Changes of Adenine Nucleotide in the Course of Mitochondrial Development of Mung Bean Cotyledon and Their Effect on Cellular Energy Status

The changes of adenine nucleotide and adenylate energy charge (AEC) during the development of mitochondria in imbibed mung bean cotyledons and the relationship between these changes and cellular energy status are studied. After cotyledons were imbibed in water for two hours, mitochondrial cristae were not observed, but for 12 hours, they appeared obviously on the inner membrane. With the structural integrity of the mitochondria, the functional mitochondria were graduately shown. For instance, the activity of H+-ATPase of cotyledons imbibed for 24 hours was about twice higher than that of 2 hours. The ATP content and the AEC value in the cotyledons imbibed for 24 hours increased sharply and the AMP decreased, but these were not observed in the mitochondria of the cotyledons imbibed either for 24 hours or 2 hours. When the cotyledons were imbibed in 1 × 10-4 mol/l or 5 × 10-4 mol/l DNP solution for 24 hours, the ATP and the AEC in the Cells exhibited a rapid decrease, but in the mitochondria they remained canstant. In the same DNP solution with cotyledons for 24 hours, the activity of mitochondrial adenylate kinase (AK) not only was not decreased but also increased by about 50% over the control. This result shows that the energy equilibration in the mitochondria seems likely to be regulated by adenylate kinase locating between inner and out membranes of the mitochondria.     

Studies on the nature of adenosine diphosphatase activity from rat liver mitochondria

Adenosine diphosphatase (ADPase) activity was studied in rat liver with [beta-32P]ADP as a substrate. Mitochondria and outer mitochondrial membrane fractions were isolated and assayed for ADPase and various marker enzymes. ADPase activity was strikingly reduced when the outer membranes were removed from the mitochondria whether by digitonin treatment or osmotic shock. Addition of the inter-membrane space subfraction to the purified outer membranes resulted in enhanced ADPase activity. Addition of the inter-mitochondrial membrane enzyme adenylate kinase to outer membranes also produced a large stimulation of activity. The ADPase activity could also be reconstituted in vitro with adenylate kinase and either mitoplast ATPase or ouabain-sensitive (Na+ + K+ + Mg2+)-ATPase. Chloroform-released ATPase, however, was not capable of producing an ADPase activity when combined with adenylate kinase. Gel permeation chromatography of Triton-solubilised outer mitochondrial membranes was unable to resolve ADPase activity from contaminating ATPase. These results suggest that the majority of ADPase activity in rat liver mitochondria consists of the coupled activity of adenylate kinase and ATPase.     

Participation of epsilonADP and epsilonATp in the reactions of oxidative phosphorylation in rat liver mitochondria

The 1,N6-ethenoadenine nucleotide analogs epsilonADP and epsilonATP, contrary to recent findings (1), are shown to be unable to penetrate the inner mitochondrial membrane of intact rat liver mitochondria and can not be used as substrates by the respiratory chain enzymes in oxidative phosphorylation. On the other hand, these analogs are able to participate in transphosphorylation reactions, being good substrates for mitochondrial phosphotransferases located in the intermembrane space, such as nucleosidediphosphate kinase and adenylate kinase.     

The effect of lactoperoxidase-catalyzed iodination on the integrity of mitochondrial membranes.

The effect of lactoperoxidase-catalyzed iodination on rat liver mitochondria was investigated. A change from the condensed to the swollen conformation is observed by electron microscopy after extensive iodination of the mitochondria. The outer membrane breaks after incorporation of 0.2 nmol or more iodine atoms per mg of mitochondrial protein releasing adenylate kinase, a soluble enzyme located in the intermembrane space. Further iodination of the mitochondria ruptures the inner membrane, releasing proteins such as glutamic dehydrogenase from the matrix space. Lipid peroxides and I₂ are not intermediates in the disruptive effect of extensive lactoperoxidase-catalyzed iodination on the membranes. During iodination at pH 6.5 almost no release of protein or glutamic dehydrogenase activity is detectable and the loss of adenylate kinase activity from the particulate is diminished. The effect of extensive iodination on mitochondrial membranes limits the amount of iodide which can be incorporated with the lactoperoxidase membrane-labeling procedure when this technique is used as a surface probe of mitochondrial membranes.     

