Energetics of heart mitochondria during acute phase of Trypanosoma cruzi infection in rats

The energetics of heart mitochondria was studied in the acute phase of Trypanosoma cruzi infection in rats. Wistar rats were infected with 2 × 10⁵ trypomastigote forms of the Y strain of T. cruzi, and heart mitochondria and submitochondrial particles isolated after 7 and 25 days of infection. Ultrastructure of mitochondria seemed to be preserved, but cytochrome c levels were significantly depressed. Respiratory control ratios (RCR) were decreased for glutamate and succinate oxidations, as a consequence of inhibition of respiration in state 3 and/or of stimulation of respiration in state 4. Stimulation of hydrolytic activity of FoF1-ATPase by energization of mitochondria was approx. 2-fold higher in relation to controls. Mitochondrial ATP concentration remained constant. In conclusion, during the acute phase of T. cruzi infection in rats there is an energy impairment at the level of heart mitochondria, but their ultrastructure and ATP concentration seem to be preserved; the maintenance of ATP may be due to an adaptative mechanism of the cell which includes inhibition of the hydrolytic activity of FoF1-ATPase.     

Exploration of nucleotide binding sites in the mitochondrial membrane by 2-azido-[α-P]ADP

The ADP/ATP carrier of beef heart mitochondria is able to bind 2-azido-[α-³²P]ADP in the dark with a K_d value of 8 μM. 2-Azido ADP is not transported and it inhibits ADP transport and ADP binding. Photoirradiation of beef heart mitochondria with 2-azido-[α-³²P]ADP results mainly in photolabeling of the ADP/ATP carrier protein; photolabeling is prevented by carboxyatractyloside, a specific inhibitor of ADP/ATP transport. Upon photoirradiation of inside-out submitochondrial particles with 2-azido-[α-³²P]ADP, both the ADP/ATP carrier and the β subunit of the membrane-bound F₁-ATPase are covalently labeled. The binding specificity of 2-azido-[α-³²P]ADP for the β subunit of F₁-ATPase is ascertained by prevention of photolabeling of isolated F₁ by preincubation with an excess of ADP.     

Involvement of ectonucleotidases and purinergic receptor expression during acute Chagas disease in the cortex of mice treated with resveratrol and benznidazole

Chagas disease (CD) is caused by the parasite Trypanosoma cruzi. CD affects people worldwide, primarily in tropical areas. The central nervous system (CNS) is an essential site for T. cruzi persistence during infection. The protozoan may pass through the blood–brain barrier and may cause motor and cognitive neuronal damage. Once in the CNS, T. cruzi triggers immune responses that the purinergic system can regulate. Treatment for CD is based on benznidazole (BNZ); however, this agent has negative side-effects and is toxic to the host. For this reason, we investigated whether resveratrol (RSV), a potent antioxidant and neuroprotective molecule, would modulate purinergic signaling and RSV alone or in combination with BNZ would prevent changes in purinergic signaling and oxidative damage caused by T. cruzi. We infected mice with T. cruzi and treated them with RSV or BNZ for 8 days. Increases in ATP and ADP hydrolysis by NTPDase in the total cortex of infected animals were observed. The treatment with RSV in infected group diminished ATP, ADP, and AMP hydrolysis compared to infected group. The combination of RSV + BNZ decreased AMP hydrolysis in infected animals compared to the INF group, exerting an anti-inflammatory effect. RSV acted as a neuroprotector, decreasing adenosine levels. Infected animals presented an increase of P2X₇ and A_2A density of purine receptors. RSV reduced P2X₇ and A_2A and increased A1 density receptors in infected animals. In addition, infected animals showed higher TBARS and reactive oxygen species (ROS) levels than control. RSV diminished ROS levels in infected mice, possibly due to antioxidant properties. In short, we conclude that resveratrol could act as a neuroprotective molecule, probably preventing inflammatory changes caused by infection by T. cruzi, even though the mice experienced high levels of parasitemia.     

