N-methyl oxidation in liver mitochondria of triiodothyronine-treated and thyroidectomized rats

The levels of sarcosine dehydrogenase and acid-nonextractable flavin in the inner matrix of mitochondria of rat liver are decreased in animals treated with triiodothyronine and are elevated in the mitochondria obtained from thyroidectomized animals. Administration of triiodothyronine does not affect the electron-transfer flavoprotein associated with the sarcosine dehydrogenase or the relative amounts of soluble and membrane-bound proteins of the mitochondria. In phosphate-washed mitochondria from either the controls or the triiodothyronine-treated rats, the O₂ uptake equals the total of the [¹⁴C]formaldehyde and [β-¹⁴C]serine isolated as reaction products of the sarcosine-[¹⁴C]methyl group. In contrast to its restraint of sarcosine or choline oxidation in preparations capable of oxidative phosphorylation, ADP does not inhibit the oxidation of these substrates in mitochondria of rats given triiodothyronine.     

The catabolism of endogenous adenine nucleotides in rat liver mitochondria

Summary The degradation of intramitochondrial adenine nucleotides to nucleosides and bases was investigated by incubating isolated rat liver mitochondria at 37°C under non-phosphorylating conditions in the presence of oligomycin and carboxyatractyloside. Within 30 min the adenine nucleotides were degraded by about 25 per cent. The main products formed were adenosine and inosine the contents of which increased five- to sevenfold.Compartmentation studies revealed that about 50 to 60 per cent of the adenosine formed remained inside the organelles whereas inosine was almost completely released into the surrounding medium. Outside the mitochondria only very small amounts of adenine nucleotides were detected. Similar incubations in the presence of [¹⁴C]-adenosine yielded no [¹⁴C]-inosine ruling out extramitochondrial adenosine deamination.It is concluded that endogenous adenine nucleotides can be degraded in mitochondria via AMP dephosphorylation and subsequent adenosine deamination. A purine nucleoside transport system mediating at least the efflux of inosine from the mitochondria is suggested.     

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.     

Carboxyatractyloside-insensitive influx and efflux of adenine nucleotides in rat liver mitochondria

Unidirectional transport (influx and efflux) of adenine nucleotides in rat liver mitochondria was examined using carboxyatractyloside to inhibit rapid exchange of matrix and external adenine nucleotides via the adenine nucleotide translocase. Influx of adenine nucleotides was concentration-dependent. ATP was the preferred substrate with a Km of 2.67 mM and V of the preferred substrate with a Km of 2.67 mM and V of 8.33 nmol/min/mg of protein. For ADP, the Km was 14.7 mM and V was 10.8 nmol/min/mg of protein. Efflux of adenine nucleotides was also concentration-dependent, varying directly as a function of the matrix adenine nucleotide pool size. Any increase in the influx of adenine nucleotides was coupled to an increase in efflux. However, as the external ATP concentration was increased, influx was stimulated to a much greater extent than was efflux. This imbalance suggested that under certain conditions adenine nucleotide movement might be coupled to the movement of an alternate anion such as phosphate. Adenine nucleotide efflux increased as the external phosphate concentration was varied from 0.5 to 4 mM. Also, increasing the external phosphate concentration caused adenine nucleotide influx to decrease, suggesting competition. In the absence of external adenines and phosphate, no efflux occurred. Both adenine nucleotide influx and efflux were depressed if Mg2+ was omitted. Adenine nucleotide efflux in the presence of external phosphate was inhibited much less by lack of Mg2+ than was efflux in the presence of external ATP. This evidence supports a model in which either adenine nucleotides (probably with Mg2+) or phosphate can move across the mitochondrial membrane on a single carrier. Net adenine nucleotide movements can occur when adenine nucleotide movement is coupled to the movement of phosphate in the opposite direction.     

The effect of acetate oxidation on the endogenous adenine nucleotides of rat heart mitochondria

When citrate cycle substrates or glutamate are oxidized by rat heart mitochondria, AMP comprises only a few percent of the endogenous pool of adenine nucleotides. However, when acetate is oxidized, greater than half or about 30% of the total mitochondrial pool of adenine nucleotides is converted to AMP in the ADP-stimulated or resting state, respectively. Supporting substrates which form GTP as a result of their oxidation partially, but not completely, reverse the accumulation of AMP which results from acetate metabolism.     

