Respiration and mitochondrial ATPase in energized mitochondria during isoproterenol-induced cell injury of myocardium.

1. Respiration of mitochondria, membrane potential and mitochondrial ATPase under energized conditions were studied in rat myocardium during cell injury induced by treatment with isoproterenol. 2. Increase in the state 4 rate of respiration and ADP:O ratio, as well as decrease in the state 3 rate and Respiratory Control Ratio (RCR) were found. 3. The optimum pH for RCR and for maximum ATPase activity was shifted to lower values. 4. The state 3 respiration was more sensitive to oligomycin inhibition. 5. The mitochondria showed lower ability to generate membrane potential. 6. An increase in the K0.5 values for catalytic sites II and III of mitochondrial ATPase at pH 7.4 and 5.5 was found. 7. These results are consistent with alterations on the integrity of mitochondrial membrane, and corroborate with the hypothesis of changes on the mitochondrial ATPase during isoproterenol-induced cell injury of myocardium.     

Functional changes in mitochondrial properties as a result of their membrane cryodestruction. II. Influence of freezing and thawing on ATP complex activity of intact liver mitochondria

The influence of the freeze-thawing rates on ATP synthetase (ATPase) complex of intact liver mitochondria was investigated. It was shown that the increase in latent ATPase activity and decrease in ATP synthetase activity resulted from an influence on the inner mitochondrial membrane. An increase in freeze-thawing rates led to the preservation of ATP synthetase activity and ATP hydrolysis reduction. Kinetic parameter changes of the ATP synthetase reaction resulted from an insignificant nonspecific increase in the inner mitochondrial membrane permeability and changes in its electrochemical potential level.     

Lability of coupling between proton translocase and ATPase of mitochondrial H+-ATPase complex]

The interrelationship between the ATPase and H+-translocase functions of mitochondrial H+-ATPase was studied. The efficiency of the functioning was estimated by the value of coupling coefficient (Kc), which is represented by a ratio of proton translocation rate versus ATP coupling hydrolysis rate. It was shown that under conditions of increased concentrations of ATP and low concentrations of oligomycin the value of Kc is decreased. The increase in the concentration of valinomycin results in an increase of Kc. It was also found that the H+-ATPase activity shows a considerable increase during incubation of mitochondria, reaching its maximum with respect to both functions 1--2 min after addition of ATP. The data obtained are indicative of a lack of tight coupling between the H+-translocase and ATPase functions of mitochondrial H+-ATPase. The mechanism of action of H+-ATPase is discussed.     

The function of mitochondrial F(O)F(1) ATP-synthase from the whiteleg shrimp Litopenaeus vannamei muscle during hypoxia

The effect of hypoxia and re-oxygenation on the mitochondrial complex F(O)F(1)-ATP synthase was investigated in the whiteleg shrimp Litopenaeus vannamei. A 660 kDa protein complex isolated from mitochondria of the shrimp muscle was identified as the ATP synthase complex. After 10h at hypoxia (1.5-2.0 mg oxygen/L), the concentration of L-lactate in plasma increased significantly, but the ATP amount and the concentration of ATPβ protein remained unaffected. Nevertheless, an increase of 70% in the ATPase activity was detected, suggesting that the enzyme may be regulated at a post-translational level. Thus, during hypoxia shrimp are able to maintain ATP amounts probably by using some other energy sources as phosphoarginine when an acute lack of energy occurs. During re-oxygenation, the ATPase activity decreased significantly and the ATP production continued via the electron transport chain and oxidative phosphorylation. The results obtained showed that shrimp faces hypoxia partially by hydrolyzing the ATP through the reaction catalyzed by the mitochondrial ATPase which increases its activity.     

Interdependence of mitochondrial ATP production and extramitochondrial ATP utilization in intact spermatozoa

The dependence of both respiration and total activity of ATP-consuming reactions on the cellular adenine nucleotide pattern was investigated in intact bovine spermatozoa. ATP consumption was manipulated by inhibition with vanadate and activation with caffeine, leading to a decrease or increase in the rate of respiration up to 70% or 20%, respectively. Oligomycin blocked the respiration to the same extent as did vanadate, suggesting that the total extramitochondrial ATP-consuming activity is vanadate-sensitive. The major part of ATP utilization must be linked to dynein ATPase, since inhibition of (Na⁺, K⁺) ATPase by ouabain showed only a small effect on respiration (−17%). Being a potent inhibitor of dynein ATPase, vanadate drastically reduced the amount of motile cells, whereas caffeine tended to increase the intensity of motion. The effects of vanadate or caffeine on respiration were paralleled by changes in cellular ATP, reflecting the response of mitochondrial respiration on the cellular ATP/ADP ratio. Respiration was found to depend on changes in the ATP/ADP ratio in the range from about 3 (+ caffeine) to 9 (+ vanadate). The range of response of ATP consumption to the ATP/ADP ratio was determined by varying the mitochondrial ATP production via the concentration of lactate which was used as substrate. The measured effects on both respiratory rate and ATP/ADP ratio suggested that ATP consumption was markedly dependent on ATP/ADP ratios below 5. It is concluded that lactate concentrations above 1 mM sufficiently supply bovine spermatozoa with substrate and the energy turnover is mainly limited by the activity of dynein ATPase rather than by the capacity of mitochondrial oxidative phosphorylation.     

