Adenine Nucleotide Exchange Research Articles

Adenylate kinase AK1 knockout heart: energetics and functional performance under ischemia-reperfusion.

Deletion of the major adenylate kinase AK1 isoform, which catalyzes adenine nucleotide exchange, disrupts cellular energetic economy and compromises metabolic signal transduction. However, the consequences of deleting the AK1 gene on cardiac energetic dynamics and performance in the setting of ischemia-reperfusion have not been determined. Here, at the onset of ischemia, AK1 knockout mice hearts displayed accelerated loss of contractile force compared with wild-type controls, indicating reduced tolerance to ischemic stress. On reperfusion, AK1 knockout hearts demonstrated reduced nucleotide salvage, resulting in lower ATP, GTP, ADP, and GDP levels and an altered metabolic steady state associated with diminished ATP-to-P(i) and creatine phosphate-to-P(i) ratios. Postischemic AK1 knockout hearts maintained approximately 40% of beta-phosphoryl turnover, suggesting increased phosphotransfer flux through remaining adenylate kinase isoforms. This was associated with sustained creatine kinase flux and elevated cellular glucose-6-phosphate levels as the cellular energetic system adapted to deletion of AK1. Such metabolic rearrangements, along with sustained ATP-to-ADP ratio and total ATP turnover rate, maintained postischemic contractile recovery of AK1 knockout hearts at wild-type levels. Thus deletion of the AK1 gene reveals that adenylate kinase phosphotransfer supports myocardial function on initiation of ischemic stress and safeguards intracellular nucleotide pools in postischemic recovery.

Conserved properties of hydrogenosomal and mitochondrial ADP/ATP carriers: a common origin for both organelles.

Mitochondria are one of the hallmarks of eukaryotic cells, exporting ATP in exchange for cytosolic ADP using ADP/ATP carriers (AAC) located in the inner mitochondrial membrane. In contrast, several evolutionarily important anaerobic eukaryotes lack mitochondria but contain hydrogenosomes, peculiar organelles of controversial ancestry that also supply ATP but, like some fermentative bacteria, make molecular hydrogen in the process. We have now identified genes from two species of the hydrogenosome-containing fungus Neocallimastix that have three-fold sequence repeats and signature motifs that, along with phylogenetic analysis, identify them as AACs. When expressed in a mitochondrial AAC- deficient yeast strain, the hydrogenosomal protein was correctly targeted to the yeast mitochondria inner membrane and yielded mitochondria able to perform ADP/ATP exchange. Characteristic inhibitors of mitochondrial AACs blocked adenine nucleotide exchange by the Neocallimastix protein. Thus, our data demonstrate that fungal hydrogenosomes and yeast mitochondria use the same pathway for ADP/ATP exchange. These experiments provide some of the strongest evidence yet that yeast mitochondria and Neocallimastix hydrogenosomes are but two manifestations of the same fundamental organelle.

Respiratory Chain-Generated Oxidative Stress Following Treatment of Leukemic Blasts with DNA-Damaging Agents

Oxidative stress occurs in diverse life forms during programmed cell death and appears to be a significant mediator since a wide range of manipulations that enhance cellular antioxidant systems are protective. Using a recently developed flow cytometry technique to assess respiratory chain function, we have investigated the mechanism of reactive oxygen generation in OCI/AML-2 leukemic blasts following treatment with cytosine arabinoside, etoposide, and γ-irradiation. Increases in mitochondrially generated reactive oxygen were seen using all three agents, in association with hyperpolarization of the mitochondrial inner membrane. Increased reactive oxygen occurred when mitochondria were energized using substrates for either complex I or complex II, indicating that the likely source is complex III (cytochrome c reductase). These findings are consistent with impaired adenine nucleotide exchange across the mitochondrial membrane, recently proposed to be an important event during the early stages of apoptosis induction (M. G. Vander Heiden et al., 1999, Mol. Cell 3, 159–167). Elevations of the antioxidants glutathione and thioredoxin occurred in association with this oxidative stress, likely the result of feedback mechanisms based on redox-sensitive transcription factors. Since glutathione and thioredoxin can protect from drug-induced apoptosis, their upregulation in response to respiratory chain-generated reactive oxygen might represent a cellular adaptation to DNA damage that promotes cell survival.

Role of oxidative phosphorylation in Bax toxicity.

