Studies of the mitochondria from Eimeria tenella and inhibition of the electron transport by quinolone coccidiostats.

Intact but fragile mitochondria were isolated from unsporulated oocysts of Eimeria tenella. The mitochondria respired in response to succinate, malate plus pyruvate, and L-ascorbate at rates of 1.00, 0.40, and 0.25 mu1 O2/min/mg protein, respectively. Spectrophotometric analyses of the cytochromes in mitochondria and whole oocysts revealed b-type and o-type cytochromes, at roughly similar levels, but no cytochrome c could be detected. The mitochondrial respiration was inhibited by cyanide, azide, carbon monoxide, antimycin A, and 2-heptyl-4-hydroxyquinoline-N-oxide, but was relatively resistant to rotenone and amytal. The quinolone coccidiostats buquinolate, amquinate, methyl benzoquate, and decoquinate were identified as very powerful inhibitiors of succinate and malate plus pyruvate supported respiration in E. tenella mitochondria. None of these four drugs exhibited any inhibitory effect on chicken liver mitochondria. Only 3 pmol of the quinolones per mg mitochondrial protein was needed to achieve 50% inhibition. The inhibition could not be reversed by coenzymes Q6 or Q10. Since the quinolones did not affect L-ascorbate-supported respiration or the activities of submitochondrial succinate dehydrogenase and NADH dehydrogenase, the site of action of the quinolone coccidiostats was tentatively identified as probably near cytochrome b in E. tenella mitochondria. Mitochondria isolated from an E. tenella amquinate-resistant mutant were much less susceptible to quinolone coccidiostats; 50% inhibition was attained by 300 pmol of the drugs/mg mitochondrial protein. The results suggest that the mechanisms of action of quinolone coccidiostats is by inhibiting the cytochrome-mediated electron transport in the mitochondria of coccidia. 2-Hydroxynaphthoquinone coccidiostats were identified as inhibitors of mitochondrial respiration of both E. tenella and chicken liver. They inhibited submitochondrial succinate dehydrogenase and NADH dehydrogenase of E. tenella, and remained equally active against the mitochondrial function of E. tenella amquinolate-resistant mutant.     

Effect of morphine in vitro on the oxidative phosphorylation in rat liver mitochondria]

The rates of respiration in the presence of ADP and of phosphorylation as an ATP-ase activity of rat liver mitochondria was inhibited was in vitro by morphine with Ki=6.5 mM. The uncoupler-stimulated respiration of the mitochondria and the activity of ATP-ase and synthesis of ATP in the submitochondrial particles were not altered in the presence of morphine. It is suggested that morphine inhibited the adenine nucleotide transport through the mitochondrial membrane     

The inhibitory effect of extracts of cigarette tar on electron transport of mitochondria and submitochondrial particles.

Acetonitrile extracts of cigarette tar inhibit state 3 and state 4 respiration of intact mitochondria. Exposure of respiring submitochondrial particles to acetonitrile extracts of cigarette tar results in a dose-dependent inhibition of oxygen consumption and reduced nicotinamide adenine dinucleotide (NADH) oxidation. This inhibition was not due to a solvent effect since acetonitrile alone did not alter oxygen consumption or NADH oxidation. Intact mitochondria are less sensitive to extracts of tar than submitochondrial particles. The NADH-ubiquinone (Q) reductase complex is more sensitive to inhibition by tar extract than the succinate-Q reductase and cytochrome complexes. Nicotine or catechol did not inhibit respiration of intact mitochondria. Treatment of submitochondrial particles with cigarette tar results in the formation of hydroxyl radicals, detected by electron spin resonance (ESR) spin trapping. The ESR signal attributable to the hydroxyl radical spin adduct requires the presence of NADH and is completely abolished by catalase and to a lesser extent superoxide dismutase (SOD). Catalase and SOD did not protect the mitochondrial respiratory chain from inhibition by tar extract, indicating that the radicals detected by ESR spin trapping are not responsible for the inhibition of the electron transport. We propose that tar causes at least two effects: (1) Tar components interact with the electron transport chain and inhibit electron flow, and (2) tar components interact with the electron transport chain, ultimately to form hydroxyl radicals.     

Thermodynamics of oxidative phosphorylation in bovine heart submitochondrial particles.

