic glutamate is permeability transition pore (PTP) opening, which critically depends on ROS production and matrix Ca2+ entry through the MCU. By increasing matrix content of adenine nucleotides, SCaMC-3 activity protects against glutamate-induced PTP opening and lowers matrix free Ca2+, resulting in protracted appearance of DCD and protection against excitotoxicity in vitro and in vivo, while the lack of lactate protection during in vivo excitotoxicity explains increased vulnerability to kainite-induced toxicity in Aralar +/− mice. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

Mitochondrial ATP-Mg/Pi carrier SCaMC-3/Slc25a23 counteracts PARP-1-dependent fall in mitochondrial ATP caused by excitotoxic insults in neurons.

Glutamate excitotoxicity is caused by sustained activation of neuronal NMDA receptors causing a large Ca(2+) and Na(+) influx, activation of poly(ADP ribose) polymerase-1 (PARP-1), and delayed Ca(2+) deregulation. Mitochondria undergo early changes in membrane potential during excitotoxicity, but their precise role in these events is still controversial. Using primary cortical neurons derived from mice, we show that NMDA exposure results in a rapid fall in mitochondrial ATP in neurons deficient in SCaMC-3/Slc25a23, a Ca(2+)-regulated mitochondrial ATP-Mg/Pi carrier. This fall is associated with blunted increases in respiration and a delayed decrease in cytosolic ATP levels, which are prevented by PARP-1 inhibitors or by SCaMC-3 activity promoting adenine nucleotide uptake into mitochondria. SCaMC-3 KO neurons show an earlier delayed Ca(2+) deregulation, and SCaMC-3-deficient mitochondria incubated with ADP or ATP-Mg had reduced Ca(2+) retention capacity, suggesting a failure to maintain matrix adenine nucleotides as a cause for premature delayed Ca(2+) deregulation. SCaMC-3 KO neurons have higher vulnerability to in vitro excitotoxicity, and SCaMC-3 KO mice are more susceptible to kainate-induced seizures, showing that early PARP-1-dependent fall in mitochondrial ATP levels, counteracted by SCaMC-3, is an early step in the excitotoxic cascade.

Effect of adenine nucleotide pool size in mitochondria on intramitochondrial ATP levels

Net adenine nucleotide transport into and out of the mitochondrial matrix via the ATP-Mg/P i carrier is activated by micromolar calcium concentrations in rat liver mitochondria. The purpose of this study was to induce net adenine nucleotide transport by varying the substrate supply and/or extramitochondrial ATP consumption in order to evaluate the effect of the mitochondrial adenine nucleotide pool size on intramitochondrial adenine nucleotide patterns under phosphorylating conditions. Above 12 nmol/mg protein, intramitochondrial ATP/ADP increased with an increase in the mitochondrial adenine nucleotide pool. The relationship between the rate of respiration and the mitochondrial ADP concentration did not depend on the mitochondrial adenine nucleotide pool size up to 9 nmol ADP/mg mitochondrial protein. The results are compatible with the notion that net uptake of adenine nucleotides at low energy states supports intramitochondrial ATP consuming processes and energized mitochondria may lose adenine nucleotides. The decrease of the mitochondrial adenine nucleotide content below 9 nmol/mg protein inhibits oxidative phosphorylation. In particular, this could be the case within the postischemic phase which is characterized by low cytosolic adenine nucleotide concentrations and energized mitochondria.

Neonatal hypoxia or maternal diabetes delays postnatal development of liver mitochondria.

The matrix adenine nucleotide pool size of rat liver mitochondria was low at birth (2.6-3.0 nmol ATP + ADP + AMP/mg mitochondrial protein). After parturition, the pool size was increased by 50-75% within 1 h, which was sufficient for full development of state 3 respiration rates. The adenine nucleotide pool size continued to increase to 100-150% of the value at birth by 2-3 h postnatal. The ATP/ADP ratio in isolated mitochondria also increased postnatally, to about double the value at birth by 3 h. There were no matrix volume changes over this postnatal period, so the increased ATP + ADP + AMP pool size and the increased ATP/ADP ratio together inferred an overall increase of about 5-fold in the matrix ATP concentration under aerobic conditions. The postnatal uptake of adenine nucleotides into mitochondria occurred at a slower rate in newborns that were hypoxic (11% 02) and in newborns of diabetic mothers (diabetes induced on day 5 of gestation by streptozotocin injection). The normal increase in matrix ATP content is responsible for the rapid stimulation of pyruvate carboxylation (an ATP-requiring matrix reaction) and this in turn contributes to the rapid postnatal onset of gluconeogenesis. The results suggest that delayed adenine nucleotide uptake into liver mitochondria may retard initiation of gluconeogenesis in newborns experiencing hypoxia, as in respiratory distress or in newborns of diabetic mothers. We speculate that this mechanism contributes to the persistent hypoglycemia that is typical of these at-risk newborns.