Respiratory chain defect of myocardial mitochondria in idiopathic dilated cardiomyopathy of Doberman pinscher dogs.

Idiopathic dilated cardiomyopathy (IDCM) is a primary myocardial disease of unknown cause. We tested the hypothesis that IDCM was associated with a myocardial metabolic defect by determining a comprehensive biochemical profile of metabolite concentrations and enzyme activities for the major metabolic pathways of the myocardium. We used the Doberman pinscher breed as a naturally occurring canine model of IDCM and compared its myocardial profile with that of healthy adult mongrels. Compared with controls, myocardium in IDCM had markedly reduced mitochondrial electron transport activity and myoglobin concentration, in association with acidosis and energy depletion following anoxic challenge: 60% decreased NADH dehydrogenase and 50% decreased ATP synthetase activities; 90% decreased myoglobin concentration; and 30% reduced ATP and 50% increased lactate and proton concentrations. Sarcoplasmic reticulum Ca(2+)-transport ATPase was decreased by 42%. There was a 15% compensatory increase in fatty acid oxidation and Krebs cycle activity. Other biochemical changes were mild by comparison with the mitochondrial defects. We conclude that IDCM is associated with a marked impairment of mitochondrial production of ATP, arising from decreased activity of the mitochondrial electron transport system, including myoglobin. These changes may be secondary to an underlying genetic defect or may indicate a deficiency of the mitochondrial respiratory chain that predisposes this breed to heart failure.     

低氧大鼠脑线粒体体外转录活性的研究

目的：探讨低氧对大鼠脑线粒体DNA表达的影响及其与能量生成的关系。方法：雄性Wistar大鼠随机分为3组：急性低氧组（AH）、慢性低氧组（CH）和对照组,其中急、慢性低氧组动物分别连续暴露于模拟海拔4000m高原3d(AH)和40d（CH）。分离脑线粒体,分别测定线粒体体外转录活性、F0F1－ATP酶活性以及ATP对线粒体体外转录的影响。结果：急性低氧大鼠脑线粒体体外转录活性及F0F1－ATP酶活性显著降低,慢性低氧时有所回升,两者呈线性相关。ATP对大鼠脑线粒体体外转录活性呈双相效应。结论：低氧时脑线粒体转录活性改变可能参与低氧抑制线粒体能量代谢的机制,ATP可能通过反馈作用对线粒体转录进行微调。     

萌发绿豆子叶衰老期间的呼吸作用与线粒体功能的改变

暗中培养的绿豆幼苗子叶在萌发后3—4天时,外观出现衰老征状,6天后子叶凋落。随子叶日龄的增加,子叶的呼吸强度一直下降,呼吸商始终小于1。当外加L—苹果酸、a—酮戊二酸、琥珀酸和NADH为底物测定离体线粒体氧化活性时,衰老子叶的线粒体对上述四种底物的氧化活性有不同程度的增加;抗氰呼吸也有所升高。子叶衰老时,线粒体的ADP／O和呼吸控制(RC值均降低);线粒体ATPase水解ATP的活性升高。衰老绿豆子叶线粒体氧化磷酸化偶联效率的降低和ATPase水解活性的增强是与线粒体结构改变相联系的一种功能变化,它导致能量亏缺,并进一步加速了衰老的恶化进程。     

The use of arylazido-beta-alanyl-ATP as a photoaffinity label for the isolated and membrane-bound mitochondrial ATPase complex.