The rates of both forward and reverse electron transfer in phosphorylating submitochondrial particles from bovine heart can be controlled by the thermodynamic phosphorylation potential (deltaGp) of the adenine nucleotide system. deltaGp is the Gibbs free energy of ATP synthesis and is defined by the relationship deltaGp = -deltaG'o + RTln([ATP]/[ADP][Pi]) where deltaG'o is the standard free energy of ATP hydrolysis. Studies of the effects of deltaGp on NADH respiration and the reduction of NAD+ by succinate show that increasing values of deltaGp cause an inhibition of forward electron transfer and a stimulation of reverse electron transfer. Between deltaGp values of 7.6 and 13.0 kcal/mol the rate of NADH respiration decreased 3-fold and the rate of NAD+ reduction by succinate increased 3-fold. Indirect phosphorylation potential titration experiments as well as direct chemical measurements indicate that steady state levels of ATP, ADP, and Pi are established during NADH respiration which correspond to a deltaGp equal to 10.7 to 11.4 kcal/mol.     

The metabolic effects and uptake of ethidium bromide by rat liver mitochondria.

The uptake of ethidium bromide by rat liver mitochondria and its effect on mitochondria, submitochondrial particles, and F₁ were studied. Ethidium bromide inhibited the State 4-State 3 transition with glutamate or succinate as substrates. With glutamate, ethidium bromide did not affect State 4 respiration, but with succinate it induced maximal release of respiration. These effects appear to depend on the uptake and concentration of the dye within the mitochondrion. In submitochondrial particles, the aerobic oxidation of NADH is much more sensitive to ethidium bromide than that of succinate. Ethidium bromide partially inhibited the ATPase activity of submitochondrial particles and of a soluble F₁ preparation. Ethidium bromide behaves as a lipophilic cation which is concentrated through an energy-dependent process within the mitochondria, producing its effects at different levels of mitochondrial function. The ability of mitochondria to concentrate ethidium bromide may be involved in the selectivity of the dye as a mitochondrial mutagen.     

On the sidedness of the ubiquinone redox cycle. Kinetic studies in mitochondrial membranes

The inhibition of NADH oxidation but not of succinate oxidation by the low ubiquinone homologs UQ-2 and UQ-3 is not due to a lower rate of reduction of ubiquinone by NADH dehydrogenase: experiments in submitochondrial particles and in pentane-extracted mitochondria show that UQ-3 is reduced at similar rates using either NADH or succinate as substrates. The fact that reduced UQ-3 cannot be reoxidized when reduced by NADH but can be reoxidized when reduced by succinate may be explained by a compartmentation of ubiquinone.Using reduced ubiquinones as substrates of ubiquinol oxidase activity in intact mitochondria and in submitochondrial particles we found that ubiquinol-3 is oxidized at higher rates in submitochondrial particles than in mitochondria. The initial rates of ubiquinol oxidation increased with increasing lengths of isoprenoid side chains in mitochondria, but decreased in submitochondrial particles. These findings suggest that the site of oxidation of reduced ubiquinone is on the matrix side of the membrane; reduced ubiquinones may reach their oxidation site in mitochondria only crossing the lipid bilayer: the rate of diffusion of ubiquinol-3 is presumably lower than that of ubiquinol-7 due to the differences in hydrophobicity of the two quinones.     

Glucagon treatment stimulates the metabolism of hepatic submitochondrial particles

Hepatic submitochondrial particles, prepared at neutral pH from rats pretreated with glucagon, exhibited stimulated rates of State 3 and uncoupled respiration when succinate or NADH were the substrates, but not when ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine were employed. Measurements of 8-anilino-1-naphthalenesulfonic acid fluorescence in the particles indicated that glucagon treatment resulted in a stimulation of energization supported by succinate respiration or ATP hydrolysis. Similarly, the energy-linked pyridine nucleotide transhydrogenase and reverse electron flow reactions driven by succinate oxidation or ATP were also stimulated. The results indicate that mitochondrial substrate transport is not the prime locus of glucagon action. It is suggested that the increased level of energization in particles prepared from glucagon-treated rats is a reflection of a stimulation of the respiratory chain, possibly between cytochromes b and c, and the ATP-forming reactions.     

