Transport of adenine nucleotides in the mitochondria of Saccharomyces cerevisiae: Interactions between the ADP/ATP carriers and the ATP-Mg/Pi carrier

The ADP/ATP and ATP-Mg/Pi carriers are widespread among eukaryotes and constitute two systems to transport adenine nucleotides in mitochondria. ADP/ATP carriers carry out an electrogenic exchange of ADP for ATP essential for oxidative phosphorylation, whereas ATP-Mg/Pi carriers perform an electroneutral exchange of ATP-Mg for phosphate and are able to modulate the net content of adenine nucleotides in mitochondria. The functional interplay between both carriers has been shown to modulate viability in Saccharomyces cerevisiae. The simultaneous absence of both carriers is lethal. In the light of the new evidence we suggest that, in addition to exchange of cytosolic ADP for mitochondrial ATP, the specific function of the ADP/ATP carriers required for respiration, both transporters have a second function, which is the import of cytosolic ATP in mitochondria. The participation of these carriers in the generation of mitochondrial membrane potential is discussed. Both are necessary for the function of the mitochondrial protein import and assembly systems, which are the only essential mitochondrial functions in S. cerevisiae.     

Specific inhibition of phosphate transport in mitochondria by N-ethylmaleimide

N-ethylmaleimide (NEM), a reagent that alkylates free sulfhydryl groups, was shown to be a highly effective inhibitor of the following coupled mitochondrial processes: oxidative phosphorylation, ATP-³²Pi exchange, Pi-induced light scattering and configurational changes, State III respiration, valinomycin-induced translocation of potassium with Pi as the anion, and calcium accumulation in presence of Pi. However, NEM was less effective or ineffective in inhibiting some processes that do not require inorganic Pi, namely electron transfer and ATPase activity, ADP binding, energized light scattering changes induced by arsenate and nonenergized light scattering changes induced by acetate. The rate of oxidative phosphorylation and of ATP-³²Pi exchange was normal in ETP_H particles prepared from NEM-treated mitochondria. Also NEM, even et levels 2–3 times greater than those required to inhibit oxidative phosphorylation in intact mitochondria, did not inhibit coupled processes in submitochondrial particles. We are proposing that NEM alkylates sulfhydryl groups in the mitochondrion that modulate Pi translocation, and that the suppression of Pi translocation blocks oxidative phosphorylation, the Pi-dependent energized configurational change in mitochondria and Pi-dependent transport processes.On leave of absence from the Department of Biochemistry, Cancer Institute Okayama University Medical School, Okayama, Japan.On leave of absence from the Department of Pathology, Nagoya University Medical School, Nagoya, Japan.     

Occurrence and significance of oxygen exchange reactions catalyzed by mitochondrial adenosine triphosphatase preparations.

The capacity of various ATPase preparations from beef heart mitochondria to catalyze exchange of phosphate oxygens with water has been evaluated. Oligomycin-sensitive ATPase preparations retain a capacity for considerable intermediate Pi equilibrium HOH exchange per Pi formed during ATP hydrolysis at relatively high ATP concentration (5 mM). Submitochondrial particles prepared by an ammonia-Sephadex procedure with 5 mM ATP showed more rapid ATPase, less oligomycin sensitivity, and less capacity for intermediate exchange. With these particles, intermediate Pi equilibrium HOH exchange per Pi formed was increased as ATP concentration was decreased. The purified, soluble ATPase from mitochondria catalyzed little or no intermediate Pi equilibrium HOH exchange at 5 mM ATP but showed pronounced increase in capacity for such exchange as ATP concentration was lowered. The ATPase also showed a weak catalysis of an ADP-stimulated medium Pi equilibrium HOH exchange. The results support the alternating catalytic site model for ATP synthesis or cleavage. They also demonstrate that a transmembrane protonmotive force is not necessary for oxygen exchange reactions. At lower ATP concentrations, ADP and Pi formed at a catalytic site appear to remain bound and continue to allow exchange of Pi oxygens until ATP binds at another site on the enzyme.     

