Modification by calcium ions of adenine nucleotide translocation in rat liver mitochondria

1. Added Ca(2+) stimulates the translocation of ATP by isolated rat liver mitochondria. 2. The apparent K(m) for added Ca(2+) in stimulating the translocation of 200mum-ATP is approx. 160mum (75mum ;free' Ca(2+)). 3. The greatest stimulation of ATP translocation by Ca(2+) occurs at the lower concentrations of ATP. 4. Sr(2+) (and to a lesser extent Ba(2+)) can replace Ca(2+) whereas Mg(2+) and Mn(2+) have only little ability to stimulate ATP translocation. 5. Translocation of dATP is also stimulated by Ca(2+) whereas that of ADP is stimulated to only a relatively small degree. 6. Studies with metabolic inhibitors and uncouplers provide evidence that stimulation by Ca(2+) and by uncouplers is additive and that the mechanism of Ca(2+) stimulation does not seem to involve the high-energy intermediate of oxidative phosphorylation. 7. In the presence of Ca(2+), ATP is able to effectively compete with ADP for translocation. 8. Added K(+) further enhances the ability of Ca(2+) to stimulate ATP translocation. 9. These findings are discussed in relation to the potential involvement of Ca(2+) in modifying enzymic reactions involved in the regulation of cell metabolism.     

Inhibition of adenine nucleotide translocation in maize seedling mitochondria by anionic detergents

Low concentrations (50–200 μ M ) of the anionic detergents cholate, deoxycholate and dodecylsulphate inhibited the activity of adenine nucleotide translocator in mitochondria from etiolated maize ( Zea mays L. hybrid Krasnodarskij 303) coleoptiles. This resulted in: (a) a decrease in the rates of oxidative phosphorylation and hydrolysis of extramitochondrial ATP; (b) a decrease in the rate of [³³P]-ATP transport through the inner mitochondrial membrane. Anionic detergents may act as competitive inhibitors of ADP and ATP transport in maize mitochondria.     

Inhibition of adenine nucleotide transport in rat liver mitochondria by long-chain acyl-coenzyme A beta-oxidation intermediates

Long-chain acyl-coenzyme A esters (LCAC), which may accumulate under different pathological conditions and especially in patients with a mitochondrial fatty acid beta-oxidation defect, have long been known as potent inhibitors of several enzymes in multiple metabolic pathways, particularly the oxidative phosphorylation system (OXPHOS). To shed more light on the inhibitory mechanisms of acyl-CoA esters upon energy metabolism, the effect of palmitoyl-CoA and its beta-oxidation intermediates on OXPHOS was studied. We have recently shown that, using rat liver mitochondria, LCAC inhibit l-glutamate driven oxygen consumption in the presence of ADP whereas no effect is found when an uncoupler is used to stimulate respiration maximally. A similar inhibitory effect of these compounds is now reported upon the distribution of ATP for intra- and extra-mitochondrial utilization. Taken together these data strongly suggest that the inhibition of ADP-induced respiration with l-glutamate as substrate by LCAC is primarily due to inhibition of the mitochondrial ADP/ATP carrier.     

The effect of butacaine on adenine nucleotide binding and translocation in rat liver mitochondria.

The effect of the local anaesthetic, butacaine, on adenine nucleotide binding and translocation in rat liver mitochondria partially depleted of their adenine nucleotide content was investigated. The range of butacaine concentrations that inhibit adenine nucleotide translocation and the extent of the inhibition are similar to the values obtained for native mitochondria. Butacaine does not alter either the total number of atractyloside-sensitive binding sites of depleted mitochondria, or the affinity of these sites for ADP or ATP under conditions where a partial inhibition of the rate of adenine nucleotide translocation is observed. The data are consistent with an effect of butacaine on the process by which adenine nucleotides are transported across the mitochondrial inner membrane rather than on the binding of adenine nucleotides to sites on the adenine nucleotide carrier. The results are briefly discussed in relation to the use of local anaesthetics in investigations of the mechanism of adenine nucleotide translocation.     

Inhibition of rat liver cholesterol esterase by local anaesthetics.

1. A number of local anaesthetics was shown to inhibit rat liver cholesterol esterase activity towards radioactively labelled cholesterol oleate. The anaesthetics inhibited in the order dibucaine greater than chlorpromazine greater than tetracaine greater than benzocaine greater than procaine greater than lidocaine greater than cocaine. 2. The mode of inhibition was seen to be non-competitive with respect to the substrate and is probably independent of any involvement of Ca2+. 3. The inhibition by tetracaine is partially reversed by sodium deoxycholate. However, all ionic and non-ionic detergents studied, sodium deoxycholate, sodium taurocholate, Triton X-100, and cetyltrimethylammonium bromide are capable of inhibiting the rat liver cholesterol esterase in a concentration dependent manner. Only sodium taurocholate stimulates enzymic activity.     

