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.     

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.     

Inhibition of citrulline synthesis by octanoate and its modulation by adenine nucleotides

Liver mitochondria from octanoate-treated rabbits showed an impaired ability to synthesize citrulline. Two methods were used to evaluate citrulline synthesis in rat liver mitochondria. Under these conditions octanoate inhibited citrulline synthesis by over 50%. When ATP was included in the assay medium the inhibitory effect of octanoate was prevented. In the absence of ATP in the suspending medium, octanoate did not significantly lower total adenine nucleotides in rat liver mitochondria. However, under these conditions octanoate caused a change in the adenine nucleotide profile such that ATP content was decreased and AMP content was increased. When ATP was present in the assay medium, octanoate caused a similar increase in AMP content. However, ATP decreased only slightly. The alterations in mitochondrial adenine nucleotide profile by octanoate and the reversal of the effect by exogenous ATP suggests that octanoate inhibits citrulline synthesis via reduced intramitochondrial ATP levels. The ability of octanoate to lower mitochondrial ATP and elevate mitochondrial AMP may be related to its intramitochondrial activation by the medium chain fatty acid activating enzyme.     

Competition between extramitochondrial and intramitochondrial ATP-consuming processes.

The relationship between intra- and extramitochondrial ATP utilization was investigated in liver mitochondria isolated from normally fed, starved and high-protein fed rats. ATP export was provoked by adding a hexokinase-glucose-trap and intramitochondrial ATP consumption by adding ammonia, bicarbonate and ornithine in order to stimulate citrulline synthesis. Both processes compete for ATP produced via oxidative phosphorylation; the rate of citrulline formation declines as the extramitochondrial [ATP]/[ADP] ratio decreases. It is concluded that ATP for adenine nucleotide translocation and that for carbamoyl phosphate synthesis are delivered from a common intramitochondrial pool of adenine nucleotides. In mitochondria from rats with a high-protein diet, citrulline synthesis greatly stimulates the rate of oxidative phosphorylation (about two thirds of state 3 respiration). Under these conditions the intramitochondrial [ATP]/[ADP] ratio is significantly reduced. The intramitochondrial [ATP]/[ADP] ratio is not in thermodynamic equilibrium with the extramitochondrial one.     

Adenine nucleotide extraction from multicellular organisms and beach sand: ATP recovery,energy charge ratios and determination of carbon/ATP ratios

Investigations were conducted comparing the efficiency of adenine nucleotide extraction from bacteria, unicellular algae, invertebrates (copepods, isopods and polychaetes), and beach sand using boiling buffers and cold acid extraction procedures. Cellular levels of ATP, ADP, and AMP obtained by these procedures were used to calculate the adenylate energy charge ratio ($\text{EC}{}_{\mathrm{<mtext>A</mtext>}}\text{= [}\text{ATP}\text{] +}\text{1}\text{2}\text{[}\text{ADP}\text{]/[}\text{ATP}\text{] + [}\text{ADP}\text{] + [}\text{AMP}\text{]}$). Although both extraction procedures efficiently extract ATP from unicellular micro-organisms, the results with multicells and beach sand indicate that the cold acid procedure preserves a greater percentage of the total adenine nucleotides ([A_T] = [ATP] + [ADP] + [AMP]) in the form of ATP, resulting in higher energy charge ratios. There were relatively large losses of ATP when multicellular organisms were extracted in boiling buffers. These data suggest that ATP hydrolysis may be important in certain fluid-solid mixtures, and also adds experimental support to the thermal gradient hypothesis.The C/ATP ratios calculated from these data indicate that multicellular organisms have C/ATP ratios < 100, as compared with the 250 ratio commonly found in micro-organisms. These results are discussed in terms of the proportion of structural (non-living) carbon vs protoplasm (living) carbon within each of these groups of organisms, as well as the relative intracellular levels of non-adenine nucleotide triphosphates. These differences in the C/ATP ratios must be considered whenever ATP measurements are used for biomass determinations.     