Testing the ?methanochondrion concept?: are nucleotides transported across internal membranes in Methanobacterium thermoautotrophicum?

In order to test the “Methanochondrion concept”, uptake of adenine nucleotides in various membrane preparations of Methanobacterium thermoautotrophicum was studied. The uptake showed properties which are in general interpreted as indicative of a transport mechanism: (i) kinetics in the time range of minutes, (ii) temperature dependence, (iii) substrate specificity and (iv) failure to remove the substrate by extensive washing.

Control of mitochondrial content of adenine nucleotides by submicromolar calcium concentrations and its relationship to hormonal effects.

Rat liver mitochondria were incubated at 30 degrees C with 4 mM ATP in a medium similar in electrolyte composition to that of hepatic cytosol. Under these conditions, a net increase in mitochondrial adenine nucleotides was observed that was dependent on the concentration of free Ca2+ [( Ca2+]) in the incubation medium. At 0.2 microM [Ca2+] or less, there was no demonstrable uptake of adenine nucleotides; at 0.4 microM [Ca2+], or greater, net uptake occurred. The calcium-dependent accumulation of nucleotides by mitochondria required Mg2+ in the incubation medium and was insensitive to carboxyatractyloside. The uptake of adenine nucleotides was enhanced by the addition of antimycin A or antimycin A together with oligomycin. Accumulation of nucleotides appeared to be associated with a small increase in mean mitochondrial volume, but the membrane potential was not affected. No uptake or loss of NAD-NADH by mitochondria was detected. Ruthenium red failed to inhibit the calcium-dependent uptake of adenine nucleotides by the mitochondria, indicating that stimulation of this process by Ca2+ does not involve transport of the cation into mitochondria by the Ca2+ uniporter. Because glucagon acts to elevate cytosolic [Ca2+] from approximately 0.2 microM to 0.6 microM, the same range affecting nucleotide uptake, it is proposed that the increase in mitochondrial adenine nucleotides that follows treatment with glucagon is mediated by the rise in cytosolic [Ca2+] produced by the hormone. This hypothesis was supported by the observation that epinephrine and A23187, agents that raise cytosolic [Ca2+], increased the content of mitochondrial adenine nucleotides in isolated hepatocytes. Furthermore, cells, incubated under calcium-depleting conditions, had a diminished response to glucagon.

Adenine nucleotide translocase-dependent anion transport in pea chloroplasts

Pea chloroplasts were found to take up actively ATP and ADP and exchange the external nucleotides for internal ones. Using carrier-free [ 14C]ATP, the rate of nucleotide transport in chloroplasts prepared from 12–14-day-old plants was calculated to be 330 μmol ATP/g chlorophyll/min, and the transport was not affected by light or temperature between 4 and 22°C. Adenine nucleotide uptake was inhibited only slightly by carboxyatractylate, whereas bongkrekic acid was nearly as effective an inhibitor of the translocator in pea chloroplasts as it was in mammalian mitochondria. There was no counter-transport of adenine nucleotides with substrates carried on the phosphate translocator including inorganic phosphate, 3-phosphoglycerate and dihydroxyacetone phosphate. However, internal or external phospho enolpyruvate, normally considered to be transported on the phosphate carrier in chloroplasts, was able to exchange readily with adenine nucleotides. Furthermore, inorganic pyrophosphate which is not transported by the phosphate carrier initiated efflux of phospho enolpyruvate as well as ATP from the chloroplast. These findings illustrate some interesting similarities as well as differences between the various plant phosphate and nucleotide transport systems which may relate to their role in photosynthesis.

Adenine nucleotide translocase activity and sensitivity to inhibitors in hepatomas. Comparison of the ADP/ATP carrier in mitochondria and in a purified reconstituted liposome system.