The effect of gonadotropins on the mitochondrial adenylate cyclase of rat testis

The formation of adenosine 3′:5′-cyclic monophosphate from ATP by testicular mitochondria of immature and mature rats was increased to the same extent by addition of either human chorionic gonadotropin or luteinizing hormone. Follicle stimulating hormone was found to be more active in stimulating adenylate cyclase activity in testicular mitochondria of immature rats. The stimulatory effect of gonadotropins were not suppressed by Ca⁺⁺ complexing agent ethylene-glycol-bis-(β-amino-ethyl ether) N,N′-tetra-acetic acid. The detergent Lubrol PX, solubilized 75–80% of the mitochondrial adenylate cyclase. The solubilized enzyme was activated by sodium fluoride but not by gonadotropins. The present results indicate a direct effect of gonadotropins on the adenylate cyclase attached to mitochondrial membranes.     

In Rat Liver Mitochondria All Nucleoside Diphosphate Kinase of the Outer Compartment Is Associated with the Outer Surface of the Outer Membrane

Data on localization of nucleoside diphosphate kinase (NDPK) in the outer mitochondrial compartment are contradictory. We have demonstrated that repeated quintuple wash of a mitochondrial pellet (protein concentration is about 2 mg/ml) solubilized only 60% of total NDPK activity. Since no release of adenylate kinase, the marker enzyme of the intermembrane space, was observed, it was concluded that the solubilized NDPK activity was associated with the outer surface of the outer mitochondrial membrane. Treatment of mitochondria with digitonin solutions in low (sucrose, mannitol) or high (KCl) ionic strength media revealed that solubilization of remaining NDPK activity basically coincided with the solubilization curve of monoamine oxidase, the marker enzyme of the outer mitochondrial membrane, but differed from solubilization behavior of adenylate kinase and malate dehydrogenase. We concluded that the remaining NDPK activity was also associated with the outer mitochondrial membrane and electrostatic interactions were not essential for NDPK binding to mitochondrial membranes. Results of polarographic determination of remaining adenylate kinase and NDPK activities of mitochondria incubated in ice for different time intervals and subjected to subsequent centrifugation suggest that all NDPK activity of the outer compartment of rat liver mitochondria is associated with the outer surface of the outer mitochondrial membrane. We suggest the existence of at least three NDPK fractions. They represent 70, 15, and 15% of total NDPK activity of the outer compartment and differ by tightness of membrane binding.     

In vivo import of yeast adenylate kinase into mitochondria affected by site-directed mutagenesis.

Site-directed mutagenesis and deletions were used to study mitochondrial import of a major yeast adenylate kinase, Aky2p. This enzyme lacks a cleavable presequence and occurs in active and apparently unprocessed form both in mitochondria and cytoplasm. Mutations were applied to regions known to be surface-exposed and to diverge between short and long isoforms. In vertebrates, short adenylate kinase isozymes occur exclusively in the cytoplasm, whereas long versions of the enzyme have mitochondrial locations. Mutations in the extra loop of the yeast (long-form) enzyme did not affect mitochondrial import of the protein, whereas variants altered in the central, N- or C-terminal parts frequently displayed increased or, in the case of a deletion of the 8 N-terminal triplets, decreased import efficiencies. Although the N-terminus is important for targeting adenylate kinase to mitochondria, other parameters like internal sequence determinants and folding velocity of the nascent protein may also play a role.     

Submitochondrial distribution of cAMP during incubation with rat liver mitochondria

Labeled cAMP incubated with rat liver mitochondria penetrates not only through outer mitochondrial membranes, but also into mitoplasts, where it is accumulated mainly in the matrix. Damage of mitochondrial membranes caused by single freezing-thawing treatment promotes no influx, but efflux of cAMP from mitoplasts. cAMP molecules penetrate inside mitochondria largely in an unchanged state in all submitochondrial fractions, as was demonstrated by the TLC method. cAMP transport into mitochondria can serve as a reason for: 1) stimulation of mitochondrial function by hormones whose effects are realized through activation of cytoplasmic adenylate cyclase and by extramitochondrial (cytosolic) cAMP; 2) existence of cAMP-dependent protein kinase and cAMP-phosphodiesterase in mitochondria.     