The Bcl-2-related protein Bax is toxic when expressed either in yeast or in mammalian cells. Although the mechanism of this toxicity is unknown, it appears to be similar in both cell types and dependent on the localization of Bax to the outer mitochondrial membrane. To investigate the role of mitochondrial respiration in Bax-mediated toxicity, a series of yeast mutant strains was created, each carrying a disruption in either a component of the mitochondrial electron transport chain, a component of the mitochondrial ATP synthesis machinery, or a protein involved in mitochondrial adenine nucleotide exchange. Bax toxicity was reduced in strains lacking the ability to perform oxidative phosphorylation. In contrast, a respiratory-competent strain that lacked the outer mitochondrial membrane Por1 protein showed increased sensitivity to Bax expression. Deficiencies in other mitochondrial proteins did not affect Bax toxicity as long as the ability to perform oxidative phosphorylation was maintained. Characterization of Bax-induced toxicity in wild-type yeast demonstrated a growth inhibition that preceded cell death. This growth inhibition was associated with a decreased ability to carry out oxidative phosphorylation following Bax induction. Furthermore, cells recovered following Bax-induced growth arrest were enriched for a petite phenotype and were no longer able to grow on a nonfermentable carbon source. These results suggest that Bax expression leads to an impairment of mitochondrial respiration, inducing toxicity in cells dependent on oxidative phosphorylation for survival. Furthermore, Bax toxicity is enhanced in yeast deficient in the ability to exchange metabolites across the outer mitochondrial membrane.

What keeps a resting T cell alive?

The controls of quiescent lymphocyte survival remainpoorly understood. Recently, adoptive transfer experiments have suggested that ongoing signal transductionthrough the T-cell receptor is required to keep both naiveand memory T cells alive. This signaling results in thepromotion of cellular survival in the absence of proliferation. One common feature of the signal transductionpathways that promote survival is the ability to stimulatecellular metabolism. It appears that there is a minimal rateof cellular metabolism to maintain intracellular homeostasis of organelles such as mitochondria and the endoplasmic reticulum. In the absence of external stimulation,cells are unable to maintain a sufficient metabolic rate tomaintain organelle homeostasis. The loss of survival factor-induced glycolysis results in progressive changes inmitochondria that culminate in cytochrome c release. Release of cytochrome c induces the activation of caspasesand results in apoptosis. The Bcl-2-related protein, BclxL, allows cells to survive at lower rates of cellularmetabolism by promoting mitochondrial adaptationthrough the ability to facilitate adenine nucleotide exchange between the mitochondrial matrix and the cytosol.These data suggest that one mechanism by which Bcl-2-related proteins maintain cell survival is by promotingadaptation of mitochondria to changing bioenergetic requirements of the cell. The ability of Bcl-2 proteins topromote cell survival in the absence of extrinsic survivalsignals may explain why Bcl-2 is critical for the survivalof naive T cells and may account for why overexpressionof either Bcl-2 or Bcl-xL leads to an expansion in thenumber of both naive and memory cells...

BDM drives protein dephosphorylation and inhibits adenine nucleotide exchange in cardiomyocytes.

Contractile dysfunction plays a key role in injury sustained by ischemic myocardium at reperfusion, whereas interventions that impede hypercontracture enhance recovery. In permeabilized adult rat cardiomyocytes, the negative inotrope 2,3-butanedione monoxime (BDM; 10-50 mM) inhibited rigor at low MgATP concentration but stimulated net ATP hydrolysis. Hydrolysis was attenuated by H-7, kaempferol, chelerythrine, and genistein. Evidently BDM opposed phosphorylation of both serine/threonine and tyrosine kinase target proteins, either directly or by enhancing protein phosphatase activity, in a futile cycle of ATP hydrolysis independent of cross-bridge cycling. Although 20 mM BDM did not affect the onset of rigor contracture in permeabilized cells at low MgATP, in intact cells exposed to the metabolic inhibitors cyanide and 2-deoxyglucose rigor onset was accelerated, indicating that BDM increases ATP depletion in quiescent cardiomyocytes. Conversely, in cells exposed to the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone, BDM delayed the onset of contracture and hence ATP depletion, consistent with an inhibition of adenine nucleotide movement across the mitochondrial inner membrane. Such effects will limit the value of BDM as a cardioprotective agent at physiological temperature.

Structure determination and characterization of Saccharomyces cerevisiae profilin.