Study of the mitochondrial phosphate carrier in the course of calcium phosphate accumulation: A requirement for Mg2+ and ADP of its sensitivity to thiol reagents

1. The accumulation of calcium phosphate driven by succinate oxidation is ADP-dependent. In its absence the accumulation stops after a short incubation time and the oxygen uptake is permanently stimulated. This uncoupled oxygen uptake is insensitive to the inhibitors of phosphate transport, like mersalyl and N-ethylmaleimide. When ADP plus Mg²⁺ are added to the medium, or when ADP is added in the initial presence of magnesium, the inhibitory action of the thiol reagents on oxygen uptake is re-established. ADP alone or Mg²⁺ alone are without any effect.2. Phosphate/phosphate exchange has been studied, in the absence of ADP, when calcium phosphate accumulation had stopped and oxygen uptake is uncoupled. Under these conditions the exchange process becomes insensitive to thiol reagents. Sensitivity is recovered solely in the presence of ADP plus Mg²⁺.3. When mitochondrial swelling is studied according to the method of Chappell, it also appears that the phosphate carrier loses it sensitivity to mersalyl in the absence of ADP, which confirms the data obtained with phosphate/phosphate exchange experiments. When ADP plus Mg²⁺ are added (or present), together with mersalyl, the action of the thiol inhibitor is recovered. ADP and magnesium are inactive separately. EGTA plus Mg²⁺ (but not EGTA plus ADP) may substitute for ADP plus Mg²⁺ in this process.4. A possible interaction between the magnesium binding site and the phosphate carrier is considered and discussed.     

The mechanism of phosphate permeation in purified bean mitochondria

The permeability properties and mechanism of Pi transport wereinvestigated in purified bean mitochondria.

1. Purified bean mitochondria are impermeable to small moleculesand ions. However, Pi, arsenate, acetate and formate can enterthe osmotically active space of bean mitochondria.
2. Nigericinor the association of valinomycin and FCCP cause mitochondrialswelling in isoosmotic potassium phosphate.
3. The SH-blockingreagents mersalyl, pHMB and NEM inhibit variousmitochondrialfunctions dependent on the translocation of Piand arsenateacross the membrane. These include the respirationstimulatedby ADP, Ca²⁺+Pi, and K⁺+valinomycin +Pi; the swellingin ammoniumphosphate medium and, in the presence of nigericin,in potassiumphosphate medium; the energy-linked yalinomycin-inducedswellingand the subsequent CICCP-induced shrinking. The uncoupler-stimulatedrespiration, as well as the other processes when acetate issubstituted for Pi, are not influenced by SH reagents.
4. Mersalyland pHMB cause complete inhibition at about 20 nmoles/mgprotein,whereas, NEM is effective at about 1 µmole/mgprotein.The inhibition by mersalyl and pHMB, but not that byNEM, issigmoidal and reversed by 2-mercaptoethanol. Non-inhibitoryamounts of mersalyl protect the Pi transport from irreversibleinhibition by NEM.
5. We concluded that a carrier-mediated transportsystem for Piis present in bean mitochondria, and that someof its propertiesare similar to the Pi carrier of animal mitochondria.

(Received June 5, 1975; )     

Transport of inorganic phosphate and C3- and C6-sugar phosphates across the envelope membranes of potato tuber amyloplasts

