Peroxidative damage to cardiac mitochondria: identification and purification of modified adenine nucleotide translocase.

Rat myocardial membranes exposed to free radical-generating systems exhibit both lipid peroxidation and protein alterations. The most sensitive protein, a 28-kDa polypeptide, was previously shown to increase slightly in apparent molecular weight before disappearing completely from the protein profile [N. L. Parinandi, C. W. Zwizinski, and H. H. O. Schmid (1991) Arch. Biochem. Biophys. 289, 118-123]. We now report that isolated cardiac mitochondria contain a 28-kDa protein which responds in the same manner to treatment with Cu2+/t-butylhydroperoxide. The protein exhibits several characteristic properties of the mitochondrial adenine nucleotide translocase. This assignment is supported by the finding that carboxyatractyloside, a specific inhibitor of the adenine nucleotide translocase, can prevent the oxidant-induced changes in the 28-kDa protein. Efficient purification schemes for the isolation of milligram quantities of unmodified and oxidatively altered adenine nucleotide translocase from rat heart mitochondria are described.     

Effects of ethacrynic acid and furosemide on phosphorylation reactions of kidney mitochondria. Inhibition of the adenine nucleotide translocase.

Previous reports that ethacrynic acid and furosemide diminish mitochondrial P : O ratios and reduce (Na+ + K+)-ATPase activity suggested that these diuretics may inhibit mitochondrial phosphorylation reactions. This possibility was initially studied by determining the effects of ethacrynic acid and furosemide on [32P]ATP exchange activity of rat kidney mitochondria. Concentrations of both drugs at 10(-4) M or greater, significantly inhibited [32P]ATP exchange. To investigate the mechanism of this inhibition, the effects of ethacrynic acid and furosemide on the ATPase activity of intract mitochondria and sonicated submitochondrial particles were determined. Both diuretics inhibited ATPase activity of intact mitochondria at 10(-4) M. In contrast, ATPase of submitochondrial particles was significantly less susceptible to inhibition by the diuretics. These results suggested that ethacrynic acid anf furosemide inhibit adenine nucleotide transport across the mitochondrial membrane. This was directly tested by determining the effects of the diretics on the mitochondrial adenine nucleotide translocase. At 5-10(-4) M, both ethacrynic acid and furosemide significantly inhibited adenine nucleotide transport. These findings suggest that ethacrynic acid and furosemide may diminish renal tubular solute reabsorption by direct inhibition of adenine nucleotide transport across the mitochondrial inner membrane.     

Effects of ethacrynic acid and furosemide on phosphory-lation reactions of kidney mitochondria. Inhibition of the adenine nucleotide translocase

Previous reports that ethacrynic acid and furosemide diminish mitochondrial P : O ratios and reduce (Na⁺ + K⁺)-ATPase activity suggested that these diuretics may inhibit mitochondrial phosphorylation reactions. This possibility was initially studied by determining the effects of ethacrynic acid and furosemide on [³²P]ATP exchange activity of rat kidney mitochondria. Concentrations of both drugs at 10⁻⁴ M or greater, significantly inhibited [³²P]ATP exchange. To investigate the mechanism of this inhibition, the effects of ethacrynic acid and furosemide on the ATPase activity of intact mitochondria and sonicated submitochondrial particles were determined. Both diuretics inhibited ATPase activity of intact mitochondria at 10⁻⁴ M. In contrast, ATPase of submitochondrial particles was significantly less susceptible to inhibition by the diuretics. These results suggested that ethacrynic acid and furosemide inhibit adenine nucleotide transport across the mitochondrial membrane. This was directly tested by determining the effects of the diuretics on the mitochondrial adenine nucleotide translocase. At 5 · 10⁻⁴ M, both ethacrynic acid and furosemide significantly inhibited adenine nucleotide transport. These findings suggest that ethacrynic acid and furosemide may diminish renal tubular solute reabsorption by direct inhibition of adenine nucleotide transport across the mitochondrial inner membrane.     

Effect of palmitoyl-CoA binding with adenine nucleotide translocase on energization of mitochondria]

Under conditions of inhibiting oxidative phosphorylation of oligomycin palmitoyl-CoA (p-CoA) decreases the rate of energy dependent reduction of acetoacetate and Ca2+-capacity of mitochondria in a phosphate medium. Energy independent osmotic swelling of mitochondria in NH4NO3, which depends on H+ permeability of the inner mitochondrial membrane is inhibited by ADP and acclereated by p-CoA. Carnitin and competitive ADP abolish all the effects of p-CoA. It is concluded that decreased energization induced by p-CoA is related to an increase in the inner mitochondrial membrane permeability b- H+ as a result of the inhibitor bindings with adenine nucleotide translocase.     

Increase in the adenine nucleotide translocase content of duckling subsarcolemmal mitochondria during cold acclimation

Intermyofibrillar and subsarcolemmal mitochondria were isolated from duckling gastrocnemius muscle. The adenine nucleotide translocase (ANT) content of subsarcolemmal mitochondria was found to be half of that present in intermyofibrillar mitochondria. In addition, cold acclimation resulted in a 1.7-fold increase in subsarcolemmal mitochondrial ANT content, with intermyofibrillar mitochondrial ANT remaining constant. This change in mitochondrial ANT content correlates with the previously reported cold-induced change in the sensitivity of mitochondria to palmitate-inhibited ATP synthesis [Roussel et al. (1998) FEBS Lett. 439, 258-262]. It is suggested that the mitochondrial ANT content enhances or reduces the fatty acid uncoupling activity in tissue, depending on the energetic state of mitochondria.     