Free pyrimidine nucleotide pool of Ehrlich ascites-tumour cells. Characteristics related to quantitative studies of RNA metabolism

The atractyloside-insensitive accumulation of adenine nucleotides by rat liver mitochondria (as opposed to the exchange-diffusion catalysed by the adenine nucleotide translocase) has been measured by using the luciferin/luciferase assay as well as by measuring [14C]ATP uptake. In foetal rat liver mitochondria ATP is accumulated more rapidly than ADP, whereas AMP is not taken up. The uptake of ATP occurs against a concentration gradient, and the rate of ATP uptake is greater in foetal than in adult rat liver mitochondria. The accumulated [14C]ATP is shown to be present within the mitochondrial matrix space and is freely available to the adenine nucleotide translocase for exchange with ATP present in the external medium. The uptake is specific for ATP and ADP and is not inhibited by adenosine 5'-[beta gamma-imido] triphosphate, GTP, CTP, cyclic AMP or Pi, whereas dATP and AMP do inhibit ATP accumulation. The ATP accumulation is also inhibited by carbonyl cyanide m-chlorophenylhydrazone, KCN and mersalyl but is insensitive to atractyloside. The ATP uptake is concentration-dependent and exhibits Michaelis-Menten kinetics. The divalent cations Mg2+ and Ca2+ greatly enhance ATP accumulation, and the presence of hexokinase inhibits the uptake of ATP by foetal rat liver mitochondria. These latter effects provide an explanation for the low adenine nucleotide content of foetal rat liver mitochondria and the rapid increase that occurs in the mitochondrial adenine nucleotide concentration in vivo immediately after birth.     

A study of spatial and temporal variability in the adenine nucleotides of lake phytoplankton during a diel cycle (Lac Pavin — France)

A study of diel variations in the adenine nucleotide concentrations of natural phytoplanktonic populations led to the following conclusions:There are significant diel variations in cellular ATP concentrations. However, this temporal variability is not as great as the spatial variability between the various sampling depths, characterized by different phytoplankton populations. Within a globally stable pool of adenine nucleotides, diel variations inside the algal cell mainly take place at the expense of ATP and AMP, while ADP concentrations remain relatively stable.The statistical relationship between algal biomass obtained from cell counts and from adenine nucleotides also confirms maximum stability for ADP and (ATP + ADP + AMP). The diel variations in adenine nucleotide cell concentrations gradually become smaller with depth. This probably reflects the importance of light in causing such variability.     

Adenine nucleotide translocation in plant mitochondria

The rapid translocation of external ADP-[^14C]by corn mitochondria is inhibited by high concentrations of atractyloside with enhanced inhibition occurring in the presence of Mg²⁺. This translocation is also inhibited by AMP or ATP but CDP, GDP, IDP or UDP have little effect. Backward exchange of internal ADP-[¹⁴C] occurs in the presence of AMP, ADP or ATP but is not promoted by other nucleoside diphosphates. It is suggested that the adenine nucleotide (AdN) carrier is specific for ADP and ATP and that apparent translocation of AMP is a result of adenylate kinase activity. The translocated ADP can be separated into 3 components: (1) atractyloside-insensitive binding; (2) carrier-bound ADP saturated at ca 30 μM external ADP; and (3) exchanged ADP saturated as ca 5 μM external ADP. It is suggested that the adenine nucleotide carrier of plant mitochondria possesses similar properties to the classical carrier of vertebrate mitochondria.     

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.     

Regulation of the mitochondrial adenine nucleotide pool size in liver: mechanism and metabolic role

The ATP-Mg/Pi carrier in liver mitochondria can catalyze the exchange of ATP-Mg on one side of the inner membrane for Pi on the other. This mechanism allows for net uptake or release of ATP-Mg from mitochondria and thus regulates the matrix ATP + ADP + AMP pool size. In isolated mitochondria, carrier activity is stimulated by submicromolar concentrations of calcium, suggesting that calcium may regulate transport rates in vivo. Whenever the carrier is active, the direction of any net changes in the matrix adenine nucleotide pool size is determined mainly by the extent to which the prevailing ATP-Mg concentration gradient deviates from an equilibrium related to delta pH through the phosphate concentration gradient. Thus it seems that in the cell, energy status (reflected by ATP:ADP ratios in the cytoplasm and matrix) determines whether calcium-mediated hormone activation of the carrier will produce an increase or a decrease in the matrix adenine nucleotide content. Consequent variations in the absolute concentrations of ATP, ADP, and AMP in the matrix may contribute to the selective regulation of those metabolic activities in the cell that have adenine nucleotide dependent steps localized to the mitochondrial compartment (gluconeogenesis, urea synthesis, mitochondrial biogenesis, and even oxidative phosphorylation).     

