In vitro alteration of the size of the liver mitochondrial adenine nucleotide pool: Correlation with respiratory functions

Mitochondrial respiration was studied as a function of the total adenine nucleotide content of rat liver mitochondria. The adenine nucleotide content was varied by treating isolated mitochondria with pyrophosphate or by incubating pyrophosphate-treated mitochondria with ATP. Mitochondria with at least 4 nmol adenine nucleotides/mg protein maintained at least 80% of the State 3 activity of control mitochondria, which had approximately 10 nmol/mg protein. However, State 3 decreased rapidly once the adenine nucleotide content fell below 4 nmol/mg protein. Between 2 and 4 nmol adenine nucleotides/mg, State 3 was not limited by the maximal capacity of electron flow as measured by the uncoupled respiration. However, at very low adenine nucleotide levels (<2 nmol/mg), the uncoupled rates of respiration were markedly depressed. State 4 was not affected by changes in the mitochondrial adenine nucleotide content. Adenine translocase activity varied in almost direct correlation with changes in the adenine nucleotide content. Therefore, adenine translocase activity was more sensitive than State 3 to changes in total adenine nucleotides over the range of 4 to 10 nmol/mg protein. The results suggest that (i) State 3 is dependent on the level of intramitochondrial adenine nucleotides, particularly in the range below 4 nmol/mg protein, (ii) adenine translocase activity is not rate-limiting for oxidative phosphorylation in mitochondria with the normal complement of adenine nucleotides, however, at low adenine nucleotide levels, depressed State 3 rates may be explained in part by the low rate of ADP translocation, and (iii) a mechanism of net ATP uptake exists in mitochondria with low internal adenine nucleotides.     

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 mm ATP at 30 ° C in 225 mm sucrose, 2 mm K₂HPO₄, 5 mm MgCl₂, and 10 mm 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 mm, respectively. Adenine nucleotide uptake was stoichiometrically dependent on Mg²⁺ 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 $\text{ATP}\text{ADP}$ 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.     

Developmental changes in the adenine nucleotide translocase in the guinea pig

Investigations of developmental changes in energy metabolism in guinea pig liver mitochondria showed that mitochondria from the newborn were well coupled, with respiratory control ratios and membrane energy potentials similar to those obtained with mitochondria from the 1-day-old and the adult. In contrast, there was a 3-fold increase in the rate of mitochondrial respiration and a 2-fold increase in adenine nucleotide content during the first 24 h of extrauterine life. There was no significant change in the ATP/ADP ratio and only a 30% increase in the uncoupled rate of respiration during this same time period. Titrations of the adenine nucleotide translocase with the specific inhibitor, carboxyatractyloside, showed that the newborn had only 50% of the adenine nucleotide translocase activity of the adult. Furthermore, by applying flux control theory to these inhibitor titrations, it was possible to demonstrate that the adenine nucleotide translocase exerted greater control over respiration in the newborn than in the adult, and at maximal rates of coupled respiration the translocase had a control strength of 0.98. The consequences of this finding on cellular energy metabolism are discussed in relation to adaptation of the newborn to extrauterine life.     

Modification of liver mitochondrial lipids and of adenine nucleotide translocase and oxidative phosphorylation by cold adaptation

The decrease in respiration rate following thyroidectomy is preceded by changes in the lipid composition of the mitochondrial membrane (Hoch, F.L., Subramanian, C., Dhopeshwarkar, G.A. and Mead, J.F. (1981) Lipids 16, 328–334) and in concert, changes in the kinetic parameters of the adenine nucleotide translocase (Mak, I.T., Shrago, E. and Elson, C.E. (1981) Fed. Proc. 40, 398). To demonstrate that physiological adaptation also involves this sequence of events, rats were housed at 8°C for 3–4 weeks. Cold adaptation resulted in a modest (5%) increase in the unsaturation index for the mitochondrial fatty acids comprised of a significant increase in arachidonic acid and a reciprocal decrease in linoleic acid. Phospholipid analysis indicated that cold adaptation increased the mitochondrial phosphatidylethanolamine and reciprocally decreased the phosphatidylcholine content. Concomitantly, cold adaptation resulted in 25–30% increases in rat liver mitochondrial respiratory activities without changing the respiratory control or ADP/O ratios. The kinetic parameters of the adenine nucleotide translocase were determined by the back-exchange method (Pfaff, E. and Klingenberg, M. (1968) Eur. J. Biochem. 6, 66–79). At 0–4 and 10°C, the V_max and K_m of the cold-adapted rat liver adenine nucleotide translocase were not distinguishable from the control values. The K_i values determined by Dixon plot studies for atractylate and palmitoyl-CoA were also comparable between the two groups. However, at 25 and 37°C, cold-adapted rat liver adenine nucleotide translocase exhibited a 20% increase in V_max and a 20% decrease in K_m for external ADP. The results suggest that one adaption to a cold environment involves hormone-mediated changes in the lipid composition in the mitochondrial membranes which in turn modulate the adenine nucleotide translocase and subsequent respiratory activities.     

