Role of ATP in the intramitochondrial sorting of cytochrome c1 and the adenine nucleotide translocator.

Import of precursor proteins across the mitochondrial inner membrane requires ATP in the matrix. However, some precursors can still cross the outer membrane in ATP-depleted mitochondria. Here we show that the adenine nucleotide translocator is imported normally into the inner membrane after the matrix has been depleted of ATP. This result supports the earlier suggestion that the translocator inserts into the inner membrane without passing through the matrix. Depletion of matrix ATP also has no detectable effect on the import and maturation of cytochrome c1, which is targeted to the intermembrane space. It thus seems probable that cytochrome c1 does not completely cross the inner membrane during its import pathway.     

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.     

Relationship of the intra- and extramitochondrial adenine nucleotide ratios during synthesis of phosphoenolpyruvate using extramitochondrial ATP

Mitochondria prepared from the livers of guinea pig, chicken, and pigeon all actively synthesize phosphoenolpyruvate from oxalacetate and GTP, utilizing phosphoenolpyruvate carboxykinase. It was previously shown (Wilson, D. F., Erecińska, M., and Schramm, V. L. (1983). J. Biol. Chem. 258, 10464-10473) that phosphoenolpyruvate carboxykinase is freely reversible and that, in conjunction with nucleoside diphosphate kinase and malate dehydrogenase, which are also present in the mitochondria, it can be used to measure the intramitochondrial [ATPfree]/[ADPfree]. In this study, synthesis of phosphoenolpyruvate by guinea pig liver mitochondria was studied under conditions for which the only source of GTP was extramitochondrial ATP via adenine nucleotide translocase and nucleoside diphosphate kinase (the mitochondria were treated with rotenone, oligomycin, uncoupler, and fluorocitrate). When the extramitochondrial [ATP]/[ADP] was greater than the intramitochondrial [ATPfree]/[ADPfree] calculated from the phosphoenolpyruvate carboxykinase reaction, there was net synthesis of phosphoenolpyruvate, but when it was less, there was net disappearance of phosphoenolpyruvate. Thus, the intramitochondrial [ATPfree]/[ADPfree] was equal to the extramitochondrial value at the point of reversal of the phosphoenolpyruvate carboxykinase reaction. This equality of the intra- and extramitochondrial adenine nucleotide ratios occurred with a measured mitochondrial membrane potential of approximately -36 mV, whereas in the previous experiments, equality was observed for conditions in which the measured membrane potential was -111 to -125 mV. Thus, adenine nucleotide translocation was not dependent on the transmembrane electrical potential and must, therefore, have occurred by electroneutral exchange.     

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/Pi 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.     

Molecular sieve filtration: a method for separate measurement of intra- and extramitochondrial adenine nucleotides

A simple technique of molecular sieve filtration is described for the separation of mitochondria (10–15 mg of protein) from the incubation medium. This method can be used for the determination of metabolites in the pellet and filtrate of the same incubation. Very little damage occurred to the mitochondria during filtration and the metabolic state was preserved. Measurements with ¹⁴C-labeled sucrose showed that only about 0.25 μl/mg protein of extramitochondrial fluid remained with the mitochondrial pellet on the filter. Results for adenine nucleotides, as well as for citrate and malate are presented.     

Physiological level of norepinephrine increases adenine nucleotides hydrolysis in rat blood serum

Extracellular adenosine 5′-triphosphate (ATP) and its breakdown products, adenosine 5′-diphosphate (ADP) and adenosine, have significant effects on a variety of biological processes. NTPDase enzymes, responsible for adenine nucleotides hydrolysis, are considered the major regulators of purinergic signaling in the blood. Previous work by our group demonstrated that ATP and ADP hydrolysis in rat blood serum are higher during the dark (activity) phase compared to the light (rest) phase. In nocturnal animals (e.g., rats), important physiological changes occur during the dark phase, such as increased circulating levels of melatonin, corticosterone, and norepinephrine (NE). This study investigated the physiological effects, in vivo and in vitro, of melatonin, dexamethasone, and NE upon nucleotides hydrolysis in rat blood serum. For in vivo experiments, the animals received a single injection of saline (control), melatonin (0.05 mg/kg), dexamethasone (0.1 mg/kg), or NE (0.03 mg/kg). For in vitro experiments, melatonin (1.0 nM), dexamethasone (1.0 μM), or NE (1.0 nM) was added directly to the reaction medium with blood serum before starting the enzyme assay. The results demonstrated that ATP and ADP hydrolysis in both in vitro and in vivo experiments were significantly higher with NE treatment compared to control (in vitro: ATP = 36.63%, ADP = 22.43%, P < 0.05; in vivo: ATP = 44.1%, ADP = 37.28%, P < 0.001). No significant differences in adenine nucleotides hydrolysis were observed with melatonin and dexamethasone treatments. This study suggests a modulatory role of NE in the nucleotidases pathway, decreasing extracellular ATP and ADP, and suggests that NE might modulate its own release by increasing the activities of soluble nucleotidases.     