Oligomeric C-terminal truncated Bax preferentially releases cytochrome c but not adenylate kinase from mitochondria, outer membrane vesicles and proteoliposomes

The mechanism by which the proapoptotic protein Bax releases cytochrome c from mitochondria is not fully understood. The present work approaches this problem using C-terminal truncated oligomeric Bax (BaxDeltaC). Micromolar concentrations of BaxDeltaC released cytochrome c from isolated rat heart and liver mitochondria, while the release of adenylate kinase was not significantly affected. BaxDeltaC also released cytochrome c but not adenylate kinase from outer membrane vesicles filled with these proteins. However, BaxDeltaC was ineffective in releasing cytochrome c when outer membrane vesicles were obtained in the presence of glycerol, conditions under which the number of contact sites was drastically reduced. BaxDeltaC did not liberate encapsulated cytochrome c and adenylate kinase from pure phospholipid vesicles or vesicles reconstituted with porin. However, when the hexokinase-porin-adenine nucleotide translocase complex from brain mitochondria was reconstituted in vesicles, BaxDeltaC released internal cytochrome c but not adenylate kinase. In all these systems, only a small portion of total cytochrome c present in either mitochondria or vesicles could be liberated by BaxDeltaC. BaxDeltaC also increased the accessibility of external cytochrome c to either oxidation by complex IV or reduction by complex III in intact liver and heart mitochondria. CONCLUSIONS: (1) BaxDeltaC selectively releases cytochrome c and enables a bidirectional movement of cytochrome c across the outer mitochondrial membrane. (2) A multiprotein complex that resembles the mitochondrial contact sites is a prerequisite for BaxDeltaC action. (3) A limited pool of cytochrome c becomes the first target for BaxDeltaC.     

ADP-Glucose Transport by the Chloroplast Adenylate Translocator Is Linked to Starch Biosynthesis

In organello starch biosynthesis was studied using intact chloroplasts isolated from spinach leaves (Spinacia oleracea). Immunoblot analysis using a specific antiserum against the mitochondrial adenylate (ADP/ATP) translocator of Neurospora crassa shows the presence of an adenylate translocator protein in the chloroplast envelope membranes, similar to that existing in mitochondria and amyloplasts from cultured cells of sycamore (Acer pseudoplatanus). The double silicone oil layer-filtering centrifugation technique was employed to study the kinetic properties of adenylate transport in the purified chloroplasts; ATP, ADP, AMP, and most importantly ADP-Glc were shown to be recognized by the adenylate translocator. Similar to the situation with sycamore amyloplasts, only ATP and ADP-Glc uptake was inhibited by carboxyatractyloside, an inhibitor of the mitochondrial adenylate translocator. Evidence is presented to show that the ADP-Glc transported into the chloroplast stroma is utilized for starch synthesis catalyzed by starch synthase (ADP-Glc:1,4-α-d-glucan 4-α-d-glucosyltransferase). The high activity of sucrose synthase producing ADP-Glc observed in the extrachloroplastic fractions suggests that starch biosynthesis in chloroplasts may be coupled with the direct import of ADP-Glc from the cytosol.     

Multiple disc gel bands of mitochondrial creatine and adenylate kinases

Abstract— The activities and electrophoretic patterns of creatine and adenylate kinases in the mitochondrial and high speed supernatant fractions of adult mouse brain were determined. Approximately 22 per cent of the activities of both kinases is firmly bound to the mitochondria. On acrylamide gel electrophoresis of creatine kinase, in addition to the major band previously described, there were several other bands found. Although present in both the mitochondrial and supernatant fractions these additional protein bands with creatine kinase activity were significantly more intense in the mitochondrial fraction. There was only onesecondary band of adenylate kinase activity in the mitochondrial fraction but additional bands were found in the soluble fraction.     

And all of a sudden it's over: mitochondrial outer-membrane permeabilization in apoptosis

Identification of pro-apoptotic activities for a variety of proteins normally resident in the mitochondrial inter-membrane space has substantiated the role of mitochondria as integral to the apoptotic process. Cytochrome c is involved in apoptosome formation and caspase activation, SMAC/Diablo deregulates the inhibitor of apoptosis proteins, apoptosis-inducing factor may play a role in chromatin condensation and release of other proteins such as adenylate kinase may adversely affect cellular metabolism and contribute to the death of a cell if the downstream apoptotic pathway is blocked. It is still unclear how these proteins are released from the mitochondria. Recent advances in our knowledge of mitochondrial outer-membrane permeabilization and the consequences of this event on mitochondria will be discussed.     