Amyloplasts have been isolated from tubers of potato plants (Solarium tuberosum. cv. Desirée). As it is difficult to isolate amyloplasts that have a high starch content, we used transformed plants in which the content of starch was reduced. This was achieved by decreasing the activity of ADP-glucose pyrophosphorylase by antisense techniques (Müller-Röber et al., 1992, EMBO. 11, 1229–1238). In the isolated plastids the activity of glutamine-oxoglutarate-aminotransferase (glutamate synthase, EC 2.6.1.53) was dependent upon the intactness of the plastids. For the supply of redox equivalents the addition of glucose-6-phosphate (Glc6P) was required. Glucose-1-phosphate (Glc1P) did not support glutamate synthesis. Plastids were treated with Triton X-100 and the solubilized proteins reconstituted into liposomes. Transport measurements with these liposomes revealed that inorganic phosphate (Pi), dihydroxyacetone phosphate (DHAP), 3-phosphoglycerate and Glc6P are transported in a counter-exchange mode. Transport of phosphoenolpyruvate was low and Glc1P was virtually not transported in exchange for Pi. Kinetic constants were determined for the Pi/Pi and Glc6P/Pi counter exchanges. For comparison, proteins of mitochondria from potato tubers and pea leaves were reconstituted into liposomes. As expected, the Pi/Pi exchange across the mitochondrial membrane was not affected by DHAP and Glc6P. Kinetic constants of the Pi/Pi counter exchange were determined for potato tuber mitochondria.Abbreviations DHAP dihydroxyacetone phosphate - Glc1P glucose-1-phosphate - Glc6P glucose-6-phosphate - PEP Phosphoenolpyruvate - 3-PGA 3-phosphoglycerate - Pi inorganic phosphate - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)-ethyl] glycine This work was supported by Deutsche Forschungsgemeinschaft.     

Energy-linked Adenosine Diphosphate Accumulation by Corn Mitochondria: I. General Characteristics and Effect of Inhibitors

Corn mitochondria show respiration-linked net accumulation of [³H]ADP in the presence of phosphate and magnesium, especially if the formation of ATP is blocked with oligomycin. Inhibition of ADP-ATP exchange by carboxyatractyloside also activates ADP accumulation, and addition of carboxyatractyloside or palmitoyl-coenzyme A to oligomycin-blocked mitochondria produces additional ADP uptake. With carboxyatractyloside the accumulated ADP is phosphorylated to ATP. With oligomycin, only a little ATP is formed. Millimolar concentrations of ADP are required for maximum uptake, and the K_m (3.77 millimolar) for ADP translocation is independent of whether oligomycin or carboxyatractyloside is used. This is not true for ADP concentrations in the 0.05 to 0.25 millimolar range. Accumulated [³H]ADP rapidly exchanges with unlabeled AMP, ADP, or ATP, but not with other diphosphate nucleotides or 2 millimolar substrate anions. [³H]AMP is not accumulated, but [³H]ATP is accumulated to about one-half the extent of [³H]ADP. Tricarboxylate substrates inhibit ADP net uptake, and inhibition by citrate is competitive with K_i = 10 millimolar. The evidence suggests the presence of a pathway, carboxyatractyloside-insensitive and different from the translocase, which operates to maintain adenine nucleotides in the matrix.     

Deficiency of uncoupler-stimulated adenosine triphosphatase activity in yeast mitochondria

Oligomycin-sensitive ATPase activity was studied in isolated yeast mitochondria. The protonophore CCCP, at a concentration which completely inhibited ATP synthesis, induced only a low rate of hydrolysis of externally added ATP, and the extent of hydrolysis was dependent upon phosphate (Pi) concentration. CCCP promoted hydrolysis of intramitochondrial ATP. However, hydrolysis of externally added ATP was total in a medium containing potassium phosphate plus valinomycin. Without ionophores, ATPase activity was only observed at high external pH or with detergent-treated mitochondria. Under state 4 conditions, external ATP had access to the catalytic nucleotide site of ATPase as shown by 32Pi-ATP exchange experiments. These results are discussed in terms of a limitation of the translocase-mediated ATP/ADP exchange in uncoupled mitochondria.     

The ATP-Mg/Pi carrier of rat liver mitochondria catalyzes a divalent electroneutral exchange.

Net transport of ATP-Mg or ADP in exchange for phosphate in isolated rat liver mitochondria has been shown to be an electroneutral process mediated by the ATP-Mg/Pi carrier. We compared the steady state distribution ratios of phosphate, ATP-Mg, and ADP at a pH of 7.4 to determine whether the divalent or monovalent form of these anions is the transported substrate. The log of the divalent ATP-Mg distribution ratio (in/out) approached the log of the divalent phosphate distribution ratio (approximately 0.85), which was approximately twice the value of the delta pH (approximately 0.40) across the inner mitochondrial membrane. This steady state relationship held under several different conditions, e.g. when the medium ATP concentration was varied or if the phosphate gradient was modified by partial uncoupling with the proton ionophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Unidirectional ADP efflux in exchange for external ADP or ATP-Mg was stimulated by an increase in matrix H+. The log of the trivalent ADP distribution ratio (approximately 1.20) approached 3 times the value of delta pH. All these data are consistent with the model of an electroneutral exchange of divalent phosphate (HPO2-4) for divalent ATP-Mg (ATP-Mg2-) or for divalent protonated ADP (HADP2-). We conclude that this transport mechanism accounts for the adenine nucleotide concentration gradient that normally exists between the matrix and external medium.     