Defective dimerization of FoF1‐ATP synthase secondary to glycation favors mitochondrial energy deficiency in cardiomyocytes during aging

Aged cardiomyocytes develop a mismatch between energy demand and supply, the severity of which determines the onset of heart failure, and become prone to undergo cell death. The FoF1‐ATP synthase is the molecular machine that provides >90% of the ATP consumed by healthy cardiomyocytes and is proposed to form the mitochondrial permeability transition pore (mPTP), an energy‐dissipating channel involved in cell death. We investigated whether aging alters FoF1‐ATP synthase self‐assembly, a fundamental biological process involved in mitochondrial cristae morphology and energy efficiency, and the functional consequences this may have. Purified heart mitochondria and cardiomyocytes from aging mice displayed an impaired dimerization of FoF1‐ATP synthase (blue native and proximity ligation assay), associated with abnormal mitochondrial cristae tip curvature (TEM). Defective dimerization did not modify the in vitro hydrolase activity of FoF1‐ATP synthase but reduced the efficiency of oxidative phosphorylation in intact mitochondria (in which membrane architecture plays a fundamental role) and increased cardiomyocytes’ susceptibility to undergo energy collapse by mPTP. High throughput proteomics and fluorescence immunolabeling identified glycation of 5 subunits of FoF1‐ATP synthase as the causative mechanism of the altered dimerization. In vitro induction of FoF1‐ATP synthase glycation in H9c2 myoblasts recapitulated the age‐related defective FoF1‐ATP synthase assembly, reduced the relative contribution of oxidative phosphorylation to cell energy metabolism, and increased mPTP susceptibility. These results identify altered dimerization of FoF1‐ATP synthase secondary to enzyme glycation as a novel pathophysiological mechanism involved in mitochondrial cristae remodeling, energy deficiency, and increased vulnerability of cardiomyocytes to undergo mitochondrial failure during aging.     

Adenylate kinase is a source of ATP for tumor mitochondrial hexokinase

It has proposed that hexokinase bound to mitochondria occupies a preferred site to wich ATP from oxidative phosphorylation is channeled directly (Bessman, S. (1966) Am. J. Medicine 40, 740–749). We have investigated this problem in isolated Zajdela hepatoma mitochondria. Addition of ADP to well-coupled mitochondria in the presence of an oxidizable substrate initiates the synthesis of glucose 6-phosphate via bound hexokinase. This reaction is only partially inhibited by oligomycin, carboxyatractyloside, carbonyl cyanide m-chlorophenylhydrazone (CCCP) ot any combination of these, suggesting a source of ATP in addition to oxidative phosphorylation. This source appears to be adenylate kinase, since Ado₂P₅, an inhibitor of the enzyme, suppresses hexokinase activity by about 50% when added alone or suppresses activity completely when added together with any of the inhibitors of oxidative phosphorylation. Ado₂P₅ does not uncouple oxidative phosphorylation nor does it inhibit ADP transport (state 3 respiration) or hexokinase. The relative amount of ATP contributed by adenylate kinase is dependent upon the ADP concentration. At low ADP concentraions, glucose phosphorylation is supported by oxidative phosphorylation, but as the adenine nucleotide translocator becomes saturated the ATP contributed by adenylate kinase increases due to the higher apparent K_m of the enzyme. Under conditions of our standard experiment ([ADP] = 0.5 mM), adenylate kinase provides about 50% of the ATP used by hexokinase in well-coupled mitochondria. In spite of this, externally added ATP supported higher rates of hexokinase activity than ADP. Our findings demonstrate that oxidative phosphorylation is not a specific or preferential source of ATP for hexokinase bound to hepatoma mitochondria. The apparent lack of a channeling mechanism for ATP to hexokinase in these mitochondria is discussed.     

Pump and displacement currents of reconstituted ATP synthase on black lipid membranes