Measurement of mitochondrial oxidative phosphorylation: Selective inhibition of adenylate kinase activity by P1,P5-di-(adenosine-5′)-pentaphosphate

A biochemical assay for the measurement of ATP synthesis coupled to electron transport in the presence of adenylate kinase was developed as an alternative to using the conventional Clark-type oxygen electrode. The assay utilizes P¹,P⁵-di-(adenosine-5′)-pentaphosphate which is shown to be a competitive inhibitor with MgADP for rat liver mitochondrial adenylate kinase (K_i = 7.04 × 10^?8m) and was found to have no effect on oxidative phosphorylation of either intact mitochondria or submitochondrial particles.     

A matrix-assisted laser desorption ionization post-source decay (MALDI-PSD) analysis of proteins released from isolated liver mitochondria treated with recombinant truncated Bid

A crucial event in the process of apoptosis is caspase-dependent generation of truncated Bid (tBid), inducing release of cytochrome c. In an in vitro reconstitution system we combined purified recombinant tBid with isolated liver mitochondria and identified the released proteins using a proteomic matrix-assisted laser desorption ionization post-source decay (MALDI-PSD) approach. In order to meet physiological conditions, the concentration of tBid was chosen such that it was unable to induce cytochrome c release in mitochondria derived from liver-specific Bcl-2-transgenic mice. Several mitochondrial proteins were identified to be released in a tBid-dependent way, among which cytochrome c, DIABLO/Smac, adenylate kinase 2, acyl-CoA-binding protein, endonuclease G, polypyrimidine tract-binding protein, a type-I RNA helicase, a WD-40 repeat-containing protein and the serine protease Omi. Western blotting confirmed the absence of adenylate kinase 3, a matrix mitochondrial protein. These results demonstrate that a physiologically relevant concentration of tBid is sufficient to induce release of particular intermembrane mitochondrial proteins belonging to a broad molecular-mass range.     

Two pathways for tBID-induced cytochrome c release from rat brain mitochondria: BAK- versus BAX-dependence

The mechanisms of truncated BID (tBID)-induced Cyt c release from non-synaptosomal brain mitochondria were examined. Addition of tBID to mitochondria induced partial Cyt c release which was inhibited by anti-BAK antibodies, implicating BAK. Immunoblotting showed the presence of BAK, but not BAX, in brain mitochondria. tBID did not release Cyt c from rat liver mitochondria, which lacked both BAX and BAK. This indicated that tBID did not act independently of BAX and BAK. tBID plus monomeric BAX produced twice as much Cyt c release as did tBID or oligomeric BAX alone. Neither tBID alone nor in combination with BAX induced mitochondrial swelling. In both cases Cyt c release was insensitive to cyclosporin A plus ADP, inhibitors of the mitochondrial permeability transition (mPT). Recombinant Bcl-xL inhibited Cyt c release induced by tBID alone or in combination with monomeric BAX. Koenig's polyanion, an inhibitor of VDAC, suppressed tBID-induced Cyt c release from brain mitochondria mediated by BAK but not by BAX. Thus, tBID can induce mPT-independent Cyt c release from brain mitochondria by interacting with exogenous BAX and/or with endogenous BAK that may involve VDAC. In contrast, neither adenylate kinase nor Smac/DIABLO was released from isolated rat brain mitochondria via BAK or BAX.     

Distinct behaviors of adenylate kinase and cytochrome c observed following induction of mitochondrial permeability transition by Ca2+ in the absence of respiratory substrate

For induction of the mitochondrial permeability transition (PT) by Ca²⁺, the addition of a respiratory substrate such as succinate is required. However, earlier studies indicated the possible induction of the mitochondrial PT by Ca²⁺ in the absence of a respiratory substrate (Hunter, D.R., and Haworth, R.A. (1979) Arch. Biochem. Biophys. 195, 453–459). In the present study, we obtained clear evidence showing that the mitochondrial PT could be induced by Ca²⁺ even in the absence of respiratory substrate. We next examined the protein release from mitochondria that accompanied the induction of PT in the absence of a respiratory substrate. Interestingly, distinct from the ordinary mitochondrial PT induced by Ca²⁺ in the presence of a respiratory substrate, which is associated with the release of mitochondrial cytochome c and adenylate kinase, the mitochondrial PT occurring in the absence of a respiratory substrate was associated with release of mitochondrial adenylate kinase but not with that of mitochondrial cytochrome c. This experimental system should be quite useful for understanding the mechanisms of protein release from mitochondria.     