Adenine nucleotide uptake was found to be lower in mitochondria from hepatoma 7777, 7800, and 9618A than in the host livers. Moreover, in the fast-growing hepatoma 7777 the sensitivity of the adenine nucleotide translocase to inhibition by carboxyatractylate and bongkrekic acid was considerably decreased. Purification of the ADP/ATP carrier from hepatoma 7777 mitochondria and its reconstitution into an artificial liposome system reversed the abnormal kinetics in that the adenine nucleotide uptake and response to inhibitors were identical in proteoliposome preparations from host liver and tumor mitochondria. Analysis of the lipids of the hepatoma inner mitochondrial membrane indicated considerable differences from normal in the levels of phospholipids and cholesterol. Most striking was the increase in cholesterol and sphingomyelin of the hepatoma 7777 inner membrane. An artificial liposome system containing cholesterol in addition to the standard phospholipids could produce alterations in kinetics of the purified ADP/ATP carrier from heart mitochondria similar to those seen in the hepatoma 7777. In general, these results support the suggestion that alterations in the lipid environment of the inner mitochondrial membrane rather than intrinsic changes in the carrier protein itself produce the aberrant observations of adenine nucleotide translocase activity in hepatoma mitochondria.

The maturation of oxidative phosphorylation in human mitochondria and its relation to increased adenine nucleotide concentration

Transition from a uterine environment to an independent existence requires the maturation of energy production. Foetalrat liver mitochondria show a rapid increase in respiratory activity aRer birth, dependent on a net uptake of adenine nucleotides (Sutton & Pollak, 1980). The present work examines the relationship between oxygen consumption in the presence of three substrates by human placental and uterine mitochondria and their adenine nucleotide contents. Placentae were obtained from uncomplicated deliveries (gestations 36-41 weeks) and non-pathological uterine tissue from hysterectomies. Mitochondria were prepared and assayed by the methods of Olivera & Meigs (1975), with the exception of the preparation and assay media. Isolation buffer contained 0.22 M - mannitol, [4 - (2 hydroxyethy1)- 1 -piperazine-ethanesulphonic acid1 and 1 mMEDTA, pH7.4, with the addition of 2mg of bovine serum albumin/ml. Assay buffer was similar but also contained lOmM-KH,PO,, 5 mM-MgCl, and fatty acid-free bovine serum albumin. Adenine nucleotides were extracted from both uterine and placental mitochondria and assayed by high-pressure liquid chromatography (Simmonds et al., 1982).

The mitochondrial localization of hexokinase in pea leaves

Up to 80% of total cellular hexokinase (EC 2.1.7.4) activity in pea (Pisum sativum L.) leaves was found to be associated with particulate fractions. Fractionation on sucrose density gradients showed this particulate activity to be associated exclusively with mitochondria. In the presence of glucose and ATP, the bound mitochondrial hexokinase could support rates of O2 uptake of up to 30% of normal ADP-stimulated rates. This stimulation of O2 uptake by hexokinase was completely sensitive to oligomycin, indicating that it resulted from an increase in the supply of ADP for mitochondrial oxidative phosphorylation. Spectrophotometric measurements of the mitochondrial hexokinase activity showed that ADP could support rapid rates of activity provided oxidizable substrates were also present to support the conversion of ADP to ATP in oxidative phosphorylation. Carboxyatractyloside, an inhibitor of adenine-nucleotide uptake by mitochondria, inhibited this ADP-supported activity, but had no effect on hexokinase activity in the presence of added ATP, demonstrating that the hexokinase enzyme was located external to the inner mitochondrial membrane. Oligomycin also inhibited ADP-supported activity but had no effect on ATP-supported hexokinase activity. Glucose (Km 53 μM) was the preferred substrate of pea-leaf mitochondrial hexokinase compared with fructose (Km 5.1 mM). Hexokinase was not solubilised in the presence of glucose-6-phosphate.

Electrophoretic control of reconstituted adenine nucleotide translocation.