Mathematical modeling of mitochondrial adenine nucleotide translocase

We have developed a mathematical model of adenine nucleotide translocase (ANT) function on the basis of the structural and kinetic properties of the transporter. The model takes into account the effect of membrane potential, pH, and magnesium concentration on ATP and ADP exchange velocity. The parameters of the model have been estimated from experimental data. A satisfactory model should take into account the influence of the electric potential difference on both ternary complex formation and translocation processes. To describe the dependence of translocation constants on electric potential we have supposed that ANT molecules carry charged groups. These groups are shifted during the translocation. Using the model we have evaluated the translocator efficiency and predicted the behavior of ANT under physiological conditions.     

Adenine nucleotide pool size,adenine nucleotide translocase activity,and respiratory activity in newborn rabbit liver mitochondria

The adenine nucleotide content (ATP+ADP+AMP) of newborn rabbit liver mitochondria was 6.0±0.5 nmol/mg mitochondrial protein at birth, increased rapidly to 14.5±1.7 nmol/mg protein by 2 h postnatal, peaked at 6 h, then decreased gradually to 7.8±0.6 nmol/mg protein by 4 days postnatal. There was a strong positive correlation (r=0.82) between the total adenine nucleotide pool size and adenine nucleotide translocase activity in these mitochondria. In contrast, glutamate + malate-supported State 3 respiratory rates remained constant from birth through the first week of life. State 4 rates also remained constant, as did the respiratory control index and uncoupled respiratory rates. The following conclusions are suggested: (1) The maximum rate of translocase activity is limited by the intramitochondrial adenine nucleotide pool size. (2) In newborn rabbit liver mitochondria, the State 3 respiratory rate is not limited by either the adenine pool size or the maximum capacity for translocase-mediated adenine exchange. (3) In contrast to rat, rabbit liver mitochondria are fully functional at birth with regard to respiratory rates and oxidative phosphorylation. (4) The rapid postnatal accumulation of adenine nucleotides by liver mitochondria, now documented in two species, may be a general characteristic of normal metabolic adjustment in neonatal mammals.     

An adenine nucleotide translocase in the procaryote Methanobacterium thermoautotrophicum

ATP synthesis, ATP hydrolysis and ADP uptake by membrane vesicles of Methanobacterium thermoautotrophicum are inhibited by inhibitors of mitochondrial $\text{ADP}\text{ATP}$ translocases. Atractyloside binds to one of the membrane proteins. These data demonstrate the presence of an eucaryotic type of $\text{ADP}\text{ATP}$ translocase in a procaryotic microorganism and stress the unique position of methanogenic bacteria in evolution.     

Effects of adenine nucleotide translocase inhibitors on dinitrophenol-induced Ca2+ efflux from pig heart mitochondria

Bongkrekic acid and atractyloside, inhibitors of adenine nucleotide translocase, do not inhibit Ca2+ uptake and H+ production by pig heart mitochondria. However, bongkrekic acid, but not atractyloside, inhibits dinitrophenol-induced Ca2+ efflux and H+ uptake. Conversely, ruthenium red blocks Ca2+ uptake and H+ production but does not prevent dinitrophenol-induced Ca2+ efflux and H+ uptake by mitochondria. These results suggest that mitochondrial Ca2+ uptake and release exist as two independent pathways. The efflux of Ca2+ from mitochondria is mediated by a bongkrekic acid sensitive component which is apparently not identical to the ruthenium red sensitive Ca2+ uptake carrier.     

stress sensitive B encodes an adenine nucleotide translocase in Drosophila melanogaster.

Adenine nucleotide translocases (ANT) are required for the exchange of ADP and ATP across the inner mitochondrial membrane. They are essential for life, and most eukaryotes have at least two different Ant genes. Only one gene had been described from Drosophila, and this had not been characterized genetically. We show that mutations in this gene correspond to the previously described loci, sesB and l(1)9Ed. Immediately adjacent to this gene is another encoding a second ANT protein, which has 78% identity to that encoded by sesB/l(1)9Ed. These two genes are transcribed from a common promoter, and their mRNAs are produced by differential splicing. Hutter and Karch suggested that the sesB ANT gene corresponded to Hmr, a gene identified by an allele that rescues otherwise inviable interspecific hybrids between Drosophila melanogaster and its sibling species. This hypothesis is not supported by our study of the ANT genes of D. melanogaster.     

Relationship of phosphoenolpyruvate transport, acyl coenzyme A inhibition of adenine nucleotide translocase and calcium ion efflux in guinea pig heart mitochondria.

The transport of phosphoenolpyruvate by the adenine nucleotide translocase system of heart mitochondria may be directly involved in the mechanism of phosphoenolpyruvate-induced calcium ion efflux. In contrast to liver mitochondria, the transport of phosphoenolpyruvate via the tricarboxylate carrier system is low or absent in heart mitochondria. The translocation of phosphoenolpyruvate which catalyzed adenine nucleotide and calcium efflux from heart mitochondria was inhibited by palmitoyl-CoA as well as atractylate and ATP. These results suggest that phosphoenolpyruvate, which is preferentially transported on the tricarboxylate carrier of liver mitochondria, is transported primarily via the adenine nucleotide translocase system in heart mitochondria. As a result of its inward transport, phosphoenolpyruvate is able to catalyze calcium ion as well as adenine nucleotide efflux from the mitochondrial matrix. Although not yet proven, either or both phosphoenolpyruvate and long chain acyl-CoA esters may act as natural physiological effectors in the regulation and distribution of intracellular calcium.