The effects of a photoaffinity derivate of ATP, arylazido-beta-alanyl-ATP, 3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl) adenosine 5'-triphosphate, on submitochondrial particles and the partially purified ATPase complex of beef heart mitochondria have been investigated. In the absence of light the ATP analogue has been found to be a substrate for the E132PA1P1-ATP exchange reaction of submitochondrial particles. When photoirradiated in the presence of arylazido-beta-alanyl-ATP, the ATPase activity and the the the [32P]Pi-ATP exchange reaction are inhibited maximally 80%. Arylazido-beta-alanyl-ATP following photolysis is a noncompetitive inhibitor with respect to ATP while arylazido-beta-alanine, the azido-containing adjunct of the ATP analogue, has no inhibitory effect under the same conditions. The inactivating effect of arylazido-beta-alanyl-ATP is prevented in part by the presence of ATP, or ADP and pyrophosphate. Photolabeling produces a covalent binding of the derivative with the F1ATPase being the major protein labeled. The binding of 0.22 mumol of arylazido-beta-alanyl-ATP/mg of mitochondrial protein is associated with a maximal inhibitory effect. The ATPase activity of the partially purified ATPase complex is also sensitive to photoirradiation in the presence of arylazido-beta-alanyl-ATP. When the ATPase complex is associated with liposomes there is an increase in the specific ATPase activity with a 10-fold increase in Vmax and a 4-fold decrease in KmATP associated with a parallel increase in the apparent affinity and maximal inhibitory effect of the arylazido-beta-alanyl-ATP. The photoinhibition of the ATPase complex in the presence of arylazido-beta-alanyl-ATP results in covalent binding of 1.6 mumol of arylazido-beta-alanyl-ATP/mg of protein. The alpha and beta subunits are the only components of the ATPase complex labeled by the [3H]arylazido-beta-alanyl-ATP. The relationship between the arylazido-beta-alanyl-ATP-labeled sites and the nucleotide binding sites on the mitochondrial ATPase is discussed.     

Simulation of cardiac work transitions, in vitro: effects of simultaneous Ca2+ and ATPase additions on isolated porcine heart mitochondria

During increases in cardiac work there are net increases in cytosolic [Ca(2+)] and ATP hydrolysis by myofiliments and ion transport ATPases. However, it is still unclear what role Ca(2+)or the ATP hydrolysis products, ADP and Pi, have on the regulation of mitochondrial ATP production. In this study, work jumps were simulated by simultaneous additions of Ca(2+) and ATPase to porcine heart mitochondria. The net effects on the mitochondrial ATP production were monitored by simultaneously monitoring respiration (mVo2), [NADH], [ADP] and membrane potential (deltapsi) at 37 degrees C. Addition of exogenous ATPase (300 mlU.ml(-1))]ATP (3.4 mM) was used to generate a 'resting' background production of ADP. This resting metabolic rate was 200% higher than the quiescent rate while [NADH] and deltapsi were reduced. Subsequent ATPase additions (1.3IU.ml(-)) were made with varying amounts of Ca(2+)(0 to 535 nM) to simulate step increases in cardiac work. Ca(2+) additions increased mVo2 and depolarized deltapsi, and were consistent with an activation of Fo/F1)ATPase. In contrast, Ca(2+) reduced the [NADH] response to the ATPase addition, consistent with Ca(2+)-sensitive dehydrogenase activity (CaDH). The calculated free ADP response to ATPase decreased \2-fold in the presence of Ca(2+). The addition of 172nM free Ca(2+)] ATPase increased mVo2 by 300% (P<0.05, n=8) while deltapsi decreased by 14.9+/-0.1 mV without changes in [NADH] (P > or =0.05, n=8), consistent with working heart preparations. The addition of Ca(2+) and ATPase combined increased the mitochondrial ATP production rate with changes in deltapsi, NADH and [ADP], consistent with an activation of CaDH and F o /F(1)ATPase activity. These balancing effects of ATPase activity and [Ca(2+)] may explain several aspects of metabolic regulation in the heart during work transitions in vivo.     

A novel mechanism of enzymic ester hydrolysis. Inversion of configuration and carbon-oxygen bond cleavage by secondary alkylsulphohydrolases from detergent-degrading micro-organisms.

Ligand-binding studies with labelled triethyltin on yeast mitochondrial membranes showed the presence of high-affinity sites (KD = 0.6 micronM; 1.2 +/- 0.3 nmol/mg of protein) and low-affinity sites (KD less than 45 micronM; 70 +/- 20 nmol/mg of protein). The dissociation constant of the high-affinity site is in good agreement with the concentration of triethyltin required for inhibition of mitochondrial ATPase (adenosine triphosphatase) and oxidative phosphorylation. The high-affinity site is not competed for by oligomycin or venturicidin, indicating that triethyltin reacts at a different site from these inhibitors of oxidative phosphorylation. Fractionation of the mitochondrial membrane shows a specific association of the high-affinity sites with the ATP synthase complex. During purification of ATP synthase (oligomycin-sensitive ATPase) there is a 5-6-fold purification of oligomycin- and triethyltin-sensitive ATPase activity concomitant with a 7-9-fold increase in high-affinity triethyltin-binding sites. The purified yeast oligomycin-sensitive ATPase complex contains approximately six binding sites for triethyltin/mol of enzyme complex. It is concluded that specific triethyltin-binding sites are components of the ATP synthase complex, which accounts for the specific inhibition of ATPase and oxidative phosphorylation by triethyltin.     