Is the Cytosolic Pi Concentration a Limiting Factor for Plant Cell Respiration?

The substrate-dependent O₂ uptake by sycamore (Acer pseudoplatanus L.) cell mitochondria in the presence of ADP and limiting Pi concentrations has been measured. The Pi concentration for half-maximum O₂ uptake rate was found to be in the range 20 to 50 micromolar for all the substrates tested. ³¹P NMR of intact sycamore cells indicated that the Pi concentration in the cytoplasm was in the range 5 to 6 millimolar, approximately 100-fold higher than the Pi concentration required for maximum O₂ uptake rates by isolated mitochondria. When sycamore cells were transferred to a culture medium devoid of Pi, the cytoplasmic Pi concentration decreased from 6 to less than 3 millimolar, but the intact cell respiration remained practically constant for at least 4 days. These results strongly suggest that, in vivo, the respiration rate of sycamore cells is not limited by the quantity of Pi supplied to the mitochondria.     

The interaction of Ga2+ with mitochondria from human myometrium.

Mitochondria prepared from human myometrium contain large amounts of endogenous Ca²⁺ (up to 200 nmol/mg of protein) even if isolated in media containing ethylene glycol-bis(β-aminoethylether)-N,N′-tetraacetic acid. The endogenous Ca⁺², however, is not irreversibly sequestered, since it can be rapidly and quantitatively discharged by uncouplers. Human myometrial mitochondria are capable of efficient energy-linked Ca²⁺ transport. In the absence of phosphate, the amount of Ca²⁺ accumulated is reduced to insignificant levels. Mg²⁺ has a strong inhibitory effect, which has been exploited to develop an inhibitor-stop method which has permitted the determination of the affinity of myometrial mitochondria for Ca²⁺ (K_m, ~5 μM) and of the maximal velocity of uptake (0.55 nmol/mg of protein/s). The respiration of human myometrial mitochondria is stimulated by Ca²⁺, with respiratory control indexes of the order of 4–5. In contrast, ADP induces an insignificant stimulation, or no stimulation at all. The response of respiration to ADP is somewhat improved if mitochondria are preincubated under conditions which decrease their endogenous Ca²⁺ content. The adenine nucleotide exchange in human myometrial mitochondria is deficient with respect to liver mitochondria.     

Reconstruction of steady state in cell-free systems. Interactions between glycolysis and mitochondrial metabolism: regulation of the redox and phosphorylation states

A reconstituted "open" system comprising respiring mitochondria and actively glycolyzing muscle extract was devised for studies of vectorially mediated interactions. Glycogen particles were the substrate for the glycolyzing enzymes. Purified soluble (F1) ATPase was added in varying quantities to establish a range of energetic steady states. The data generally confirm our recent conclusions (Wu and Davis, (1981) Arch. Biochem. Biophys. 208, 85-89) on the relative efficacy of the adenine nucleotides and their ratios, and of inorganic phosphate on flux through rate-controlling steps of glycolysis. When mitochondrial ATP synthesis was blocked, glycolytic flux was relatively rapid, and the lactate/pyruvate ratio increased with time to values up to greater than 300. If functional mitochondria were present, glycolytic flux was very strongly suppressed, provided the energy state (ATP/ADP) was high, and the phosphate concentration[Pi] was low. Adenine nucleotide control of glycolysis was to a large extent lost when the steady-state ATP/ADP was below about 10, or if [Pi] was elevated. In the two-phase system containing respiring mitochondria and components of the malate-aspartate shuttle, the ATP/ADP and both extra- and intramitochondrial NAD+/NADH ratios were maintained constant, and to various perturbable levels with varying energy load (ATPase). The gradient in reduction potentials attained values (delta Gredox) of up to about 2.5 kcal. The extramitochondrial redox state was not positively correlated with the external phosphorylation potential ([ATP]/[ADP] X [Pi]). The following conclusions are drawn on the basis of the present data, together with other reports (Davis, Bremer, and Akerman (1980) J. Biol. Chem. 255, 2277-2283) and (Klingenberg and Rottenberg (1977) Eur. J. Biochem. 73, 125-130): (a) the gradient in reduction potential is driven by the membrane potential (delta psi), mediated by the electrogenic glutamate-aspartate exchange, and the poise or set point of this gradient is a function of delta psi; and (b) the gradient of ATP/ADP ratios across the membrane is also driven principally by delta psi, mediated by the electrogenic ATP-ADP exchange. Hence, segregation of phosphorylation and reduction potentials is linked through a mutually shared electrical driving force.     