Increased adenine nucleotides in liver mitochondria after mannoheptulose injection in vivo

In adult rats, mannoheptulose injection causes a transient decrease in the serum insulin-to-glucagon ratio and a concomitant increase in serum glucose concentration. These effects attain a maximum 1 h after the injection and then decline toward normal. Correlated with the hormone changes is a dramatic increase in the adenine nucleotide content (ATP + ADP + AMP) of liver mitochondria, which peaks to over 50% of control values at 1 h. The increase in mitochondrial adenine nucleotides must occur by uptake from the cytosol, because the adenine nucleotide content of the whole tissue remains constant. The accumulation of adenine nucleotides by the mitochondria probably occurs over the recently characterized carboxyatractyloside-insensitive transport pathway that allows exchange of ATP-Mg for Pi. The actual mechanism by which net uptake is regulated after mannoheptulose injection has not yet been elucidated; however, changes in the Km or Vmax of the carrier and an increase in the tissue ATP/ADP ratio were eliminated as possibilities. The increase in matrix adenine nucleotide content in response to hormone changes brought about by mannoheptulose was much greater and more reproducible than what is achieved with glucagon injection. Mannoheptulose treatment may therefore be preferable as a model for further study of hormone effects on mitochondrial function.     

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.     

Decreased exchange of adenine nucleotides in human placental mitochondria

The purpose of this work was to study the exchange of adenine nucleotides in mitochondria isolated from human placenta tissue. The results indicate that ADP and ATP are translocated at a lower rate than those reported for rat liver mitochondria. It is proposed that the limited transport is due to the particular lipid composition of placental mitochondria membrane, which induces an arrest in membrane fluidity with the consequent restriction in adenine nucleotide translocase mobility.     

Energy transduction and adenine nucleotides in mitochondria from rat liver after hypoxic perfusion.

The characteristics of mitochondria isolated from perfused livers of rats under hypoxic or oxic conditions were studied. The electron transfer activity was about 60% of normal after hypoxic perfusion for 3 h, but respiratory control was abolished almost completely. These parameters recovered considerably on subsequent oxic perfusion. The adenine nucleotide contents and their net uptake decreased in hypoxia, closely correlated with the energy transduction. Energy-dependent nicotinamide nucleotide transhydrogenase activity and NAD reduction by succinate in submitochondrial particles were most severely inhibited after hypoxic perfusion and were also correlated with adenine nucleotide contents in the particles. These results are discussed in terms of the involvement of adenine nucleotides in energy-transducing systems in mitochondrial membranes.     

Changes in intramitochondrial adenine nucleotides in blowfly flight-muscle mitochondria

1. With freshly isolated blowfly mitochondria 38% of the intramitochondrial adenine nucleotide was present as AMP. 2. On incubation with oxidizable substrates the AMP and ADP concentrations fell and that of ATP rose; with pyruvate together with proline the ATP concentration reached its maximum value at 6min; with glycerol phosphate the phosphorylation of endogenous nucleotide was more rapid. 3. Addition of the uncoupling agent carbonyl cyanide phenylhydrazone caused a rapid fall of ATP and a parallel rise in ADP, then ADP was converted into AMP. 4. This was in contrast with rat liver mitochondria endogenous AMP concentrations, which were always lower than those of blowfly mitochondria and changed little under different metabolic conditions. 5. Evidence is presented that adenylate kinase (EC 2.7.4.3) has a dual distribution in blowfly mitochondria, a part being located in the matrix space and a part in the space between the outer and inner mitochondrial membranes, as in liver and other mitochondria. 6. The possible regulatory role of changing AMP concentrations in the mitochondrial matrix was investigated. Partially purified pyruvate carboxylase (EC 6.4.1.1) and citrate synthase (EC 4.1.3.7) were inhibited 30% by 2mm-AMP, whereas pyruvate dehydrogenase (EC 1.2.4.1) was unaffected. 7. AMP activated the NAD(+)-linked isocitrate dehydrogenase (EC 1.1.1.41) activity of blowfly mitochondria in the absence of ADP, but in the presence of ADP, AMP caused inhibition. 8. It is suggested that AMP may exert a controlling effect on the oxidative activity of blowfly mitochondria.     

Modulation of branched-chain 2-oxoacid dehydrogenase activity by adenine nucleotides in isolated rat liver mitochondria

Feeding a 17.5% amino acid diet to rats results in inactivation of the hepatic branched-chain 2-oxoacid dehydrogenase complex. Reactivation occurs when preincubating mitochondria in the presence of 0.3 mM ATP, ADP, and AMP. The effect of AMP is assumed to be due to de novo formation of ADP. NaF (25 mM) blocks reactivation suggesting the involvement of a protein phosphatase in the activation process. At high nucleotide concentrations (3 mM) the enzyme is inactive. In the presence of Mg2+ ions nucleotide induced activation is further increased. Mg2+ ions themselves influence the equilibrium state of the enzyme complex. Low concentrations (1 mM) favor inactivation while high concentrations (10 mM) stimulate activation of the enzyme suggesting that Mg2+ ions may act by regulating the associated kinase and phosphatase.