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.     

Pleiotropic effects of the yeast Sal1 and Aac2 carriers on mitochondrial function via an activity distinct from adenine nucleotide transport

In Saccharomyces cerevisiae, SAL1 encodes a Ca2+ -binding mitochondrial carrier. Disruption of SAL1 is synthetically lethal with the loss of a specific function associated with the Aac2 isoform of the ATP/ADP translocase. This novel activity of Aac2 is defined as the V function (for Viability of aac2 sal1 double mutant), which is independent of the ATP/ADP exchange activity required for respiratory growth (the R function). We found that co-inactivation of SAL1 and AAC2 leads to defects in mitochondrial translation and mitochondrial DNA (mtDNA) maintenance. Additionally, sal1Delta exacerbates the respiratory deficiency and mtDNA instability of ggc1Delta, shy1Delta and mtg1Delta mutants, which are known to reduce mitochondrial protein synthesis or protein complex assembly. The V function is complemented by the human Short Ca2+ -binding Mitochondrial Carrier (SCaMC) protein, SCaMC-2, a putative ATP-Mg/Pi exchangers on the inner membrane. However, mitochondria lacking both Sal1p and Aac2p are not depleted of adenine nucleotides. The Aac2R252I and Aac2R253I variants mutated at the R252-254 triplet critical for nucleotide transport retain the V function. Likewise, Sal1p remains functionally active when the R479I and R481I mutations were introduced into the structurally equivalent R479-T480-R481 motif. Finally, we found that the naturally occurring V-R+ Aac1 isoform of adenine nucleotide translocase partially gains the V function at the expense of the R function by introducing the mutations P89L and A96 V. Thus, our data support the view that the V function is independent of adenine nucleotide transport associated with Sal1p and Aac2p and this evolutionarily conserved activity affects multiple processes in mitochondria.     

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.     

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.     

Adenine nucleotide efflux in mitochondria induced by inorganic pyrophosphate

The efflux of mitochondrial adenine nucleotide which is induced by addition of PP_i to suspensions of rat liver mitochondria has been investigated. This efflux of adenine nucleotide is greatly stimulated by the uncoupler FCCP at 1 μM, V_max being 6.7 nmol/min per mg protein as compared to 2.0 nmol/min per mg protein in its absence. The depletion process is inhibited by carboxyatractyloside. The K_m for PP_i of 1.25 mM is essentially unchanged when uncoupler is added. Quantitation of the individual adenine nucleotide species (ATP, ADP and AMP) and their relationship to the rate of efflux suggests that ADP is the predominant species being exchanged for PP_i.     

Citrulline synthesis: Regulation by alterations in the total mitochondrial adenine nucleotide content