Deletion of mitochondrial ATPase inhibitor in the yeast Saccharomyces cerevisiae decreased cellular and mitochondrial ATP levels under non-nutritional conditions and induced a respiration-deficient cell-type.

T(1), a mutant yeast lacking three regulatory proteins of F(1)F(o)ATPase, namely ATPase inhibitor, 9K protein and 15K protein, grew on non-fermentable carbon source at the same rate as normal cells but was less viable when incubated in water. During the incubation, the cellular ATP content decreased rapidly in the T(1) cells but not in normal cells, and respiration-deficient cells appeared among the T(1) cells. The same mutation was also induced in D26 cells lacking only the ATPase inhibitor. Overexpression of the ATPase inhibitor in YC63 cells, which were derived from the D26 strain harboring an expression vector containing the gene of the ATPase inhibitor, prevented the decrease of cellular ATP level and the mutation. Isolated T(1) mitochondria exhibited ATP hydrolysis for maintenance of membrane potential when antimycin A was added to the mitochondrial suspension, while normal and YC63 mitochondria continued to show low hydrolytic activity and low membrane potential. Thus, it is likely that deletion of the ATPase inhibitor induces ATPase activity of F(1)F(o)ATPase to create a dispensable membrane potential under the non-nutritional conditions and that this depletes mitochondrial and cellular ATP. The depletion of mitochondrial ATP in turn leads to occurrence of aberrant DNA in mitochondria.     

ATP10, a yeast nuclear gene required for the assembly of the mitochondrial F1-F0 complex

A yeast nuclear gene (ATP10) is reported whose product is essential for the assembly of a functional mitochondrial ATPase complex. Mutations in ATP10 induce a loss of rutamycin sensitivity in the mitochondrial ATPase but do not affect respiratory enzymes. This phenotype has been correlated with a defect in the F0 sector of the ATPase. The wild type ATP10 gene has been cloned by transformation of an atp 10 mutant with a yeast genomic library. The gene codes for a protein of Mr = 30,293. The primary structure of the ATP10 product is not related to any known subunit of the yeast or mammalian mitochondrial ATPase complexes. To further clarify the role of this new protein in the assembly of the ATPase, an antibody was prepared against a hybrid protein expressed from a trpE/ATP 10 fusion gene. The antibody recognizes a 30-kDa protein present in wild type mitochondria. The protein is associated with the mitochondrial membrane but does not co-fractionate either with F1 or with the rutamycin-sensitive F1-F0 complex. These data suggest that the ATP10 product is not a subunit of the ATPase complex but rather is required for the assembly of the F0 sector of the complex.     

Both ATP and an energized inner membrane are required to import a purified precursor protein into mitochondria.

We have investigated the energy requirement of mitochondrial protein import with a simplified system containing only isolated yeast mitochondria, energy sources and a purified precursor protein. This precursor was a fusion protein composed of 22 residues of the cytochrome oxidase subunit IV pre-sequence fused to mouse dihydrofolate reductase. Import of this protein required not only an energized inner membrane, but also ATP. ATP could be replaced by GTP, but not by CTP, TTP or non-hydrolyzable ATP analogs. Added ATP did not increase the membrane potential of respiring mitochondria; it supported import even if the proton-translocating mitochondrial ATPase and the entry of ATP into the matrix were blocked. We conclude that ATP exerts its effect on mitochondrial protein import outside the inner membrane.     

Regulation of mitochondrial matrix pH and adenosine 5'-triphosphatase activity during ischemia in slow heart-rate hearts. Role of Pi/H+ symport