The initial velocity of adenine nucleotide exchange catalyzed by the reconstituted ADP-ATP carrier from beef heart mitochondria was measured under the influence of membrane potential and with different nucleotide distributions between the internal liposomal and the external buffer volume. Both Vmax and Km of adenine nucleotide uptake not only changed due to the applied potential but also depended on the respective nucleotide distribution. The rate equations for the ADP-ATP exchange under the various conditions were derived. These equations were simplified by assuming two alternative situations; either (a) af affinity type model, where the membrane potential influences only the affinity of the adenine nucleotide carrier toward ATP and ADP, or (b) a velocity type or distribution model, where the membrane potential modulates the rate constants of the ADP-ATP exchange. On the basis of several simplifications in the reconstituted system, the rate equations could be solved and the rate constants and dissociation constants of the exchange in the "energized" and in the "deenergized" state could be calculated. These values were used to derive prediction tables for normalized exchange rates under different nucleotide distributions, which were then compared with the experimental data. Only the exchange rates predicted by the velocity-type model agreed with the measured values. On the basis of this model a definite asymmetry caused by the membrane potential could be seen. Whereas this asymmetry is not very pronounced in the case of carrier-ADP complexes, about 40 times more ATP-loaded binding sites face the outside of the vesicles in the energized state.

Regulation of the mitochondrial adenine nucleotide pool size

A mechanism by which normal adult rat liver mitochondria may regulate the matrix adenine nucleotide content was studied in vitro. If mitochondria were incubated with 1 m m ATP at 30 ° C in 225 m m sucrose, 2 m m K 2HPO 4, 5 m m MgCl 2, and 10 m m Tris-Cl (pH 7.4), the adenine nucleotide pool size increased at a rate of 0.44 ± 0.02 nmol/mg mitochondrial protein/min. The rate of adenine nucleotide accumulation under these conditions was concentration dependent and specific for ATP or ADP; AMP was not taken up. The rate of net ADP uptake was 50–75% slower than that for ATP. The K m values for net uptake of ATP and ADP were 2.08 and 0.36 m m, respectively. Adenine nucleotide uptake was stoichiometrically dependent on Mg 2+ and stimulated by inorganic phosphate. Net uptake was inhibited by n-ethylmaleimide, or mersalyl, but not by n-butylmalonate. Nigericin inhibited net uptake, but valinomycin did not. In the presence of uncouplers, net uptake was not only inhibited, but adenine nucleotide efflux was observed instead. Like uptake, uncoupler-induced efflux of adenine nucleotides was inhibited by mersalyl, indicating that a protein was required for net flux in either direction. Carboxyatractyloside, bongkrekic acid, or respiratory substrates reduced the rate of adenine nucleotide accumulation, however, this did not appear to be a direct inhibition of the transport process, but rather was probably related indirectly to an increase in the matrix ATP ADP ratio. The collective properties of the transport mechanism(s) for adenine uptake and efflux were different from those which characterize any of the known transport systems. It is proposed that uptake and efflux operate to regulate the total matrix adenine nucleotide pool size: a constant pool size is maintained if the rates of uptake and efflux are equal. Transient alterations in the relative rates of uptake and efflux may occur in response to hormones or other metabolic signals, to bring about net changes in the pool size.

Net uptake of adenine nucleotides by newborn rat liver mitochondria

In newborn rat liver, the adenine nucleotide content (ATP + ADP + AMP) of mitochondria increases severalfold within 2 to 3 h of birth. The net increase in mitochondrial adenines suggests a novel mechanism by which mitochondria are able to accumulate adenine nucleotides from the cytosol ( J. R. Aprille and G. K. Asimakis, 1980, Arch. Biochem. Biophys. 201, 564. ). This was investigated further in vitro. Isolated newborn liver mitochondria incubated with 1 m M ATP for 10 min at 30 °C doubled their adenine nucleotide content with effects on respiratory functions similar to those observed in vivo: State 3 respiration and adenine translocase activity increased, but uncoupled respiration was unchanged. The mechanism for net uptake of adenine nucleotides was found to be specific for ATP or ADP, but not AMP. Uptake was concentration dependent and saturable. The apparent K m ′s for ATP and ADP were 0.85 ± 0.27 m M and 0.41 ± 0.20 m M, respectively, measured by net uptake of [ 14C]ATP or [ 14C]ADP. The specific activities of net ATP and ADP uptake averaged 0.332 ± 0.062 and 0.103 ± 0.002 nmol/min/mg protein, respectively. ADP was a competitive inhibitor of net ATP uptake. If P i was omitted from the incubations, net uptake of ATP or ADP was reduced by 51%. Either mersalyl or N-ethylmaleimide severely inhibited the accumulation of adenine nucleotides. Net ATP uptake was stoichiometrically dependent on MgCl 2, suggesting that Mg 2+ is accumulated along with ATP (or ADP). Uptake was energy dependent as indicated by the following results: Net AdN uptake (especially ADP uptake) was stimulated by the addition of an oxidizable substrate (glutamate) and inhibited by FCCP (an uncoupler). Antimycin A had no effect on net ATP uptake but inhibited net ADP uptake, suggesting that ATP was able to serve as an energy source for its own accumulation. If carboxyatractyloside was added to inhibit the exchange translocase, thereby preventing rapid access of exogenous ATP to the matrix, net ATP uptake was inhibited; carboxyatractyloside had no effect on ADP uptake. It was concluded that the net uptake of adenine nucleotides from the extramitochondrial space occurs by a specific transport process distinct from the classic adenine nucleotide exchange translocase. The accumulation of adenine nucleotides may regulate matrix reactions which are allosterically affected by adenines or which require adenines as a substrate.