Summary Purified ATP synthase (F ₀ F ₁) fromRhodospirillum rubrum was reconstituted into asolectin liposomes which were than adsorbed to a planar lipid bilayer. After the addition of an inactive photolabile ATP derivative (caged ATP), ATP was released after illumination with UV light, which led to a transient current in the system. The transient photocurrent indicates that the vesicles and the planar membrane are capacitatively coupled. Stationary pump currents were obtained after addition of protonophores. These currents are specifically inhibited by oligomycin and stimulated threefold by inorganic phosphate (P _i ). In analogy oligomycin-sensitive pump currents in the reverse direction coupled to net ATP synthesis were induced by a light-induced concentration jump of ADP out of caged ADP, demonstrating the reversibility of the pump. For this, a preformed proton motive force and P _i were necessary.In a second series of experiments, proteoliposomes containing both ATP synthase and bacteriorhodopsin were adsorbed to a planar bilayer. The system was excited by a laser flash. The resulting photocurrents were measured with a time resolution of 2 sec. In the presence of ADP, the signal was modulated by the electrical activity of ATP synthase. ADP-induced charge displacements in ATP synthase, with time constants of 11 and 160 sec were obtained. The kinetics of the charge movements were slowed down byF ₀ specific inhibitors (DCCD or oligomycin) and were totally absent if ADP binding toF ₁ is prevented by the catalytic site-blocking agent NBD-Cl. The charge displacement of ATP synthase is coupled only to the membrane potential induced by the electrical activity of bacteriorhodopsin. The charge movements are interpreted as conformational transitions during early steps of the reaction cycle of ATP synthase.     

The activity of phosphate-dependent glutaminase from the rat small intestine is modulated by ADP and is dependent on integrity of mitochondria

The effect of adenine nucleotides and phosphate on rat small intestine phosphate-dependent glutaminase (PDG) activity was investigated in intact mitochondria. Disruption of the integrity of mitochondria by sonication or freeze-thawing resulted in loss of enzyme activity. ADP was the strongest adenine nucleotide activator of the enzyme giving a V_max that was over 5-fold of that for AMP or ATP. The sigmoid activation curve of PDG by ADP became hyperbolic in presence ATP. ADP also lowered the K_m for glutamine and increased V_max and these effects were further enhanced by the presence of ATP. Activation of PDG by phosphate and ADP was not completely additive suggesting some antagonism between the activators. There was no clear relationship between changing ATP/ADP ratios and PDG activity in presence of a constant concentration of phosphate. However, ratios of approximately 1:4 and 4:1 gave the highest and lowest activities, respectively. The pH dependence of PDG activity was affected by phosphate concentration and results suggest that the divalent ion is the activating species.     

Toward an Adequate Scheme for the ATP Synthase Catalysis

The suggestions from the author's group over the past 25 years for how steps in catalysis by ATP synthase occur are reviewed. Whether rapid ATP hydrolysis requires the binding of ATP to a second site (bi-site activation) or to a second and third site (tri-site activation) is considered. Present evidence is regarded as strongly favoring bi-site activation. Presence of nucleotides at three sites during rapid ATP hydrolysis can be largely accounted for by the retention of ADP formed and/or by the rebinding of ADP formed. Menz, Leslie and Walker ((2001) FEBS Lett., 494, 11-14) recently attained an X-ray structure of a partially closed enzyme form that binds ADP better than ATP. This accomplishment and other considerations form the base for a revised reaction sequence. Three types of catalytic sites are suggested, similar to those proposed before the X-ray data became available. During net ATP synthesis a partially closed site readily binds ADP and P_i but not ATP. At a closed site, tightly bound ADP and P_i are reversibly converted to tightly bound ATP. ATP is released from a partially closed site that can readily bind ATP or ADP. ATP hydrolysis when protonmotive force is low or lacking occurs simply by reversal of all steps with the opposite rotation of the subunit. Each type of site can exist in various conformations or forms as they are interconverted during a 120° rotation. The conformational changes with the ATP synthase, including the vital change when bound ADP and P_i are converted to bound ATP, are correlated with those that occur in enzyme catalysis in general, as illustrated by recent studies of Rose with fumarase. The _B structure of Walker's group is regarded as an unlikely, or only quite transient, intermediate. Other X-ray structures are regarded as closely resembling but not identical with certain forms participating in catalysis. Correlation of the suggested reaction scheme with other present information is considered.     