The hormonal stimulation of ureogenesis in isolated hepatocytes through increases in mitochondrial ATP production

Isolated hepatocytes incubated with 2 mm ornithine-10 mm glutamine as substrates and challenged with either glucagon, epinephrine, or phenylephrine exhibited stimulated rates of urea production, and mitochondria isolated from these cells displayed an increased rate of energy-dependent citrulline formation. There was no change in the total carbamyl phosphate synthetase I activity, nor mitochondrial content of the positive effector N-acetyl glutamate after acute hormonal treatment. The time of onset of ureogenesis and its sensitivity to glucagon were compared with stimulation of glucose production from lactate-pyruvate. No apparent differences in time of onset or sensitivity of the responses were observed indicating both pathways may be stimulated by a common mechanism. Mitochondria prepared from cells treated with catecholamines exhibited increased rates of State 3 respiration and increased uncoupler-dependent ATPase activity, in addition to the increased rates of citrulline formation. There was also an elevated intramitochondrial content of ATP and an increased $\text{ATP}\text{ADP}$ ratio. The catecholamine-induced stimulation of ureogenesis was mediated by an α-adrenergic cyclic AMP independent mechanism. The addition of the α-adrenergic antagonist, dihydroergotamine, blocked both the epinephrine-induced stimulation of ureogenesis and also the stimulated functions in the isolated mitochondria. dl-Propranolol, a β-antagonist, inhibited the rise in cyclic AMP due to epinephrine, but had no effect on any of the other reactions measured. The effects of catecholamines on citrulline formation and urea production are correlated with the increased capacity of the mitochondria to generate ATP. It is suggested that both glucagon and catecholamines, acting via independent mechanisms, stimulate electron transport and the activity of the ATP-forming enzyme complex. The consequent elevated intramitochondrial ATP levels and $\text{ATP}\text{ADP}$ ratio enhance the rate of citrulline formation and hence ureogenesis.     

Activation of 2,4-dinitrophenol-stimulated ATPase activity in cauliflower and corn mitochondria.

Cauliflower mitochondria do not have a 2,4-dinitrophenol-stimulated ATPase unless they are permitted a brief period of respiration (respiratory priming) or are preincubated for an extensive period with ATP (self-priming). Both priming processes are dependent on Mg²⁺, and are collapsed by 2,4-dinitrophenol in the absence of ATP. Corn mitochondria, which have an endogenous DNP-ATPase, contain significantly more Mg²⁺ and adenine nucleotides than cauliflower mitochondria. Primed cauliflower mitochondria have Mg²⁺ content comparable to corn mitochondria. Cauliflower mitochondria will actively accumulate adenine nucleotides through atractyloside-insensitive sites. It appears that priming consists of creating an electrochemical potential which is needed for accumulation of Mg²⁺ or adenine nucleotides or for charge compensation of the $\text{ATP}{}^{\mathrm{4?}}\text{ADP}{}^{\mathrm{3-?}}$ exchange.     

Reversible inactivation by NADH and ADP on Chlorella fusca nitrate reductase

The active form of $\text{Chlorella fusca}$ nitrate reductase can be reversibly converted into its inactive form by reduction with NADH in the presence of ADP. Under the experimental conditions used, no inactivation occurs when nitrate is simultaneously present or when the nucleotides act isolately, the inactivating effect being maximal at a concentration of ADP (0.3 mM) equimolecular with that of NADH. The inactive enzyme thus attained can be completely reactivated by reoxidation with ferricyanide. The redox state of the pyridine nucleotide and the phosphorylation degree of the adenine nucleotide are critical for the inactivation process to ensue, since neither NAD⁺ nor AMP or ATP do exert any effect. ADP is also a powerful, although rather unspecific, protector against thermal inactivation of the NADH-diaphorase moiety of the NADH-nitrate reductase complex.     