The structure of profilin from the budding yeast Saccharomyces cerevisiae has been determined by X-ray crystallography at 2.3 A resolution. The overall fold of yeast profilin is similar to the fold observed for other profilin structures. The interactions of yeast and human platelet profilins with rabbit skeletal muscle actin were characterized by titration microcalorimetry, fluorescence titrations, and nucleotide exchange kinetics. The affinity of yeast profilin for rabbit actin (2.9 microM) is approximately 30-fold weaker than the affinity of human platelet profilin for rabbit actin (0.1 microM), and the relative contributions of entropic and enthalpic terms to the overall free energy of binding are different for the two profilins. The titration of pyrene-labeled rabbit skeletal actin with human profilin yielded a Kd of 2.8 microM, similar to the Kd of 2.0 microM for the interaction between yeast profilin and pyrene-labeled yeast actin. The binding data are discussed in the context of the known crystal structures of profilin and actin, and the residues present at the actin-profilin interface. The affinity of yeast profilin for poly-L-proline was determined from fluorescence measurements and is similar to the reported affinity of Acanthamoeba profilin for poly-L-proline. Yeast profilin was shown to catalyze adenine nucleotide exchange from yeast actin almost 2 orders of magnitude less efficiently than human profilin and rabbit skeletal muscle actin. The in vivo and in vitro properties of yeast profilin mutants with altered poly-L-proline and actin binding sites are discussed in the context of the crystal structure.

Minimal model of beta-cell mitochondrial Ca2+ handling.

We develop a simplified, but useful, mathematical model to describe Ca2+ handling by mitochondria in the pancreatic beta-cell. The model includes the following six transport mechanisms in the inner mitochondrial membrane: proton pumping via respiration and proton uptake by way of the F1Fzero-ATPase (adapted from D. Pietrobon and S. Caplan. Biochemistry 24: 5764-5778, 1985), a proton leak, adenine nucleotide exchange, the Ca2+ uniporter, and Na+/Ca2+ exchange. Each mechanism is developed separately into a kinetic model for the rate of transport, with parameters taken from experiments on isolated mitochondrial preparations. These mechanisms are combined in a modular fashion first to describe state 4 (nonphosphorylating) and state 3 (phosphorylating) mitochondria with mitochondrial NADH and Ca2+ concentrations as fixed parameters and then to describe Ca2+ handling with variable mitochondrial Ca2+ concentration. Simulations are compared to experimental measurements and agree well with the threshold for Ca2+ uptake, measured mitochondrial Ca2+ levels, and the influence of Ca2+ on oxygen uptake. In the absence of Ca2+ activation of mitochondrial dehydrogenases, the simulations predict a significant reduction in the rate of production of ATP that involves a "short circuit" via Ca2+ uptake through the uniporter. This effect suggests a potential role for mitochondrial Ca2+ handling in determining the ATP-ADP ratio in the pancreatic beta-cell.

Crystal Structure of the Nucleotide Exchange Factor GrpE Bound to the ATPase Domain of the Molecular Chaperone DnaK

The crystal structure of the adenine nucleotide exchange factor GrpE in complex with the adenosine triphosphatase (ATPase) domain of Escherichia coli DnaK [heat shock protein 70 (Hsp70)] was determined at 2.8 angstrom resolution. A dimer of GrpE binds asymmetrically to a single molecule of DnaK. The structure of the nucleotide-free ATPase domain in complex with GrpE resembles closely that of the nucleotide-bound mammalian Hsp70 homolog, except for an outward rotation of one of the subdomains of the protein. This conformational change is not consistent with tight nucleotide binding. Two long alpha helices extend away from the GrpE dimer and suggest a role for GrpE in peptide release from DnaK.

Purification and Characterization of a 66-kDa Protein from Rabbit Reticulocyte Lysate Which Promotes the Recycling of Hsp 70