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

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

Platanetin and 7-iodo-acridone-4-carboxylic acid are not specific inhibitors of respiratory NAD(P)H dehydrogenases in potato tuber mitochondria

Progress in understanding the role of NAD(P)H oxidation in plant respiration is restricted by the lack of access to specific inhibitors of each of the unknown number of NAD(P)H dehydrogenases in the inner mitochondrial membrane. Platanetin (3,5,7,8-tetrahydroxy-6-isoprenyl flavone) is known to be an inhibitor of extermal NADH oxidation by plant mitochondria, while 7-iodo-acridone-4-carboxylic acid (IACA) is an inhibitor of an internal, rotenone-insensitive NAD(P)H dehydrogenase isolated from yeast mitochondria.
Here we show that platanetin inhibits external NAD(P)H oxidation by intact potato ( Solanum tuberosum L. cv. Bintje) tuber mitochondria, deamino-NADH oxidation by Complex I assayed using inside-out submitochondrial particles from these mitochondria, and rotenone-insensitive NAD(P)H oxidation by these submitochondrial particles. IACA was found to inhibit the oxidation of external NADH and succinate by intact mitochondria with similar efficiency. However, IACA also inhibited NADPH and duroquinol oxidation by intact mitochondria as well as deamino-NADH and NAD(P)H oxidation by inside-out submitochondrial particles. This indicates that IACA has several sites of inhibition in the electron transport chain. The lack of specificity of both platanetin and IACA prevents these inhibitors from being used to shed more light on the identity of the NAD(P)H dehydrogenases in plant mitochondria.     

Effects of ethacrynic acid and furosemide on phosphorylation reactions of kidney mitochondria. Inhibition of the adenine nucleotide translocase.

Previous reports that ethacrynic acid and furosemide diminish mitochondrial P : O ratios and reduce (Na+ + K+)-ATPase activity suggested that these diuretics may inhibit mitochondrial phosphorylation reactions. This possibility was initially studied by determining the effects of ethacrynic acid and furosemide on [32P]ATP exchange activity of rat kidney mitochondria. Concentrations of both drugs at 10(-4) M or greater, significantly inhibited [32P]ATP exchange. To investigate the mechanism of this inhibition, the effects of ethacrynic acid and furosemide on the ATPase activity of intract mitochondria and sonicated submitochondrial particles were determined. Both diuretics inhibited ATPase activity of intact mitochondria at 10(-4) M. In contrast, ATPase of submitochondrial particles was significantly less susceptible to inhibition by the diuretics. These results suggested that ethacrynic acid anf furosemide inhibit adenine nucleotide transport across the mitochondrial membrane. This was directly tested by determining the effects of the diretics on the mitochondrial adenine nucleotide translocase. At 5-10(-4) M, both ethacrynic acid and furosemide significantly inhibited adenine nucleotide transport. These findings suggest that ethacrynic acid and furosemide may diminish renal tubular solute reabsorption by direct inhibition of adenine nucleotide transport across the mitochondrial inner membrane.     

The hydrophobic cationic cyanine dye inhibits oxidative phosphorylation by inhibiting ADP transport, not by electrophoretic transfer, into mitochondria

The effect of the divalent cationic cyanine dye tri-S-C4(5) on oxidative phosphorylation in rat liver mitochondria was examined. The dye at about 100 n mols per mg mitochondrial protein inhibited state 3 respiration and ATP synthesis almost completely. However, it had no effect on submitochondrial particles, like other hydrophobic cations. The dye inhibited the transport of ADP into mitochondria mediated by the adenine nucleotide translocator. Thus, the inhibition of oxidative phosphorylation by the cationic dye was concluded to be due to its action on the adenine nucleotide translocator, not to its electrophoretic transfer into the inner space of mitochondria according to the inside-negative electrochemical potential.     

Effects of ethacrynic acid and furosemide on phosphory-lation reactions of kidney mitochondria. Inhibition of the adenine nucleotide translocase

Previous reports that ethacrynic acid and furosemide diminish mitochondrial P : O ratios and reduce (Na⁺ + K⁺)-ATPase activity suggested that these diuretics may inhibit mitochondrial phosphorylation reactions. This possibility was initially studied by determining the effects of ethacrynic acid and furosemide on [³²P]ATP exchange activity of rat kidney mitochondria. Concentrations of both drugs at 10⁻⁴ M or greater, significantly inhibited [³²P]ATP exchange. To investigate the mechanism of this inhibition, the effects of ethacrynic acid and furosemide on the ATPase activity of intact mitochondria and sonicated submitochondrial particles were determined. Both diuretics inhibited ATPase activity of intact mitochondria at 10⁻⁴ M. In contrast, ATPase of submitochondrial particles was significantly less susceptible to inhibition by the diuretics. These results suggested that ethacrynic acid and furosemide inhibit adenine nucleotide transport across the mitochondrial membrane. This was directly tested by determining the effects of the diuretics on the mitochondrial adenine nucleotide translocase. At 5 · 10⁻⁴ M, both ethacrynic acid and furosemide significantly inhibited adenine nucleotide transport. These findings suggest that ethacrynic acid and furosemide may diminish renal tubular solute reabsorption by direct inhibition of adenine nucleotide transport across the mitochondrial inner membrane.     