Net uptake of adenine nucleotides in isolated rat liver mitochondria

Net uptake of adenine nucleotides in isolated rat liver mitochondria

The Increasing Adenine Nucleotide Concentration and the Maturation of Rat Liver Mitochondria during Neonatal Development

The levels of adenosine triphosphate, diphosphate, and monophosphate in liver and isolated liver mitochondria were examined in foetal, neonatal, and suckling rats. It was shown that while the total adenine nucleotide level in liver varied only slightly during development, there was a steady increase in the concentration of mitochondrial adenine nucleotides. This increase was most dramatic just after birth. Experiments using pups obtained by caesarean section one day prior to normal birth and kept in a humidicrib for up to two hours, showed that the mitochondrial adenine nucleotide level doubles during this period. This increase is associated with the maturation of the mitochondrial inner membrane as measured by the enhancement of respiratory control. The results indicate that in addition to the adenine nucleotide translocator—which effects the exchange of adenosine triphosphate and diphosphate—there must be a second transport mechanism present, at least in perinatal mitochondria, which is responsible for the net uptake of adenine nucleotides. The adenine nucleotides in this study were measured, using a modified luciferin—luciferase assay. In this method the preincubation of sample with appropriate enzymes to convert adenosine monophosphate and diphosphate to adenosine triphosphate, was carried out in the same scintillation vial as the final assay. This eliminated a second sampling step and thereby increased the convenience, speed, and accuracy of this very sensitive method.

The extent of mitochondrial F 1-ATPase and adenine nucleotide carrier activity with ϵ-ATP

1. The use of 1, N 6-ethenoadenosine 5′-triphosphate (ϵ-ATP), a synthetic, fluorescent analog of ATP, by whole rat liver mitochondria and by submitochondrial particles produced via sonication has been studied. 2. Direct [ 3H]adenine nucleotide uptake studies with isolated mitochondria, indicate the ϵ-[ 3H]ATP is not transported through the inner membrane by the adenine nucleotide carrier and is therefore not utilized by the 2,4-dinitrophenol-sensitive F 1-ATPase (EC 3.6.1.3) that functions in oxidative phosphorylation. However, ϵ-ATP is hydrolyzed by a Mg 2+-dependent, 2,4-dinitrophenol-insensitive ATPase that is characteristic of damaged mitochondria. 3. ϵ-ATP can be utilized quite well by the exposed F 1-ATPase of sonic submitochondrial particles. This ϵ-ATP hydrolysis activity is inhibited by oligomycin and stimulated by 2,4-dinitrophenol. The particle F 1-ATPase displays similar K m values for both ATP and ϵ-ATP; however, the V with ATP is approximately six times greater than with ϵ-ATP. 4. Since ϵ-ATP is a capable substrate for the submitochondrial particle F 1-ATPase, it is proposed that the fluorescent properties of this ATP analog might be employed to study the submitochondrial particle F 1-ATPase complex, and its response to various modifiers of oxidative phosphorylation.