The total adenine nucleotide content of rat liver mitochondria was varied in vitro over a wide range in order to investigate a possible relationship between net changes in the total matrix ATP + ADP + AMP content and the overall rate of citrulline synthesis. Isolated mitochondria were specifically depleted of matrix adenine nucleotides by incubating with inorganic pyrophosphate (G. K. Asimakis and J. R. Aprille, 1980, Arch. Biochem. Biophys.203, 307–316); alternatively, matrix adenine nucleotides were increased by incubating mitochondria with 1 mm ATP at 30 °C. No exogenous ATP or ADP was included in the subsequent incubations for the determination of citrulline synthesis. Rates varied from 0.1 to 1.6 μmol citrulline/mg protein/h as a linear function of total adenine nucleotide content in the range 2–15 nmol (ATP + ADP + AMP)/mg protein. Further increases in the matrix ATP + ADP + AMP content caused no further increase in citrulline synthesis rates. Changes in the total adenine nucleotide content were reflected in proportional changes in both the ATP and ADP content of the matrix. The $\text{ATP}\text{ADP}$ ratio did not change significantly. Therefore, the variations in citrulline synthesis were most simply explained as the effect of different concentrations of ATP on the activity of carbamoyl-phosphate synthetase. It was concluded that net changes in the total adenine nucleotide content can contribute to the control of citrulline synthesis. These findings are significant in the context of recent evidence which shows that the matrix adenine nucleotide pool size is under hormonal control.     

Cyclic AMP-induced Mg2+ release from rat liver hepatocytes, permeabilized hepatocytes, and isolated mitochondria.

The addition of norepinephrine, epinephrine, or forskolin to collagenase-dispersed rat liver hepatocytes increase cAMP and result in a 15% loss in total cell Mg2+ within 5 min. Conversely, carbachol and vasopressin induce a 10-15% increase of total cell Mg2+. Permeabilized hepatocytes also mobilize a large pool of Mg2+ when stimulated by ADP or cAMP. This stimulation is completely inhibited by atractyloside and bongkrekic acid, two different specific inhibitors of the mitochondrial adenine nucleotide translocase. cAMP directly mobilizes Mg2+ efflux from isolated rat liver mitochondria. 50 nM cAMP or 250 microM ADP induces in 5 min a mitochondrial loss of about 6 nmol of Mg2+/mg of protein and a stimulation of ATP efflux. The effect of cAMP is specific, is not reproduced by other cyclic or noncyclic nucleotides, and is inhibited by inhibitors of the adenine nucleotide translocase. These data indicate that cAMP is a messenger for a major mobilization of Mg2+ in hepatocytes. A major target for the effect of cAMP are mitochondria, which lose up to 20-25% of their total Mg2+ in 5 min, both within the cell and after isolation. Evidence is presented suggesting that the adenine nucleotide translocase is the target of the cAMP-dependent Mg2+ efflux and that cAMP may change the operation of the translocase. This, in turn, could change within the matrix the substrate of choice of the translocase from ATP to ATP.Mg.     

Inhibition of oxidative phosphorylation by a Ca2+-induced diminution of the adenine nucleotide translocator

The mechanism through which internal Ca²⁺ inhibits oxidative phosphorylation of rat heart mitochondria has been explored. In parallel to a Ca²⁺-induced diminution of the activity of the adenine nucleotide translocator, an efflux of internal adenine nucleotides is observed. The efflux of adenine nucleotides depends on the amount of Ca²⁺ accumulated by the mitochondria and on the time that Ca²⁺ remains in the mitochondria; this efflux is atractyloside insensitive. These results suggest that internal Ca²⁺, by inducing a lowering of the internal concentration of adenine nucleotides, diminishes the rate of exchange of adenine nucleotides via the translocase, and in consequence of oxidative phosphorylation. Under conditions in which the Ca²⁺-induced release of adenine nucleotides takes place, no gross changes of the permeability properties of the membrane are observed. As revealed by studies with arsenate, respiratory activity and the function of the ATPase in the direction of ATP synthesis are not affected by internal Ca²⁺.     

Control of mitochondrial respiration. The contribution of the adenine nucleotide translocator depends on the ATP- and ADP-consuming enzymes