Effects of phthalate esters on the respiration of rat liver mitochondria.

The effects of phthalate esters on the oxidation of succinate, glutamate, beta-hydroxybutyrate and NADH by rat liver mitochondria were examined and it was found that di-n-butyl phthalate (DBP) strongly inhibited the succinate oxidation by intact and sonicated rat mitochondria, but did not inhibit the State 4 respiration with NAD-linked substrates such as glutamate and beta-hydroxybutyrate. However, oxygen uptake accelerated by the presence of ADP and substrate (State 3) was inhibited and the rate of oxygen uptake decreased to that without ADP (State 4). It was concluded that phthalate esters were electron and energy transport inhibitors but not uncouplers. Phthalate esters also inhibited NADH oxidation by sonicated mitochondria. The degree of inhibition depended on the carbon number of alkyl groups of phthalate esters, and DBP was the most potent inhibitor of respiration. The activity of purified beef liver glutamate dehydrogenase [EC 1.4.1.3] was slightly inhibited by phthalate esters.     

Effects of Optically Active 1-(α-Methylbenzyl)-3-(3,4-dichlorophenyl)urea on Reactions of Mitochondria and Chloroplasts

Effects of the R- and S-isomers and racemate of 1-(alpha-methylbenzyl)-3-(3,4-dichlorophenyl)urea (MBPU) were measured on phosphorylation and electron transport in mung bean (Phaseolus aureus L.) mitochondria and spinach (Spinacia oleracea L.) chloroplasts.In chloroplasts, S-MBPU inhibited basal and methylamine-uncoupled electron transport with ferricyanide as the oxidant, both photoreduction and coupled photophosphorylation with water as the electron donor and with ferricyanide and nicotinamide adenine dinucleotide phosphate (NADP) as oxidants, and cyclic photophosphorylation with phenazine methosulfate as the electron mediator under an argon gas phase. With ascorbate 2,6-dichloro-phenolindophenol as the electron donor, phosphorylation coupled to NADP reduction was inhibited, but the reduction of NADP was not inhibited. The R-isomer of MBPU, like the S-isomer, inhibited all of the photophosphorylation reactions studied. However, unlike the S-isomer, the R-isomer either did not inhibit or was a very weak inhibitor of all photoreduction reactions. The effects of the MBPUs on the chloroplast reactions can be explained by action at two different sites: an optically specific site near photosystem II and the oxygen evolution pathway, and a second optically nonspecific site associated with the generation of ATP.In mitochondria, both the R- and S-isomers stimulated state 4 respiration, inhibited state 3 respiration, and released oligomycin-inhibited respiration with malate, succinate, and NADH as substrates. Both enantiomers were equally active in all studies with malate and succinate as substrates. However, with NADH as substrate, R-MBPU was a stronger inhibitor of state 3 respiration and a weaker stimulator of state 4 respiration than S-MBPU.     

Cytokinin modification of mitochondrial function

6-Benzylaminopurine, 6-(Δ²-isopentenylamino)purine, 6-furfurylaminopurine, rotenone, and antimycin A inhibited oxidation of NADH by mitochondrial sonicates or submitochondrial particles (but not by intact mitochondria) from pea (Pisum sativum L., cult. Alaska) stems and mung bean (Vigna radiata L. Wilczak) hypocotyls. The above purine cytokinins can interfere with electron transport from NADH to the cytochrome system in the inner mitochondrial membrane. Adenine did not inhibit oxidation by sonicated mitochondria, and zeatin was almost ineffective. Zeatin scarcely inhibited state 3 malate respiration by intact mitochondria, but the O-formyl and O-n-propionyl esters of zeatin and the O-acetyl ester of 2-chlorozeatin were more active. Perhaps zeatin is ineffective because it does not get into the inner membranes of the isolated mitochondria, whereas the esters and other cytokinins mentioned above do. N-4-(2-chloropyridyl)-N′-Phenylurea, which has cytokinin-like effects on plant growth and development, inhibited NADH oxidation by sonicated mitochondria. It also inhibited malate, succinate, and NADH oxidation by intact mitochondria; in contrast, the latter two oxidations were not decreased by purine cytokinins.     