Deep Origin of Plastid/Parasite ATP/ADP Translocases

Abstract Membrane proteins that transport ATP and ADP have been identified in mitochondria, plastids, and obligate intracellular parasites. The mitochondrial ATP/ADP transporters are derived from a broad-specificity transport family of eukaryotic origin, whereas the origin of the plastid/parasite ATP/ADP translocase is more elusive. Here we present the sequences of five genes coding for ATP/ADP translocases from four species of Rickettsia. The results are consistent with an early duplication and divergence of the five ATP/ADP translocases within the rickettsial lineage. A comparison of the phylogenetic depths of the mitochondrial and the plastid/parasite ATP/ADP translocases indicates a deep origin for both transporters. The results provide no evidence for a recent acquisition of the ATP/ADP transporters in Rickettsia via horizontal gene transfer, as previously suggested. A possible function of the two types of ATP/ADP translocases was to allow switches between glycolysis and aerobic respiration in the early eukaryotic cell and its endosymbiont.     

Species specific release of sulfate from adenylyl sulfate by ATP sulfurylase or ADP sulfurylase in the green sulfur bacteria Chlorobium limicola and Chlorobium vibrioforme

High activities of ATP sulfurylase were found in the soluble protein fraction of two Chlorobium limicola strains, whereas ADP sulfurylase was absent. ATP sulfurylase was partially purified and characterized. It was a stable soluble enzyme with a molecular weight of 230,000, buffer-dependent pH optima at 8.6 and 7.2 and an isoelectric point at pH 4.8. No physiological inhibitor was found. Inhibition was observed with p-CMB and heavy metals. Sulfur compounds had no effect on enzyme activity. The stoichiometry of the reaction was proven. In contrast, an ADP sulfurylase, but no ATP sulfurylase, was found in Chlorobium vibrioforme. This enzyme was very labile with a molecular weight of about 120,000 and buffer-dependent pH optima at 9.0 and 8.5. Under test conditions the apparent K _m value was determined to be 0.28 mM for adenylyl sulfate and 8.0 mM for phosphate.Abbreviations APS adenylyl sulfate - p-CMB parachloromercuribenzoate - PP_i inorganic pyrophosphate     

Regulation of the structure and catalytic properties of plasma membrane H<Superscript>+</Superscript>-ATPase involved in adaptation of two reed ecotypes to their different habitats

The properties and kinetics of ATP and p-nitrophenyl phosphate (PNPP) hydrolysis activities of plasma membrane H⁺-ATPase from the two reed ecot ypes, swamp reed (SR) and dune reed (DR), were investigated. The pH optimum of the plasma membrane H⁺-ATPase in both reed ecotypes was similar but the sensitivity of the enzyme to the reaction medium pH seemed to be higher in DR than that in SR. Compared to SR, the DR exhibited a higher V_max value for ATP hydrolysis whereas the K_m value was almost similar in both reed ecotypes. The PNPP hydrolysis of the plasma membrane H⁺-ATPase was also studied in both reed ecotypes at increasing PNPP concentrations. K_m and V_max for PNPP hydrolysis showed great differences in the two reed ecotypes and in DR the K_m and V_max values were 2- and 10-fold, respectively, higher than those in SR. The ATP hydrolysis activity of the plasma membrane was markedly inhibited by hydroxylamine in both reed ecotypes, and the percentage inhibition of ATP hydrolysis rate seemed higher in DR than that in SR. In addition, the structure or property of the C-terminal end of the plasma membrane H⁺-ATPase were also different in the two reed ecotypes. These data suggest that different isoforms of the plasma membrane H⁺-ATPase might be developed and involved in the adaptation of the plant to the long-term drought-prone habitat.This research was supported by Natural Science Foundation of China (No. 30270238 & No. 30470274) and the National Key Basic Research Special Funds of China (G1999011705).     

Investigation of the enzymatic activity of the Na,K-ATPase via isothermal titration microcalorimetry

Isothermal titration microcalorimetry (ITC) is shown here to be a sensitive and accurate method for assaying the steady-state enzyme activity of the Na⁺,K⁺-ATPase. Single ATP injection experiments yield an apparent enthalpy change for the ATP hydrolysis reaction catalyzed by the enzyme of −51 (± 1) kJ mol⁻¹. This value is independent of the amount of ADP accumulated in the sample cell, which indicates that under the experimental conditions studied here (saturating Na⁺ and K⁺ concentrations) ADP does not inhibit enzyme activity by reversal of the phosphorylation reaction and resynthesizing ATP. Multiple ATP injection titration experiments in which varying concentrations of ADP were initially included in the sample cell could be adequately explained by a Michaelis-Menten kinetic model incorporating noncompetitive inhibition. This suggests that ADP inhibits the enzyme by binding to more than one enzyme intermediate and inhibiting forward reactions of the enzyme. Values of K_m and K_I obtained for the fits agree with literature values obtained by other methods. Because ITC is a direct method of continually monitoring enzyme activity, it is a valuable supplement to less direct or noncontinuous methods such as colorimetric, enzyme-coupled or radioactivity-based assays.     