Inhibition of phosphoenolpyruvate transport via the tricarboxylate and adenine nucleotide carrier systems of rat liver mitochondria

The translocation of phosphoenolpyruvate by the tricarboxylate carrier system in rat liver mitochondria was shown to be inhibited by atractyloside and long chain fatty acyl CoA esters as well as benzene, 1, 2, 3 tricarboxylate. By contrast benzene 1, 2, 3 tricarboxylate did not inhibit atractyloside sensitive adenine nucleotide translocation catalyzed by phosphoenolpyruvate. These results indicate that although phosphoenoppyruvate is preferentially transported by the tricarboxylate carrier system, it may also be transported by the adenine nucleotide translocase. The inhibition of the adenine nucleotide and tricarboxylate carrier systems by atractyloside and long chain acyl CoA esters indicates a close functional interrelation-ship of these transport carriers in the inner mitochondrial membrane. Moreover, the potent inhibition of phosphoenolpyruvate, citrate, and adenine nucleotide transport by long chain acyl CoA's provides further evidence that these esters are natural effectors which participate in the regulation of gluconeogenesis, lipogenesis, and energy-linked respiration.     

The adenine nucleotide translocator in foetal, suckling and adult rat liver mitochondria

The adenine nucleotide content of rat liver mitochondria was shown to increase significantly after birth. On the other hand, it was found that the ligand-binding properties of the adenine nucleotide translocator were essentially the same in foetal, suckling and adult rat liver mitochondria. These results are compatible with the proposal that the accumulation of adenine nucleotides which occurs during mitochondrial biogenesis and maturation is effected by a pathway different from the adenine nucleotide translocator.     

Proton permeation, adenine nucleotide translocation and respiratory control in mitochondria cross-linked by dimethylsuberimidate

Rat liver mitochondria or isolated mitoplasts were treated with the cross-linking agent, dimethylsuberimidate, under conditions (pH 7.5; 0°C) which were not detrimental for the coupling quality of the mitochondria and the effect was evaluated on a kinetic basis. When about 25% of the NH₂-groups reacted, the mitochondria or the mitoplasts acquired complete osmotic stability. Succinate oxidation in state 4 was inhibited by about 30–35%. This effect was also observed when the organelles were amidinated by methylacetimidate, a monofunctional imidate which caused no osmotic stabilization. Uncouplers stimulated succinate oxidation in cross-linked mitochondria to the same extent as in the control, in contrast stimulation by ADP was suppressed. Accordingly, the rate of decay of the respiration-dependent cross-membrane proton gradient was only decreased by 25%, whereas the ATPase and adenine nucleotide translocase were strongly inhibited. In the cross-linked mitochondria, the extent of inhibition of the ATPase and of the translocase was found to be the same whether the assays were performed at 30°C (like the respiratory assay) or at 0°C. The effect of methylacetimidate treatment on these activities at the two temperatures was different. At 30°C, the ATPase was not inhibited and the extent of inhibition of ATP translocation was small. At 0°C, the two activities were nearly as much inhibited as in cross-linked mitochondria. Our results suggest that a considerable rigidity can be introduced in the coupling membrane by cross-linking, without a major loss in the initial step of energy conservation. However, the energy conserved in the proton gradient cannot be utilized for ATP synthesis, probably because of the restricted mobility of adenine nucleotide translocase in the cross-linked mitochondria.     

The effect of calcium on the translocation of adenine nucleotides in rat liver mitochondria.

The effect of Ca²⁺ on the adenine nucleotide translocase activity of intact rat liver mitochondria has been studied. The results indicate that in mitochondria which have been allowed to accumulate Ca²⁺, the activity of the translocase is strongly diminished; half-maximal inhibition is attained when approximately 40 nmol of Ca²⁺ are accumulated/mg of mitochondrial protein. Inhibition of electron transport or uncoupling prevents the Ca²⁺-induced inhibition of translocase activity; inhibition of Ca²⁺ uptake by ruthenium red also prevents the inhibition of the exchange. These experiments indicate that internal, but not external Ca²⁺ is responsible for the inhibition of adenine nucleotide translocase activity. Inhibition of the exchange activity by Ca²⁺ occurs even in conditions in which external adenine nucleotide concentrations are rate-limiting.     

Possible role of adenine nucleotide transport in regulating the respiration of rat liver mitochondria]

Studies with liver mitochondria from rats which starved for 48 hours showed the rate of ADP-stimulated respiration to be 20% lower than in the presence of an uncoupler. This effect was eliminated by preincubation of mitochondria with carnitine. Mitochondria from fed rats were characterized by a considerable decrease of states 3 and 4 respiration. In this case carnitine produced no effect. Preincubation of mitochondria from the liver of fed rats with alpha-ketoglutarate resulted in a substantial increase of the states 3 and 4 respiratory rates. There proved to exist at least two types of regulation of adenine nucleotide transport through the inner mitochondrial membrane depending on the metabolic state of the organism, i.e. by inhibition of adenine-nucleotide translocase by cytoplasmic acyl-CoAs and by control of intramitochondrial adenine nucleotide pool.     