We have purified to apparent homogeneity a 66-kDa protein from rabbit reticulocyte lysate which is associated with hsp 70. Our characterization of this 66-kDa protein demonstrates that its physiological role is to promote the recycling of hsp 70 by catalyzing the dissociation of hsp 70-bound ADP in exchange for ATP. We have therefore termed the 66-kDa protein RF-hsp 70, a recycling factor for hsp 70. RF-hsp 70 promotes stoichiometric binding of ATP to hsp 70, and it increases about 5-fold the rate of dissociation of hsp 70.ADP in the presence of ATP. This process represents adenine nucleotide exchange, since dissociation of ADP does not occur unless ATP is added; dATP, GTP, and ITP cannot substitute for ATP. The mechanism of action of RF-hsp 70 is to lower the KD of hsp 70 for ATP about 6-7-fold to a value that is close to the KDof hsp 70 for ADP. RF-hsp 70 also stimulates the ATPase activity of hsp 70, including the 42-kDa amino-terminal portion of hsp 70 generated by chymotrypsin, demonstrating that RF-hsp 70 interacts with that part of hsp 70 known to contain the ATP/ADP binding site. Confirming its recycling function, RF-hsp 70 stimulates about 7-10-fold the ability of hsp 70 to reactivate heat-denatured firefly luciferase. In addition, RF-hsp 70 acts catalytically to recycle hsp 70, since, at 0.2 times the molar concentration of hsp 70, RF-hsp 70 increases the rate of renaturation of luciferase by hsp 70 about 3-4-fold. The action of RF-hsp 70 is also partially species-specific since it is most effective with rabbit reticulocyte hsp 70, less effective with bovine brain hsp 70, even less effective with human hsp 70, and ineffective with broad bean hsp 70.

Nucleotide Sequence of a cDNA Encoding the ADP/ATP Carrier from Wheat (Triticum turgidum)

The adenine nucleotide carrier is the most abundant mitochondrial membrane protein. It catalyzes the exchange of adenine nucleotides between mitochondrial matrix and cytosol and plays a central role in the integration of mitochondrial metabolism. We have characterized an ADP/ ATP carrier cDNA from wheat (Triticum turgidum) with a view toward gaining an understanding of the expression and biogenesis of this protein in higher plants. The cDNA sequence has been obtained from a wheat single-stranded cDNA by PCR utilizing sequence information from the Zea mays homolog (Bathgate et al., 1989). Overlapping cDNA fragments were obtained. To extend the sequence to the 5’ end a modified cDNA tailed with a poly(A) was used. The cDNA of the ADP/ATP carrier is 1035 bp in length. The open reading frame encodes the mature protein consisting of 331 amino acids (Table I). The amino acid sequence of the wheat ADP/ATP carrier is 90.3 and 79.6% identical to that of the ADP/ATP carrier from maize (Bathgate et al., 1989) and rice (Hashimoto et al., 1993). Most of the differences are located at the N-terminal region. Since the function of this region is not known, the significance of these differences is not understood.

Coordinated inhibition of actin-induced platelet aggregation by plasma gelsolin and vitamin D-binding protein

Actin is an abundant intracellular protein that is released into the blood during tissue injury and its injection into rats causes microthrombi to form in the vasculature. This report and others have shown that actin filaments are able to aggregate platelets in an adenosine diphosphate (ADP)-dependent manner. The effects on this process of two plasma actin-binding proteins, vitamin D-binding protein (DBP) and gelsolin, were examined separately and together. The addition of DBP, a monomer-binding protein, to actin filaments did not affect their ability to induce platelet aggregation. However, severing of actin filaments with gelsolin resulted in an increased degree of platelet aggregation. Preincubation of F-actin with both gelsolin and DBP resulted in a significant inhibition of aggregation. The effects of DBP and gelsolin on actin-induced aggregation paralleled their effects on exchange of actin-bound adenine nucleotides. DBP inhibited 1, N6- ethenoadenosine 5′ triphosphate (epsilon-ATP) exchange with G-actin but not with F-actin. Gelsolin increased epsilon-ATP exchange with F-actin, which was largely abrogated by the addition of DBP. These results suggest that gelsolin's severing (and subsequent capping) of actin filaments not only results in an increase in the number of pointed filament ends but also in the dissociation of actin monomers containing ADP. Phalloidin, which stabilizes actin filaments while decreasing both monomer and nucleotide exchange, inhibited actin-induced aggregation, as well, indicating that depolymerization of actin filaments is not required to inhibit aggregation. Platelet activation by either G- or F- actin may thus be regulated by the local concentrations of the plasma actin-binding proteins gelsolin and DBP. Together, these proteins inhibit platelet aggregation in a manner that can be explained by their effects on actin's filament structure and the accessibility of its bound ADP. Depletion of DBP or gelsolin may allow actin released from injured tissues to stimulate purinergic receptors on platelets, and perhaps other cells, via its bound adenine nucleotides.