Effect of Escherichia coli endotoxin on adenine nucleotide translocation in canine heart mitochondria

The in vitro effect of Escherichia coli endotoxin on the translocation of adenine nucleotides in dog heart mitochondria was studied. Mitochondrial adenine nucleotides were labeled with ¹⁴C by incubating mitochondrial preparations in the presence of [¹⁴C]ADP. The exchange reaction was initiated by addition of unlabeled ADP, proceeded for 5 to 60 s at 4 °C, and was terminated by addition of atractyloside. The results showed that preincubation of mitochondria with endotoxin (50 μg/mg protein) for 10 min at 23 °C decreased the exchange reaction by 21.2% (P < 0.05). The inhibitory effect of endotoxin was increased with increasing concentrations of endotoxin with an I₅₀ value of 45 μg/mg protein. The initial rate and the total extent of exchange were both affected. Double reciprocal plots showed that only the V but not the K_m for ADP was affected by endotoxin, indicating that the inhibition was noncompetitive in nature. The exchange of adenine nucleotide remained depressed by endotoxin in the presence of either oligomycin or antimycin A, indicating that the inhibitory effect of endotoxin was independent of the action of endotoxin on oxidative phosphorylation. The leakage of labeled adenine nucleotides from mitochondria at 23 °C was increased by 100% by endotoxin (100 μg/mg protein) in the absence of added unlabeled ADP, and this increase in the leakage could not be blocked by atractyloside. The endotoxin-induced changes in adenine nucleotide exchange and leakage were either partially or completely prevented by hydrocortisone, heparin, dibucaine, or EDTA. Since most of these agents have in common an effect on lipid metabolism, it is suggested that endotoxin-induced alterations in the exchange and leakage of adenine nucleotides in heart mitochondria are protected through a mechanism involving membrane lipid reorganization.     

Adenine nucleotide and phosphate transport systems of mitochondria. Relative location of sulfhydryl groups based on the use of the novel fluorescent probe eosin-5-maleimide

Eosin-5-maleimide is impermeable to the inner mitochondrial membrane, exhibiting essentially no reactivity with matrix glutathione or with beta-hydroxybutyrate dehydrogenase located on the matrix surface of the inner membrane. In intact mitochondria, eosin-5-maleimide is unreactive with the ADP/ATP antiporter even under conditions which promote maximal labeling by N-[3H]ethylmaleimide (i.e., ADP present). However, eosin-5-maleimide readily labels the ADP/ATP antiporter in "inverted" inner membrane vesicles even in the presence of N-ethylmaleimide. Labeling is prevented if the vesicles are prepared from mitochondria pretreated with carboxyatractyloside. In contrast to the ADP/ATP antiporter, essential sulfhydryl groups of the Pi/H+ symporter are accessible to eosin-5-maleimide in intact mitochondria with optimal inhibition of phosphate transport being observed at 25 degrees C. Eosin-5-maleimide also prevents labeling of the Pi/H+ symporter by N-[3H]ethylmaleimide. These results show that essential sulfhydryl groups of the ADP/ATP antiporter and the Pi/H+ symporter have differing reactivities and locations in functionally intact mitochondria. With respect to eosin-5-maleimide, sulfhydryl groups of the ADP/ATP carrier occur in two distinct classes, both of which are inaccessible in intact mitochondria. Only one class, depending on conditions, can be exposed in submitochondrial particles. In contrast, sulfhydryl group(s) of the Pi/H+ symporter behave as a single reactive class which is readily accessible in mitochondria at 25 degrees C.     