Effect of Escherichia coli endotoxin on adenine nucleotide translocation in canine heart mitochondria

The in vitro effect of Escherichia coli endotoxin on the translocation of adenine nucleotides in dog heart mitochondria was studied. Mitochondrial adenine nucleotides were labeled with ¹⁴C by incubating mitochondrial preparations in the presence of [¹⁴C]ADP. The exchange reaction was initiated by addition of unlabeled ADP, proceeded for 5 to 60 s at 4 °C, and was terminated by addition of atractyloside. The results showed that preincubation of mitochondria with endotoxin (50 μg/mg protein) for 10 min at 23 °C decreased the exchange reaction by 21.2% (P < 0.05). The inhibitory effect of endotoxin was increased with increasing concentrations of endotoxin with an I₅₀ value of 45 μg/mg protein. The initial rate and the total extent of exchange were both affected. Double reciprocal plots showed that only the V but not the K_m for ADP was affected by endotoxin, indicating that the inhibition was noncompetitive in nature. The exchange of adenine nucleotide remained depressed by endotoxin in the presence of either oligomycin or antimycin A, indicating that the inhibitory effect of endotoxin was independent of the action of endotoxin on oxidative phosphorylation. The leakage of labeled adenine nucleotides from mitochondria at 23 °C was increased by 100% by endotoxin (100 μg/mg protein) in the absence of added unlabeled ADP, and this increase in the leakage could not be blocked by atractyloside. The endotoxin-induced changes in adenine nucleotide exchange and leakage were either partially or completely prevented by hydrocortisone, heparin, dibucaine, or EDTA. Since most of these agents have in common an effect on lipid metabolism, it is suggested that endotoxin-induced alterations in the exchange and leakage of adenine nucleotides in heart mitochondria are protected through a mechanism involving membrane lipid reorganization.     

Respiration-driven proton translocation in rat liver mitochondria

1. Pulses of acidity of the outer aqueous phase of rat liver mitochondrial suspensions induced by pulses of respiration are due to the translocation of H⁺ (or OH⁻) ions across the osmotic barrier (M phase) of the cristae membrane and cannot be attributed to the formation (with acid production) of a chemical intermediate that subsequently decomposes. 2. The effective quantity of protons translocated per bivalent reducing equivalent passing through the succinate-oxidizing and β-hydroxybutyrate-oxidizing spans of the respiratory chain are very close to 4 and 6 respectively. These quotients are constant between pH5·5 and 8·5 and are independent of changes in the ionic composition of the mitochondrial suspension medium provided that the conditions permit the accurate experimental measurement of the proton translocation. 3. Apparent changes in the →H⁺/O quotients may be induced by conditions preventing the occurrence of the usual backlash; these apparent changes of →H⁺/O are attributable to a very fast electrically driven component of the decay of the acid pulses that is not included in the experimental extrapolations. 4. Apparent changes in the →H⁺/O quotients may also be induced by the presence of anions, such as succinate, malonate and phosphate, or by cations such as Na⁺. These apparent changes of →H⁺/O are due to an increase in the rate of the pH-driven decay of the acid pulses. 5. The uncoupling agents, 2,4-dinitrophenol, carbonyl cyanide p-trifluoromethoxyphenylhydrazone and gramicidin increase the effective proton conductance of the M phase and thus increase the rate of decay of the respiration-driven acid pulses, but do not change the initial →H⁺/O quotients. The increase in effective proton conductance of the M phase caused by these uncouplers accounts quantitatively for their uncoupling action; and the fact that the initial →H⁺/O quotients are unchanged shows that uncoupler-sensitive chemical intermediates do not exist between the respiratory-chain system and the effective proton-translocating mechanism. 6. Stoicheiometric acid–base changes associated with the activity of the regions of the respiratory chain on the oxygen side of the rotenone- and antimycin A-sensitive sites gives experimental support for a suggested configuration of loop 3.     

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 mM 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 mM and 0.41 ± 0.20 mM, 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 MgCl₂, suggesting that Mg²⁺ 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.     

Adenine nucleotide translocation in liver mitochondria of hypothyroid rats.

In measurements using a disc filtration method, liver mitochondria obtained from hypothyroid rats translocate external ADP at 0 °C via the atractyloside-sensitive carrier much more slowly than do mitochondria from normal rats, confirming the findings of Portnay et al. (Biochem. Biophys. Res. Commun. 55, 17, 1973). The hypothyroid mitochondria contain 60% more ATP + ADP than do mitochondria from normals, but the excess nucleotides are not exchangeable and so do not contribute to translocation. A decrease in the first-order rate constant accounts for the decreased velocity. Neither a decrease in the number of translocator sites nor changes in ADP phosphorylation or ATPase activity seem to account for the abnormal kinetics of translocation. Although the filtration method limits the maximal translocation rate observed in normal mitochondria at temperatures above 17 °C that induce a fluid membrane state, no such transition is seen in mitochondria from hypothyroid rats up to 35 °C, indicating that the translocator is in an altered environment in hypothyroidism. Injecting a hypothyroid rat once with l-thyroxine corrects the abnormal compartmentation and produces a temperature-rate relationship like that in normal mitochondria in 3 days, a period which would accommodate the hormone actions reported on translation, membrane phospholipid synthesis, or fatty acid desaturation.