Coordinated Inhibition of Actin-Induced Platelet Aggregation by Plasma Gelsolin and Vitamin D-Binding Protein

AbstractActin is an abundant intracellular protein that is released into the blood during tissue injury and its injection into rats causes microthrombi to form in the vasculature. This report and others have shown that actin filaments are able to aggregate platelets in an adenosine diphosphate (ADP)- dependent manner. The effects on this process of two plasma actin-binding proteins, vitamin D-binding protein (DBP) and gelsolin, were examined separately and together. The addition of DBP, a monomer-binding protein, to actin filaments did not affect their ability to induce platelet aggregation. However, severing of actin filaments with gelsolin resulted in an increased degree of platelet aggregation. Preincubation of F-actin with both gelsolin and DBP resulted in a significant inhibition of aggregation. The effects of DBP and gelsolin on actin-induced aggregation paralleled their effects on exchange of actin-bound adenine nucleotides. DBP inhibited 1, N6-ethenoadenosine 5′ triphosphate (ϵ-ATP) exchange with G-actin but not with F-actin. Gelsolin increased ϵ-ATP exchange with F-actin, which was largely abrogated by the addition of DBP. These results suggest that gelsolin’s severing (and subsequent capping) of actin filaments not only results in an increase in the number of pointed filament ends but also in the dissociation of actin monomers containing ADP. Phalloidin, which stabilizes actin filaments while decreasing both monomer and nucleotide exchange, inhibited actin-induced aggregation, as well, indicating that depolymerization of actin filaments is not required to inhibit aggregation. Platelet activation by either G- or F-actin may thus be regulated by the local concentrations of the plasma actin-binding proteins gelsolin and DBP. Together, these proteins inhibit platelet aggregation in a manner that can be explained by their effects on actin’s filament structure and the accessibility of its bound ADP. Depletion of DBP or gelsolin may allow actin released from injured tissues to stimulate purinergic receptors on platelets, and perhaps other cells, via its bound adenine nucleotides.

Site-directed Mutagenesis of the Yeast Mitochondrial ADP/ATP Translocator: Six Arginines and One Lysine are Essential

The ADP/ATP translocator mediates adenine nucleotide exchange across the inner mitochondrial membrane. ADP/ATP exchange is essential when yeast are grown on a non-fermentable carbon source such as glycerol, but it is not required for growth on glucose. Failure to grow on glycerol is therefore a phenotypic indicator of protein function, and it has been used here to screen site-directed mutants to identify functionally important amino acids in the yeast adenine nucleotide translocator (AAC2). Single mutations of all four charged amino acids in the transmembrane segments of AAC2 (K38A, R96D, R96H, R96L, R96P, R204L, R294A) resulted in loss of function, as did mutations in the matrix arginine cluster (R252I, R253I, R254I). Seven other residues were mutated without affecting growth on glycerol (C73S, C244S, C271S, K179M, K182I, P247G, W235F). The non-functional mutants have been used to select intragenic suppressors to gain further insight into the structure of this membrane transport protein.

Transport of inorganic pyrophosphate across the spinach chloroplast envelope

Spinach-leaf chloroplasts take up PPi at a rate of 1.9 mumol/h per mg of chlorophyll (Chl) in the dark and 1.6 mumol/h per mg of Chl in the light. The Km for PPi transport is 32 microM in the dark and 6 microM in the light. Uptake is inhibited by pyridoxal phosphate, 4,4'-di-isothiocyanatostilbene-2,2'-disulphonic acid and imidodiphosphate, but not by NaF or EDTA. PPi does not appear to cross the chloroplast envelope in exchange for Pi, suggesting that it is not transported by the phosphate translocator. Exchange of PPi and adenine nucleotides across the chloroplast envelope is very slow and PPi does not competitively inhibit ATP uptake, suggesting that little, if any, PPi is transported by the adenine-nucleotide translocator. These results are consistent with the presence of a specific, high-affinity PPi translocator in the spinach chloroplast envelope. It is proposed that in vivo PPi is taken up into the chloroplast from the cytosol to replenish the Pi pool in the stroma.

Protective effect of the plasticizer di(2-ethylhexyl) phthalate against damage of the mitochondrial membrane induced by calcium: possible participation of the adenine nucleotide translocator

The effect of di(2-ethylhexyl) phthalate (DEHP) on the response of isolated rat liver mitochondria to Ca 2+ was investigated. DEHP was found to inhibit more than 60% of the auto-accelerating release of respiration induced by 100 μM Ca 2+, being maximally inhibitory at 40 μM. Prior addition of DEHP also partially inhibited Ca 2+-induced swelling of the mitochondrial matrix. However, DEHP did not change the net rate of Ca 2+ uptake measured by the steady-state infusion method. DEHP also reduced the rate of adenine nucleotide exchange across the mitochondrial membrane. Another alkyl phthalate and alkyl citrates had similar effects on Ca 2+-induced membrane damage, but their potencies depended on the lengths of their alkyl chains. These results suggest that the effects of DEHP and other alkyl esters on mitochondrial functions are mainly based on their actions on membrane lipids surrounding adenine nucleotide translocator (AdNT), resulting in alteration of the interaction between these phospholipids and AdNT, and consequent modification of the state of the protein.