The consequence of the complexity of the metabolic network on the amount of control strength of adenine nucleotide translocator was investigated with isolated rat liver mitochondria. Two experimental systems were compared: (i) mitochondria in the presence of yeast hexokinase (hexokinase system) and (ii) the same system plus additional pyruvate kinase (pyruvate kinase system). In both systems the control strength was analysed for the adenine nucleotide translocator by inhibitor titration studies with carboxyatractyloside and for the hexokinase or pyruvate kinase by changing their relative activities. Experimental results were compared with computer simulation of these systems and that of a third one, where the extramitochondrial ATP / ADP ratio was held constant by perifusion (perifusion system). The results demonstrate quite different flux-dependent control strength of the translocator in the three systems. In the hexokinase system the control strength of the translocator on mitochondrial respiration was zero up to respiration rates of about 60 nmol O₂/mg protein per min. For higher rates, the control strength increased until the maximum value (0.45) was reached in the fully active state. Here, the same value was also found in the pyruvate kinase system. In all other states of respiration the translocator exerts a higher control strength in the pyruvate kinase system than in the hexokinase system. This different behaviour was attributed to the various changes in the adenine nucleotide pattern caused by partial inhibition of the translocator in the hexokinase and pyruvate kinase system. The data clearly show that the sharing of control strength depends not only on the respiration rate but also on the complexity of the metabolic system.     

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.     

Changes in cardiac nucleotide metabolism in Huntington's disease

ABSTRACT

Huntington's disease (HD) is a monogenic neurodegenerative disorder with a significant peripheral component to the disease pathology. This includes an HD-related cardiomyopathy, with an unknown pathological mechanism. In this study, we aimed to define changes in the metabolism of cardiac nucleotides using the well-established R6/2 mouse model. In particular, we focused on measuring the activity of enzymes that control ATP and other adenine nucleotides in the cardiac pool, including eNTPD, AMPD, e5′NT, ADA, and PNP. We employed HPLC to assay the activities of these enzymes by measuring the concentrations of adenine nucleotide catabolites in the hearts of symptomatic R6/2 mice. We found a reduced activity of AMPD (12.9 ± 1.9 nmol/min/mg protein in control; 7.5 ± 0.5 nmol/min/mg protein in R6/2) and e5′NT (11.9 ± 1.7 nmol/min/mg protein in control; 6.7 ± 0.7 nmol/min/mg protein in R6/2). Moreover, we detected an increased activity of ADA (1.3 ± 0.2 nmol/min/mg protein in control; 5.2 ± 0.5 nmol/min/mg protein in R6/2), while no changes in eNTPD and PNP activities were observed. Analysis of cardiac adenine nucleotide catabolite levels revealed an increased inosine level (0.7 ± 0.01 nmol/mg dry tissue in control; 2.7 ±0.8 nmol/mg dry tissue in R6/2) and a reduced concentration of cardiac adenosine (0.9 ± 0.2 nmol/mg dry tissue in control; 0.2 ± 0.08 nmol/mg dry tissue in R6/2). This study highlights a decreased rate of degradation of cardiac nucleotides in HD mouse model hearts, and an increased capacity for adenosine deamination, that may alter adenosine signaling.     

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.     

Adenine nucleotide transport in hepatoma mitochondria. Characterization of factors influencing the kinetics of ADP and ATP uptake

Initial velocity measurements of [³H]ADP and [³H]ATP uptake have been made with mitochondria isolated from Morris hepatomas of differing growth rates, and factors known to influence the rates of nucleotide exchange have been examined in an effort to determine whether the elevated rates of aerobic glycolysis in these tumors can be attributed to altered carrier activity. These studies included the determination of the apparent kinetic constants for nucleotide uptake as a function of the mitochondrial energy state and the dependence of transport rates on temperature. Also included in these studies were measurements of the mitochondrial levels of endogenous inhibitors, divalent cations and internal adenine nucleotides. Results obtained showed that with mitochondria isolated from the various tumor lines, the apparent kinetic constants for nucleotide uptake are different from those of control rat or regenerating liver mitochondria; the apparent V_max values for both ADP and ATP uptake are significantly lower. Furthermore, under conditions of a high-energy state, the K_m and V_max values for ATP uptake are greater than the K_m and V_max value for ADP uptake but that under uncoupled conditions, the opposite is observed. Comparison of the levels of mitochondrial Ca²⁺, Mg²⁺, long-chain acyl-CoA ester and adenine nucleotide from the various mitochondria showed that important differences exist between liver and hepatoma mitochondria in the levels of Ca²⁺, long-chain acyl-CoA ester and AMP. Mitochondrial Ca²⁺ levels are elevated 3–5-fold in all tumor lines, and for Morris 7777 hepatoma (a rapidly growing tumor) by a remarkable 70-fold; whereas the levels of acyl-CoA ester and AMP are significantly lower in the more rapidly growing tumors. Arrhenius plots for nucleotide uptake in mitochondria from liver and hepatoma are characterized as being biphasic, having similar activation energies above and below the break point temperature (28–38 and 6–16 kcal/mol, respectively). However, the transition temperature for mitochondria from the various hepatomas is uniformly 4–5°C lower than mitochondria from control liver. The latter difference may reflect a variation in membrane composition, most probably lipid components. It is concluded that the presence of elevated levels of Ca²⁺ and lower levels of AMP in hepatoma mitochondria and difference of membrane compositions may play an important role in limiting adenine nucleotide transport activity in vivo and that the impaired carrier activity may contribute to higher rates of aerobic glycolysis observed in these tumors.     