Control of oxidative phosphorylation by the extramitochondrial ATP/ADP ratio

The control of mitochondrial ATP synthesis by the extramitochondrial adenine nucleotide pattern was investigated with rat liver mitochondria. It is demonstrated that any stationary state between the two limit states of maximum activity (state 3) and of resting activity (state 4) can be obtained by a hexokinase-glucose trap as an ADP-regenerating system. These intermediate states are characterized by stationary respiratory rates, stationary redox levels of the cytochromes b and c and stationary levels of extramitochondrial ATP and ADP between the rates and levels of the limit states. At a constant concentration of inorganic phosphate the activity of mitochondria between the limit states is controlled by the extramitochondrial ATP/ADP ratio independent of the total concentration of adenine nucleotides present. The control range was found to be between ratios of about 5 and 100 at 10 mM phosphate. At lower ratios the mitochondria are in their maximum phosphorylating state. With succinate + rotenone and glutamate + malate the same control range was observed, indicating that it is independent of the nature of substrate oxidized.The results suggest that in the control range the mitochondrial activity is limited by the competition of ADP and ATP for the adenine nucleotide translocator.     

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.     

Changes in adenine nucleotide levels and respiration during 1-methyladenine induced maturation of starfish oocytes

The effects of 1-methyladenine on oxygen consumption and adenine nucleotide levels were examined in oocytes of Pisaster ochraceus and Patiria miniata. Oocytes of both genera to which 1-methyladenine was added consumed more oxygen than control oocytes beginning 1 to $\text{1}\text{1}\text{2}\text{h}$ after 1-methyladenine addition. The increase in oxygen consumption was correlated with maturation changes in the oocytes and particularly with germinal vesicle breakdown. Pre-fertilization oxygen consumption of eggs did not differ significantly from post-fertilization oxygen consumption of eggs in either genus for $\text{2}\text{1}\text{2}\text{h}$ after fertilization. ATP and AMP concentrations within the oocytes decreased during 1-methyladenine induced maturation, while the ADP concentration increased. It was suggested that increases in ADP concentration and decreases in ATP concentration within maturing starfish oocytes occurred in response to greater energy demands. The simultaneous increase in oocyte oxygen consumption was interpreted as an indicator of increased oxidative phosphorylation acting to restore initial nucleotide concentrations.     

Rate control of phosphorylation-coupled respiration by rat liver mitochondria

Liver mitochondria provided with an oxidizable substrate, ATP, oxygen, and an ADP-generating system (soluble F₁-ATPase) were used to reevaluate the rate-controlling step(s) intrinsic to all of the processes of mitochondrial oxidative phosphorylation. The quantity termed “control strength” (C), previously defined as the fractional change in flux through a (system) induced by a fractional change in the concentration of an individual enzyme in the system, has been used to evaluate rate-influencing steps in this overall process by carefully defining the dimensions of the “system” under analysis. If the system is defined by a suspension of mitochondria provided with substrates, plus an extrinsic ADP-generating process (ATPase), the value of C of the latter for the overall process of phosphorylation-linked respiration is near 1.0 until the capacity of the mitochondria to phosphorylate ADP is approached, after which C for the soluble ATPase becomes zero as the maximum capacity for phosphorylation is attained. Carboxyatractyloside was found only marginally to inhibit respiration stimulated by ATPase, even when a large percentage of adenine nucleotide translocase molecules were immobilized. The relative lack of effect of carboxyatractyloside on phosphorylating respiration is explained by the readjustment of the concentration of one of the substrates (ADP) and an inhibitor (ATP), which results from inhibition of adenine nucleotide translocase. The residual blunted inhibition of respiration is explained by product inhibition of the ADP-regenerating ATPase, and not necessarily to any intrinsically mitochondrial intermediate process. The system being evaluated can be redefined to include only the processes intrinsic to mitochondria. This can be achieved by providing exactly comparable substrate concentrations to the mitochondria under comparable incubation conditions. Under these conditions, the adenine nucleotide translocase is the principal, if not the only, rate-controlling step in the overall process of oxidative phosphorylation until a new rate-limitation is attained (ATP synthesis). These data are consistent with the conclusion that, at intermediate rates of phosphorylation-coupled respiration, the extramitochondrial $\text{ATP}\text{ADP}$ ratio regulates this process through its kinetic effects on the catalytic properties of the adenine nucleotide translocase.