Growth defects in intramitochondrial energy depleted cells: Role of mitochondrial biogenesis

Simultaneous inhibition of oxidative phosphorylation by rho − mutation and adenine nucleotide exchange by op 1 mutation or bongkrekic acid results in intramitochondrial energy depletion and cessation of growth in yeast. Effect of energy depletion of mitochondria on mitochondrial biogenesis was studied in intact yeast cells. Immunoblot analysis revealed an overall decrease in cellular content of two mitochondrial proteins — ADP ATP translocase and β subunit of mitochondrial ATPase — together with their lower ability to reach the proper intramitochondrial compartment. Both effects indicate disturbed biogenesis of energy depleted mitochondria. Quantitative differences in growth abilities and mitochondrial damage observed in two studied systems — op 1rho − double mutants and rho − cells treated with bongkrekic acid — can be explained by different degree of intramitochondrial energy depletion due to leakiness of op 1 mutation in op 1rho − cells.

The stereochemical course of phospho group transfer catalyzed by rat liver 6-phosphofructo-2-kinase.

Adenosine [gamma-(S)-16O,17O,18O]triphosphate was used as substrate in the phosphokinase reaction catalyzed by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. The product D-fructose 2,6-[2-16O,17O,18O]bisphosphate was then used as substrate in the alkaline phosphatase-mediated transfer of the phospho groups to (S)-butane-1,3-diol, where the configuration at phosphorus has been determined. Although there was approximately equal transfer by alkaline phosphatase of the labeled 2- and the unlabeled 6-phospho groups, the subsequent assignment of configuration of the chiral phospho group from the 2-position was unambiguous. It was found that 6-phosphofructo-2-kinase proceeds by a pathway that results in net inversion of the configuration at phosphorus. The simplest interpretation of this result is that the phospho group is transferred directly between substrates in a ternary complex by an "in-line" mechanism not involving a phosphoenzyme intermediate. This conclusion is consistent with the fact that the enzyme cannot be labeled by [gamma-32P]ATP and with steady-state kinetic data that suggest an ordered sequential mechanism. This finding also indicates that the adenine nucleotide and sugar phosphate isotope exchange reactions catalyzed by the enzyme are not relevant to the normal catalytic pathway of the kinase.

Effect of the plasticizer di-(2-ethylhexyl)phthalate on oxidative phosphorylation in rat liver mitochondria: modification of the function of the adenine nucleotide translocator.

The effect of di-(2-ethylhexyl)phthalate (DEHP) on oxidative phosphorylation of isolated rat liver mitochondria was investigated. DEHP at concentrations of 20-1000 microM had no effect on state 4 respiration, but at 40 microM, DEHP decreased the rate of state 3 respiration by about 20%. Although DEHP had no effect on electron transport through the respiratory chain, it decreased the rate of adenosine triphosphate (ATP) synthesis. Its inhibition of ATP synthesis showed a similar concentration dependence to that of state 3 respiration. Furthermore, DEHP at 40 microM inhibited the uptake of [3H]adenosine diphosphate into mitochondria. DEHP also retarded the action of cationic uncoupling agents, which are known to modify the 29000-dalton protein involved in adenine nucleotide exchange. These results suggest that DEHP affects the activity of adenine nucleotide exchange and consequently partially decreases the rate of state 3 respiration. The action of DEHP on the 29000-dalton protein involves a protective effect against mitochondrial damage induced by hydrophobic cations or heavy metal cations.

Decreased exchange of adenine nucleotides in human placental mitochondria

1. 1. The purpose of this work was to study the exchange of adenine nucleotides in mitochondria isolated from human placenta tissue. 2. 2. The results indicate that ADP and ATP are translocated at a lower rate than those reported for rat liver mitochondria. 3. 3. It is proposed that the limited transport is due to the particular lipid composition of placental mitochondria membrane, which induces an arrest in membrane fluidity with the consequent restriction in adenine nucleotide translocase mobility.