Effect of reserpine on adenosine uptake and metabolism, and subcellular transport of platelet adenosine triphosphate in washed rabbit platelets

There is a slow exchange of adenine nucleotides between the metabolically active (cytoplasmic) pool and the releasable amine storage organelle pool of blood platelets. Reserpine is known to inhibit serotonin uptake into platelet storage organelles. Therefore, we have determined whether reserpine also inhibits the uptake of adenine nucleotides from the cytoplasm into the storage organelles of rabbit platelets. Transport of adenine nucleotides from the metabolically active pool into the releasable amine storage granule pool was followed by labeling the metabolically active pool of adenine nucleotides by incubating the platelets with [ 14C]adenosine or [ 14C]adenine. Practically complete release of amine storage granule constituents was induced at various times in aliquots of the labeled platelet suspensions by treatment with a high concentration of thrombin (0.45 units/ml. The fraction of the total labeled [ 14C]ATP released was taken as a measure of ATP transport from the metabolically active pool into the releasable pool. Reserpine (0.2 and 2 μM) decreased the rate of ATP transport into the storage granules by about 50 per cent. Platelets obtained from rabbits that had received 5 mg/kg of reserpine intraperitoneally 18 hr prior to the collection of blood released less ATP and ADP than control platelets from animals that had not received any drugs. This was not due to inhibition of the release reaction by reserpine. Since reserpine reduces the amount of adenine nucleotides in the storage granules, we conclude that if it affects the rate of efflux of adenine nucleotides from the granules at all, this effect must be slight compared with the inhibition of the uptake into the granules. Reserpine was also found to decrease the incorporation of [8- 14C]adenosine into platelet adenine nucleotides by inhibiting adenosine uptake into the platelets noncompetitively ( K i = 2 μM). Inosine uptake was also inhibited by reserpine. The effect of reserpine on adenosine uptake was reversible. In contrast, the effect of reserpine on ATP transfer from the metabolically active pool into the releasable pool was irreversible. This is in keeping with earlier observations that some reserpine binds to platelets reversibly and some binds irreversibly.

The influence of some cardioactive drugs on the energy-dependent uptake of calcium, potassium and adenine nucleotides by mitochondria

A number of cardioactive substances were studied for their influence on isolated mitochondria obtained from rat liver and rabbit heart. Among several drugs tested, quinidine, dl-propranolol, l-alprenolol, dl-oxyfedrin and tetracaine appeared to be the most active in decreasing or blocking the rate of energy-dependent uptake of calcium, potassium and adenine nucleotides in an incubation medium containing substrate (succinate or glutamate/fumarate), MgCl 2, phosphate and approximately 5 mg of mitochondrial protein. The effective concentrations (5 × 10 −5 m to 10 −3 m) were comparable to those employed by other investigators to block the effects on the ATP-dependent calcium accumulation and the ATPase-activity of isolated sarcotubular fractions of heart and skeletal muscle by means of quinidine and propranolol. It is concluded that the rate-inhibiting effects of the active drugs on the velocity and the extent of calcium, potassium, and adenine nucleotide uptake could determine their effect on cardiac contractility.

Adenine nucleotide translocation of mitochondria. Identification of carrier sites.

The present study demonstrates the existence of specific binding sites for ADP and ATP at the mitochondrial membrane which can be attributed to the adenine nucleotide carrier. Parameters of the binding and various other properties of the carrier sites are determined. The differentiation of the carrier sites is based on the assumption that atractyloside removes adenine nucleotides competitively from these sites. For the accurate evaluation and discrimination of the carriers three parallel binding samples are compared: (a) total binding; (b) atractyloside addition before adenine nucleotide, which prevents binding to the carrier and the exchange with endogenous adenine nucleotides; (c) addition of atractyloside after adenine nucleotide, which displaces adenine nucleotide from the carrier but not from the exchanged pool.Total uptake of adenine nucleotides is thus differentiated into three portions: (a) binding to the carrier sites (= specific binding); (b) exchange; (c) binding to unspecific, atractyloside‐insensitive sites. Procedures have been developed for depleting mitochondria of endogenous adenine nucleotides to increase the proportion of the atractyloside‐removable binding in relation to the magnitude of the exchangeable endogenous adenine nucleotide pool. Kinetics of the binding at atractyloside‐removable sites are very rapid compared to “binding” by exchange with endogenous adenine nucleotides, even at 0°. The specificity of the carrier binding, as measured by competitive replacement of [14C]ADP with unlabelled nucleotides, shows binding confined to ATP, ADP, dADP and, to a lesser extent, adenosine tetraphosphate. The high specificity is in agreement with that measured for the adenine nucleotide exchange. From the concentration dependence of the specific adenine nucleotide binding, non‐linear Scatchard plots are obtained consistent with two sites of unequal affinity (Kd′= 0.3 μM; Kd”= 7 μM). The total number of carrier sites is C0/cyt. a= 1.3 (mole/mole). The ratio of high/low affinity types is C′/C”= 1:5. The corresponding values for rat heart are: Kd′= 0.1 μM, Kd”= 4 μM; C0= 2.2; C′/C”= 1:4. With ATP two binding sites of unequal affinity are also obtained. Both Kd′ and Kd” for ATP are 1.5 to 2 times higher than for ADP. ATP binding is not affected by uncouplers. In arsenate depleted rat liver mitochondria a single binding site of high affinity (Kd= 0.5 μM) is found with C0/cyt. a= 1.2 (mole/mole). This is only half the content (per cyt. a) found in rat heart. Some differentiation between specific and unspecific binding is also possible on the basis of SH and histidine reagents. Only the unspecific binding is sensitive to p‐chloromercuribenzoate. The specific binding is selectively sensitive to methylene blue‐catalyzed photo‐oxidation. This fact, and the strong decrease in atractyloside‐removable binding between pH 7.0 and 7.5, suggests the involvement of a histidine group in the specific binding. Sonic particles retain some ability for uptake of adenine nucleotides, but this is no longer atractyloside‐sensitive. The concentration dependence of total adenine nucleotide binding gives non‐linear Scatchard plots which can be fitted by two binding sites with unequal affinities. The possible identity of the atractyloside‐“insensitive” sites in sonic particles with the specific sites in intact mitochondria is discussed. Treatment with detergents also largely removes atractyloside‐sensitive and atractyloside‐removable binding. The occurrence of two sites with different affinities, it is suggested, reflects inner and outer localizations of carriers. High affinity sites may be those which are in contact with residual endogenous adenine nucleotides and therefore apparently saturated early. The models may be discussed in terms of a carrier distribution in a mobile system or in terms of a double‐faced dimer carrier.