α-Synuclein overexpression impairs mitochondrial function by associating with adenylate translocator

α-Synuclein (α-syn), a protein involved in the pathogenesis of Parkinson's disease (PD), is known to accumulate in mitochondria, disrupt mitochondrial function. However, the molecular mechanisms that link these pathological responses have not been investigated. In rats overexpressing α-syn in the substantia nigra (SN) through adeno-associated virus (AAV) transduction, about 50% of tyrosine hydroxylase positive neurons were lost after 24 weeks. Overexpression of α-syn was also associated with morphological deformation of mitochondria and depolarization of the mitochondrial membrane potential (ΔΨm). Both co-immunoprecipitation and confocal microscopy demonstrated that mitochondrial α-syn associated with adenylate translocator (ANT), a component of the mitochondrial permeability transition pore (mPTP). The depolarization of ΔΨm was partially reversed in vitro by bongkrekic acid (BKA), an inhibitor of ANT, suggesting that the molecular association between α-syn and ANT facilitated ΔΨm depolarization. Concomitant with α-syn accumulation in mitochondria, abnormal mitochondrial morphology, ΔΨm depolarization, and loss of TH-positive neurons, there was a decrease in apoptosis-inducing factor (AIF) within the mitochondrial matrix, suggesting possible translocation to the cytosol. Our findings suggest that overexpression of α-syn may cause mitochondrial defects in dopaminergic neurons of the substantia nigra through an association with adenylate translocator and activation of mitochondria-dependent cell death pathways. Disruption of normal mitochondrial function may contribute to the loss of dopaminergic neurons in Parkinson's disease.     

Association of ATP citrate lyase with mitochondria

(1) The association of ATP citrate lyase with mitochondria was studied with isolated rat hepatocytes and mitochondria. (2) When hepatocytes were treated with digitonin, about 25% of the lyase activity was released like a mitochondrial enzyme. (3) The effect of temperature on release of lyase from hepatocytes was different from that on the release of other cytosolic or mitochondrial enzymes. (4) The fraction of total hepatic lyase in mitochondrial preparations made with exogenous MgCl₂ was 30 times greater than that for a cytosolic marker enzyme, phosphoglycerate kinase. (5) Lyase substrates enhanced the release of the enzyme both from hepatocytes and from isolated mitochondria. (6) The metabolic significance of association of ATP citrate lyase with mitochondria is discussed. (7) Data obtained in the course of these experiments indicate that less than 3% of adenylate kinase is cytosolic.     

The role of adenylate kinase in dynamic compartmentation of adenine nucleotides in the mitochondrial intermembrane space.

To investigate the existence of dynamic adenine nucleotide (AdN) compartment in the mitochondrial intermembrane space, we used reconstituted systems consisting of (i) functional intact liver and heart mitochondria and (ii) pyruvate kinase plus phosphoenolpyruvate, both competing for ADP either formed in the intermembrane space by adenylate kinase or added directly into, or regenerated by ATPase within, the extramitochondrial space. It is shown that ADP formation in the mitochondrial intermembrane space is a prerequisite for a dominating oxidative phosphorylation in reconstituted systems, suggesting dynamic ADP compartmentation in that space.     

Presence of a pre-apoptotic complex of pro-caspase-3, Hsp60 and Hsp10 in the mitochondrial fraction of jurkat cells

Activation of pro-caspase-3 is a central event in the execution phase of apoptosis and appears to serve as the convergence point of different apoptotic signaling pathways. Recently, mitochondria were found to play a central role in apoptosis through release of cytochrome c and activation of caspases. Moreover, a sub-population of pro-caspase-3 has been found to be localized to this organelle. In the present study, we demonstrate that pro-caspase-3 is present in the mitochondrial fraction of Jurkat T cells in a complex with the chaperone proteins Hsp60 and Hsp10. Induction of apoptosis with staurosporine led to the activation of mitochondrial pro-caspase-3 and its dissociation from the Hsps which were released from mitochondria. The release of Hsps occurred simultaneously with the release of other mitochondrial intermembrane space proteins including cytochrome c and adenylate kinase, prior to a loss of mitochondrial transmembrane potential. In in vitro systems, recombinant Hsp60 and Hsp10 accelerated the activation of pro-caspase-3 by cytochrome c and dATP in an ATP-dependent manner, consistent with their function as chaperones. This finding suggests that the release of mitochondrial Hsps may also accelerate caspase activation in the cytoplasm of intact cells.