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.     

Investigation of the dependence of the intramitochondrial [ATP]/[ADP] ratio on the respiration rate

The relationship between the respiration rate and the intra- and extramito-chondrial adenine nucleotides was investigated in isolated rat liver mitochondria.

For the determination of adenine nucleotide patterns in both compartments a new procedure was developed, based on the evaluation of these metabolites from incubation of various amounts of mitochondria under identical stationary states of oxidative phosphorylation. These identical states were adjusted by addition of appropriate amounts of hexokinase to a glucose-containing incubation mixture.

Adenine nucleotides were measured in aliquots of the total extract of the incubation mixture without any separation. The concentrations of the adenine nucleotides in both compartments were obtained from a plot of the total concentration of these species versus mitochondrial protein. Disturbances of this method by unspecific efflux of adenine nucleotides could be excluded.

The results obtained for the total adenine nucleotide content (12 nmol · mg⁻¹ protein) and the intramitochondrial [ATP]/[ADP] ratio (about 4 in the resting state) are in good agreement with data obtained by other methods.

Strong evidence is provided for a decrease of the intramitochondrial [ATP]/[ADP] ratio with increasing rate of oxygen consumption. Therefore it is not necessary to assume a microcompartmentation of the intramitochondrial adenine nucleotide pool in respect to the ATPase reaction and the adenine nucleotide translocation.     

Dependence of mitochondrial oxidative phosphorylation on activity of the adenine nucleotide translocase

The coupled reactions of electron transport and ATP synthesis for the first two sites of mitochondrial oxidative phosphorylation have been previously reported to be near equilibrium in isolated respiring pigeon heart (Erecińska, M., Veech, R. L., and Wilson, D. F. (1974) Arch. Biochem. Biophys. 160, 412-421) and rat liver mitochondria (Forman, N. G., and Wilson, D. F. (1982) J. Biol. Chem. 257, 12908-12915). Measurements are presented in this paper which demonstrate that the same relationship exists for both forward and reverse electron transport in rat heart mitochondria. This conclusion implies that adenine nucleotide translocation, a partial reaction of the system, is also near equilibrium, contrasting with proposals that the translocase is rate-limiting for oxidative phosphorylation. To resolve this controversy, the respiratory rates of suspensions of isolated rat liver and rat heart mitochondria were controlled by varying either the added [ATP]/[ADP][Pi] ratios ratios or [ADP] (by varying hexokinase in a regenerating system). Titrations with carboxyatractyloside, a high affinity inhibitor of the translocase which is noncompetitive with ADP, were carried out to assess the dependence of the respiratory rate on translocase activity. Plots of respiratory rate versus [carboxyatractyloside] were all strongly sigmoidal. In liver mitochondria, 40%-70% and in heart mitochondria 66% of the sites could be blocked with carboxyatractyloside before a 10% decrease in the respiratory rate was observed. Further analysis showed that liver and heart mitochondria have translocase/cytochrome a ratios of 1.52 and 3.20, respectively, and that at 23 degrees C the maximal turnover numbers for the translocases were 65 s-1 and 23 s-1. In all states of controlled respiration (no added inhibitor), a substantial excess of translocase activity was present, suggesting that the translocase was not normally rate-limiting in oxidative phosphorylation.