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).     

Special features of Pi transport in pig heart and rat liver mitochondria as revealed by 6,6'-dithiodinicotinic acid (CPDS).

CPDS (6,6'-dithiodinicotinic acid), a non permeant thiol agent which affects several mitochondrial functions in a way different to that of mersalyl [18-19] revealed striking differences between the phosphate translocating systems of pig heart and rat liver mitochondria. Pi entry was measured either by swelling in 0.12 M ammonium phosphate or by rapid centrifugation in 32Pi medium. Pi efflux was measured after preloading of mitochondria with 32Pi, by exchange against Pi or malate; the "ATP-FCCP" system has been tested previously [19]. In pig heart mitochondria, Pi entry seems to proceed exclusively via the Pi/OH- carrier; CPDS completely inhibits this transport and the energy-linked functions. In contrast n-butyl-malonate does not affect the Pi-entry and the energy-linked functions. The Pi efflux is not affected either by CPDS or mersalyl, which do not produce a swelling in the "ATP-uncoupler system". In rat liver mitochondria, CPDS inhibits only the Pi/OH- carrier; both CPDS and n-butylmalonate are necessary to inhibit completely Pi entry. CPDS as well as mersalyl provokes a swelling in the presence of the "APT-uncoupler system". The results suggest two distinct functions of phosphate transport in both types of mitochondria.     

Reversibility of H+-ATPase and H+-Pyrophosphatase in Tonoplast Vesicles from Maize Coleoptiles and Seeds

Tonoplast-enriched vesicles isolated from maize (Zea mays L.) coleoptiles and seeds synthesize ATP from ADP and inorganic phosphate (Pi) and inorganic pyrophosphate from Pi. The synthesis is consistent with reversal of the catalytic cycle of the H⁺-ATPase and H⁺-pyrophosphatase (PPase) vacuolar membrane-bound enzymes. This was monitored by measuring the exchange reaction that leads to ³²Pi incorporation into ATP or inorganic pyrophosphate. The reversal reactions of these enzymes were dependent on the proton gradient formed across the vesicle membrane and were susceptible to the uncoupler carbonyl cyanide p(trifluoromethoxy)-phenylhydrazone and the detergent Triton X-100. Comparison of the two H⁺ pumps showed that the H⁺-ATPase was more active than H⁺-PPase in coleoptile tonoplast vesicles, whereas in seed vesicles H⁺-PPase activity was clearly dominant. These findings may reflect the physiological significance of these enzymes in different tissues at different stages of development and/or differentiation.     

Intermediate Reactions of Oxidative Phosphorylation in Mitochondria From Cabbage

Respiratory control ratios between 2.0 and 9.0 were obtained by comparison of the respiratory rates of cabbage mitochondria in the presence and in the absence of individual components of the system used to provide ADP and by comparing the rates before and after exhaustion of added ADP. These results indicate that respiration in cabbage mitochondria is controlled by the availability of ADP, which serves as the phosphate acceptor.Pentachlorophenol (PCP), 2,4-dinitrophenol (DNP), gramicidin and oleic acid inhibited phosphorylation to a greater extent than respiration in the cabbage mitochondria, but these reagents did not stimulate respiration in the absence of a phosphate acceptor. Respiration was stimulated by DNP only in the presence of added ATP.2,4-Dinitrophenol, pentachlorophenol, dicumarol and gramicidin did not stimulate ATPase activity either in the presence or absence of added Mg(2+). Oleic acid stimulated ATPase activity in the presence of added Mg(2+), but did not stimulate respiration even in the presence of added ATP.The ATP-(32)Pi exchange rate was increased many fold in the presence of added Mg(2+). Oleic acid and 2,4-dinitrophenol inhibited the exchange almost completely.     

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.