Adenine nucleotide translocation of mitochondria. 1. Specificity and control.

The adenine nucleotide translocation in mitochondria has previously been established as an exchange between exogenous and endogenous adenine nucleotides across the inner membrane. The specificity and the control of the exchange are examined with the following major results: The adenine nucleotide translocation is relatively specific for exogenous ADP and ATP, AMP being nearly inactive. Among other nucleotides tested, only dADP and dATP exchange with a noticeable activity. In the controlled state ADP exchanges 2–4 times faster than ATP. If simultaneously added, ADP and ATP compete for the exchange, with ADP being about tenfold more active than ATP. The specificity of the exit of adenine nucleotides in the exchange is similar to the specificity of the entrance with the difference that ADP and ATP are released with equal activity in proportion to their intramitochondrial content. AMP is released only after a slow conversion to ADP. Therefore the short time exchange is limited by the endogenous content of ADP plus ATP. The exchange is influenced by the metabolic state of the mitochondria. The ATP exchange is more variable than the ADP exchange. Two effects are elucidated: (a) the influence of the metabolic state on the relative content of AMP which inhibits both the ADP and ATP exchange (b) the coupling of the energy transfer system which inhibits only the ATP exchange. An example for case (a) is the inhibition of the ADP and ATP exchange by arsenate and an example for case (b) is the strong increase of the ATP exchange on uncoupling. The following effects are relevant to the mechanism of the control of the exchange by ATP. The stimulation of the ATP exchange by uncoupler has the same concentration dependence as the uncoupling of oxidative phosphorylation (Km [CCP] = 0.08 μM, where CCP = carbonyl‐cyanide‐phenylhydrazone). Oligomycin does not abolish the uncoupler effect on the ATP exchange. “Endogenous uncoupling” on aging of mitochondria also stimulates the ATP exchange. Valinomycin plus K+ only slightly stimulate the ATP exchange. Anaerobiosis stimulates the ATP exchange to a smaller extent than uncoupling.In competition with ADP the effects of energy transfer on ATP exchange are more strongly revealed. On uncoupling the more than tenfold preference for ADP is fully abolished. It is concluded that basically the exchange for ADP and ATP has equal specificity in forward and reverse reaction. In the controlled state a superimposed force makes the specificity asymmetric and inhibits the entrance of ATP. This control of the ATP exchange is concluded to be based on the anionic character of the adenine nucleotides. Thus the ATP4‐ex–ADP3‐in exchange is inhibited unless the charge difference is compensated for by an uncoupler stimulated H+ movement across the membrane. Furthermore an electric potential gradient appears to be effective in the controlled state which is abolished on uncoupling.