The intramitochondrial volume measured using sucrose as an extramitochondrial marker overestimates the true matrix volume determined with mannitol.

The matrix volume of isolated liver and heart mitochondria has been estimated at various osmolarities and in various osmotic supports using 36Cl- and [14C]sucrose, D-mannitol, D-3-methoxyglucose and choline as extramitochondrial markers. The use of 3-methoxyglucose was only possible at 0 degree C since it entered mitochondria at physiological temperatures. All extramitochondrial markers used gave linear plots of apparent matrix volume against the reciprocal of the osmolarity, but the slope of this plot was greater when sucrose was used than with the other extramitochondrial markers. When extrapolated to infinite osmolarity the mean matrix volume was zero when mannitol was used, but about 0.6 microliter/mg of protein for sucrose and Cl- and -0.4 microliter/mg of protein when choline was used. At physiological osmolarity (about 330 m-osmol) the mean matrix volume of de-energized liver mitochondria in KCl medium estimated using mannitol was 0.46 microliter/mg of protein, whereas that obtained using sucrose was 1.68 microliters/mg of protein. Values in mannitol, choline and sucrose media were similar when mannitol but not sucrose was used as extramitochondrial marker. It is argued that the 3H2O/[14C]mannitol space more accurately reflects the true mitochondrial matrix volume than does the 3H2O/[14C]sucrose space. The consequences of this for measurements of the protonmotive force and the intramitochondrial concentration of metabolites are discussed.     

Presence of ectonucleotidases in cultured chromaffin cells: hydrolysis of extracellular adenine nucleotides

The granular ATP released from chromaffin cells during the secretory response can be hydrolyzed by ectonucleotidases that are present in the plasma membrane of these cells. The ecto-ATPase activity showed a Km for ATP of 250 +/- 18 microM and a VMAX value of 167 +/- 25 nmol/10(6) cells x min (1.67 mumol/mg protein x min) for cultured chromaffin cells, while the ecto-ADPase activity showed a Km value for ADP of 375 +/- 40 microM and a VMAX of 125 +/- 20 nmol/10(6) cells x min (1.25 mumol/mg protein x min). The ecto 5'-nucleotidase activity of cultured chromaffin cells was more specific for the purine nucleotides, AMP and IMP, than for the pirimidine nucleotides, CMP and TMP. The Km for AMP was 55 +/- 5 microM and the VMAX value was 4.3 +/- 0.8 nmol/10(6) cells x min (43 nmol/mg protein x min). The nonhydrolyzable analogs of ADP and ATP, alpha, beta-methylene-adenosine 5'-diphosphate and adenylyl-(beta, gamma-methylene)-diphosphonate were good inhibitors of ecto 5'-nucleotidase activity, the KI values being 73.3 +/- 3.5 nM and 193 +/- 29 nM, respectively. The phosphatidylinositol-specific phospholipase C released the ecto-5'-nucleotidase from the chromaffin cells in culture, thus suggesting an anchorage through phosphatidylinositol to plasma membranes. The presence of ectonucleotidases in chromaffin cells may permit the recycling of the extracellular ATP exocytotically released from these neural cells.     

Studies on the availability of intramitochondrial carbamoylphosphate for utilization in extramitochondrial reactions in rat liver

The following observations with isolated mitochondria prepared from rat liver demonstrate that Carbamoylphosphate can readily traverse the mitochondrial membrane: (a) Citrulline synthesis occurs within isolated intact mitochondria at the expense of exogenously added ornithine and [¹⁴C]carbamoylphosphate, providing evidence that the initochondrial membrane does not exclude extramitochondrial car bamoylphosphate from penetrating the intramitochondrial matrix, (b) The [¹⁴C]carbamoylphosphate synthesized within isolated intact mitochondria from NaH¹⁴CO₃ by the action of the N-acetyl-l-glutamate-activated carbamoylphosphate synthetase (CPS-I) is equally available for consumption in intramitochondrial and extramitochondrial reactions, as judged by the coupled activity of CPS-I with either intramitochondrial ornithine carbamoyltransferase or extramitochondrial aspartate carbamoyltransferase. The possibility that the coupled action of CPS-I with intramitochondrial ornithine carbamoyltransferase might prevent the export of carbamoylphosphate into the extramitochondrial medium was also examined. The addition of ornithine to the reaction mixture, at concentrations which are optimal for citrulline production, did not reduce carbamoylphosphate export below$\text{1}\text{3}$ of the total amount of carbamoylphosphate synthesized. These results indicate that the carbamoylphosphate generated intramitochondrially is not compartment ally excluded from participating in cytoplasmic reactions, and raise the possibility that the intramitochondrial carbamoylphosphate synthetase, CPS-I, may be a significant source of the carbamoylphosphate incorporated into hepatic pyrimidines by the cytoplasmic enzymes of the orotate pathway.