Preparation and some properties of submitochondrial particles from tightly coupled mung bean mitochondria

Osmotic shock was found to be better than freezing and thawing, a French press, or sonic oscillation for the preparation of submitochondrial particles from mung bean (Phaseolus aureus) hypocotyl mitochondria. Particles prepared by osmotic shock rapidly oxidize reduced nicotinamide adenine dinucleotide and succinate, but they oxidize malate slowly. NADH oxidation was slightly stimulated by cytochrome c, ATP, and ADP; succinate oxidation was markedly increased by ATP, slightly by ADP and cytochrome c; and malate oxidation required the addition of NAD⁺ NADH oxidation is inhibited weakly by amytal, completely by antimycin A and KCN, but not by rotenone. Chlorsuccinate, malonate, antimycin A, and KCN inhibit succinate oxidation. The action of antimycin A and KCN is incomplete, while chlorsuccinate and malonate were competitive inhibitors. Antimycin A combined stoichiometrically with particle protein in the ratio of 0.23 millimicromole per milligram of protein.     

Studies on the electron transfer pathway, topography of iron-sulfur centers, and site of coupling in NADH-Q oxidoreductase

Electron transfer activities and steady state reduction levels of Fe-S centers of NADH-Q oxidoreductase were measured in mitochondria, submitochondrial particles (ETPH), and complex I after treatment with various reagents. p-Chloromercuribenzenesulfonate destroyed the signal from center N-4 (gx = 1.88) in ETPH but not in mitochondria, showing that N-4 is accessible only from the matrix side of the inner membrane. N-Bromosuccinimide also destroyed the signal from N-4 but without inhibiting rotenone-sensitive electron transfer to quinone, suggesting a branched pathway for electron transfer. Diethylpyrocarbonate caused oxidation of N-3 and N-4 in the steady state without changing N-1, suggesting N-1 is before N-3 and N-4. Difluorodinitrobenzene and dicyclohexylcarbodiimide inhibited oxidation of all Fe-S centers and tetranitromethane inhibited reduction of all Fe-S centers. Titrations of the rate of superoxide (O2-) generation in rotenone-treated submitochondrial particles were similar with the ratio [NADH]/[NAD] and that of 3-acetyl pyridine adenine nucleotide in spite of different midpoint potentials of the two couples. On reaction with inhibitors the inhibition of O2- formation was similar to that of ferricyanide reductase rather than quinone reductase. The rate of O2- formation during ATP-driven reverse electron transfer was 16% of the rate observed with NADH. The presence of NAD increased the rate to 83%. The results suggest that bound, reduced nucleotide, probably E-NAD., is the main source of O2- in NADH dehydrogenase. The effect of ATP on the reduction levels of Fe-S centers in well-coupled ETPH was measured by equilibrating with either NADH/NAD or succinate/fumarate redox couples. With NADH/NAD none of the Fe-S centers showed ATP induced changes, but with succinate/fumarate all centers showed ATP-driven reduction with or without NAD present. The effect on N-2 was smaller than that on N-1, N-3, and N-4. These observations indicate that the major coupling interaction is between N-2 and the low potential centers, N-1, N-3, and N-4. Possible schemes of coupling in this segment are discussed.     

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.     

Protection of rat cardiac myocytes by fructose-1,6-bisphosphate and 2,3-butanedione

Earlier studies by our group showed that fructose-1,6-bisphosphate (FBP) enhances the hypothermic preservation of rat cardiac myocytes and the functional recovery of animal hearts after hypothermic storage. However, the mechanisms involved were not clear. We extended the cardiomyocyte studies by testing whether the FBP effects were due to chelation of extracellular calcium, leading to lower intracellular levels. We also tested effects of 2,3-butanedione monoxime (BDM), pyruvate, and adenine nucleotide precursors. Cardiomyocytes were incubated in ischemic suspension at 3 °C, and aliquots examined over 48 to 72 hours for retention of rod-shaped morphology, a measure of viability. Cytosolic Ca(2+) levels were measured in some experiments. FBP at 5 mM reduced the death rate even when added after one or two days of incubation. It caused cytosolic calcium levels that were 33% lower than controls in freshly-isolated cells and 70% lower after one day of incubation. EGTA protected against cell death similarly to FBP. These results indicated that one of the mechanisms by which FBP exerts protective effects is through chelation of extracellular calcium. BDM was strongly protective and reduced cytosolic calcium by 30% after one day of incubation. As with FBP, BDM was effective when added after one or two days of incubation. BDM may be useful in combination with FBP in preserving heart tissue. Pyruvate, adenine, and ribose provided little or no protection during hypothermia.     

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.