Identification and partial purification of a heart mitochondrial membrane proteinase

Membrane-bound proteinase activity was demonstrated by a solid-phase assay system in both beef heart and rat liver mitochondria. The activity was sensitive to SH reagents and assorted proteinase inhibitors. Although stimulated by nonionic detergents, it became labile when solubilized by detergents. The proteinase activity from heart mitochondria copurified with the ADP:ATP translocator protein. Gel electrophoresis of this preparation revealed the translocator polypeptide as well as a number of minor components. In solubilized mitochondria the ADP:ATP translocator polypeptide slowly disappeared upon standing at 0°C as revealed by polyacrylamide gel electrophoresis under denaturing conditions. The loss of this polypeptide was prevented by addition of proteinase inhibitors as well as the translocator affinity ligand, carboxyatractylate. These observations confirm the presence of an integral membrane proteinase in mitochondria and suggest a structural and enzymatic interaction between the proteinase and the ADP:ATP translocator.Abbreviations PMSF phenylmethanesulfonyl fluoride - TPCK l-1-tosylamido-2-phenylethylchloromethyl ketone - TLCK 1-chloro-3-tosylamido-7-amino-l-2-heptanone - NEM N-ethylmaleimide - PCMBS p-chloromercuriphenylsulfonic acid - SDS sodium dodecyl sulfate - MOPS morpholinopropane sulfonate - [I₅₀] concentration of inhibitor required to give 50% inhibition     

Increase of membrane permeability of mitochondria isolated from water stress adapted potato cells

In order to gain some insight into mitochondria permeability under water stress, intact coupled mitochondria were isolated from water stress adapted potato cells and investigations were made of certain transport processes including the succinate/malate and ADP/ATP exchanges, the plant mitochondrial ATP-sensitive potassium channel (PmitoK_ATP) and the plant uncoupling mitochondrial protein (PUMP). The V _maxL values measured for succinate/malate and ADP/ATP carriers, as photometrically investigated, as well as the same values for the Pmito_ATP and the PUMP were found to increase; this suggested that mitochondria adaptation to water stress can cause an increase in the membrane permeability.     

Loss of mitochondrial ATP synthase subunit beta (Atp2) alters mitochondrial and chloroplastic function and morphology in Chlamydomonas

Mitochondrial F₁F_O ATP synthase (Complex V) catalyses ATP synthesis from ADP and inorganic phosphate using the proton-motive force generated by the substrate-driven electron transfer chain. In this work, we investigated the impact of the loss of activity of the mitochondrial enzyme in a photosynthetic organism. In this purpose, we inactivated by RNA interference the expression of the ATP2 gene, coding for the catalytic subunit β, in the green alga Chlamydomonas reinhardtii. We demonstrate that in the absence of β subunit, complex V is not assembled, respiratory rate is decreased by half and ATP synthesis coupled to the respiratory activity is fully impaired. Lack of ATP synthase also affects the morphology of mitochondria which are deprived of cristae. We also show that mutants are obligate phototrophs and that rearrangements of the photosynthetic apparatus occur in the chloroplast as a response to ATP synthase deficiency in mitochondria. Altogether, our results contribute to the understanding of the yet poorly studied bioenergetic interactions between organelles in photosynthetic organisms.     

Regulation of Energy Transduction and Electron Transfer in Cytochrome c Oxidase by Adenine Nucleotides

Cytochrome c oxidase from bovine heart contains seven high-affinity binding sites for ATP or ADP and three additional only for ADP. One binding site for ATP or ADP, located at the matrix-oriented domain of the heart-type subunit VIaH, increases the H⁺/e^– stoichiometry of the enzyme from heart or skeletal muscle from 0.5 to 1.0 when bound ATP is exchanged by ADP. Two further binding sites for ATP or ADP, located at the cytosolic and the matrix domain of subunit IV, increases the K _M for Cytochrome c and inhibit the respiratory activity at high ATP/ADP ratios, respectively. We propose that thermogenesis in mammals is related to subunit VIaL of cytochrome c oxidase with a H⁺/e^– stoichiometry of 0.5 compared to 1.0 in the enzyme from bacteria or ectotherm animals. This hypothesis is supported by the lack of subunit VIa isoforms in cytochrome c oxidase from fish.     

Targeting host mitochondria: A role for the Trypanosoma cruzi amastigote flagellum

Trypanosoma cruzi is the kinetoplastid protozoan parasite that causes human Chagas disease, a chronic disease with complex outcomes including severe cardiomyopathy and sudden death. In mammalian hosts, T. cruzi colonises a wide range of tissues and cell types where it replicates within the host cell cytoplasm. Like all intracellular pathogens, T. cruzi amastigotes must interact with its immediate host cell environment in a manner that facilitates access to nutrients and promotes a suitable niche for replication and survival. Although potentially exploitable to devise strategies for pathogen control, fundamental knowledge of the host pathways co‐opted by T. cruzi during infection is currently lacking. Here, we report that intracellular T. cruzi amastigotes establish close contact with host mitochondria via their single flagellum. Given the key bioenergetic and homeostatic roles of mitochondria, this striking finding suggests a functional role for host mitochondria in the infection process and points to the T. cruzi amastigote flagellum as an active participant in pathogenesis. Our study establishes the basis for future investigation of the molecular and functional consequences of this intriguing host–parasite interaction.     

Analysis of the energy metabolism after incubation of Saccharomyces cerevisiae with sulfite or nitrite

After addition of 5 mM sulfite or nitrite to glucose-metabolizing cells of Saccharomyces cerevisiae a rapid decrease of the ATP content and an inversely proportional increase in the level of inorganic phosphate was observed. The concentration of ADP shows only small and transient changes. Cells of the yeast mutant pet 936, lacking mitochondrial F₁ATPase, after addition of 5 mM sulfite or nitrite exhibit changes in ATP, ADP and inorganic phosphate very similar to those observed in wild type cells. They key enzyme of glucose degradation, glyceraldehyde-3-phosphate dehydrogenase was previously shown to be the most sulfiteor nitrite-sensitive enzyme of the glycolytic pathway. This enzyme shows the same sensitivity to sulfite or nitrite in cells of the mutant pet 936 as in wild type cells. It is concluded that the effects of sulfite or nitrite on ATP, ADP and inorganic phosphate are the result of inhibition of glyceraldehyde-3-phosphate dehydrogenase and not of inhibition of phosphorylation processes in the mitochondria. Levels of GTP, UTP and CTP show parallel changes to ATP. This is explained by the presence of very active nucleoside monophosphate kinases which cause a rapid exchange between the nucleoside phosphates. The effects of the sudden inhibition of glucose degradation by sulfite or nitrite on levels of ATP, ADP and inorganic phosphate are discussed in terms of the theory of Lynen (1942) on compensating phosphorylation and dephosphorylation in steady state glucose metabolizing yeast.Abbreviations ATP adenosine triphosphate - ADP adenosine diphosphate - AMP adenosine monophosphate - P_i inorganic orthophosphate Dedicated to Prof. Dr. Hans Grisebach on the occasion of his sixtieth birthday     

Reduced nicotinamide-adenine-dinucleotide-pyrophosphorylase: A novel enzyme in seeds

An enzyme which splits reduced NAD has been partially purified from pea (Pisum sativum, Kelvedon Wonder) seeds. The activity requires orthophosphate and the products are ADP and probably NMN (dihydro NMN?). The enzyme splits the NADH₂ at the pyrophosphate bond and incorporates the phosphate into the AMP residue. NAD, NADP or NADPH₂ could not replace NADH₂. The enzyme is unstable during storage, is activated by Mg²⁺ and by Mn²⁺, and inhibited by Ca²⁺. K⁺, Li⁺ and NH₄⁺ have no effect. The possible role of this enzyme in the synthesis of ATP in seeds at the early stage of germination is discussed.