During ischemia in so-called slow heart-rate hearts, there is a marked inhibition of the mitochondrial ATPase mediated by inhibitor protein binding to the enzyme (Rouslin, W., and Pullman, M. E. (1987) J. Mol. Cell. Cardiol. 19, 661-668). This ischemia-induced ATPase inhibition is triggered by a drop in mitochondrial matrix pH (Rouslin, W. (1987) J. Biol. Chem. 262, 3472-3476) which occurs as a result of the cell acidification which develops rapidly during the ischemic process. One effect of the ATPase inhibition is a marked slowing of the net rate of tissue ATP hydrolysis and, thus, a prolongation of cell viability during ischemia. In the present study, we demonstrate that matrix acidification in intact mitochondria from slow heart-rate hearts appears to be mediated by the Pi transporter. Pi/H+ symport appears to be the primary process which mediates matrix acidification and thus ATPase inhibition in intact slow heart-rate heart mitochondria made acidotic in vitro and, presumably, also in mitochondria in situ during the ischemic process. In contrast, intact mitochondria from a so-called fast heart-rate species, which exhibited only a low level of ischemia-induced ATPase inhibition in situ (Rouslin, W. (1987) Am. J. Physiol. 252, H622-H627), failed to exhibit a Pi- and pH-dependent mitochondrial ATPase inhibition mechanism in vitro. The Pi-dependent mitochondrial ATPase inhibition mechanism reported here for slow heart-rate hearts is consistent with a role for Pi as a coordinating signal promoting the conservation of cell ATP during myocardial ischemia.     

Attenuation of cardiac mitochondrial dysfunction by melatonin in septic mice

The existence of an inducible mitochondrial nitric oxide synthase has been recently related to the nitrosative/oxidative damage and mitochondrial dysfunction that occurs during endotoxemia. Melatonin inhibits both inducible nitric oxide synthase and inducible mitochondrial nitric oxide synthase activities, a finding related to the antiseptic properties of the indoleamine. Hence, we examined the changes in inducible nitric oxide synthase/inducible mitochondrial nitric oxide synthase expression and activity, bioenergetics and oxidative stress in heart mitochondria following cecal ligation and puncture-induced sepsis in wild-type (iNOS(+/+)) and inducible nitric oxide synthase-deficient (iNOS(-/-)) mice. We also evaluated whether melatonin reduces the expression of inducible nitric oxide synthase/inducible mitochondrial nitric oxide synthase, and whether this inhibition improves mitochondrial function in this experimental paradigm. The results show that cecal ligation and puncture induced an increase of inducible mitochondrial nitric oxide synthase in iNOS(+/+) mice that was accompanied by oxidative stress, respiratory chain impairment, and reduced ATP production, although the ATPase activity remained unchanged. Real-time PCR analysis showed that induction of inducible nitric oxide synthase during sepsis was related to the increase of inducible mitochondrial nitric oxide synthase activity, as both inducible nitric oxide synthase and inducible mitochondrial nitric oxide synthase were absent in iNOS(-/-) mice. The induction of inducible mitochondrial nitric oxide synthase was associated with mitochondrial dysfunction, because heart mitochondria from iNOS(-/-) mice were unaffected during sepsis. Melatonin treatment blunted sepsis-induced inducible nitric oxide synthase/inducible mitochondrial nitric oxide synthase isoforms, prevented the impairment of mitochondrial homeostasis under sepsis, and restored ATP production. These properties of melatonin should be considered in clinical sepsis.     

Interleukin-3 withdrawal induces an early increase in mitochondrial membrane potential unrelated to the Bcl-2 family. Roles of intracellular pH, ADP transport, and F(0)F(1)-ATPase

Cytokines such as interleukin-3 (IL-3) promote the survival of hematopoietic cells through mechanisms that are not well characterized. Withdrawal of IL-3 from an IL-3-dependent pro-B cell line induced early stress-related events that preceded cell death by more than 40 h. Intracellular pH rose above pH 7.8, peaking 2-3 h post-IL-3 withdrawal, and induced a transient increase in mitochondrial membrane potential (Delta Psi(m)) detected using several different dyes. Similar events were observed following IL-7 withdrawal from a different dependent cell line. Bcl-2, Bax, and caspases were unrelated to these early events. Intracellular alkaline pH inhibited the mitochondrial import of ADP, which would limit ATP synthesis. Total cellular ATP sharply declined during this early period, presumably as a consequence of suppressed ADP import. This was followed by an increase in reduced pyridine nucleotides. The transient increase in Delta Psi(m) was blocked by oligomycin, an inhibitor of F(0)F(1-)ATPase that may have undergone reversal caused by the abnormal ADP-ATP balance within mitochondria. These findings suggest a novel sequence of early events following trophic factor withdrawal in which alkaline pH inhibits ADP import into mitochondria, reversing the F(0)F(1-)ATPase, which in turn consumes ATP and pumps out protons, raising Delta Psi(m).     

Unique uncoupler-stimulation pattern of mitochondrial ATPase activity of tumor cells, brain, and fetal liver

Mitochondria were prepared from various kinds of normal tissues and tumor cells of mice, and their ATPase activities were measured in the presence of an uncoupler. The ATPase activities of all mitochondria were stimulated by the uncoupler when it was added to the mitochondrial suspension just before or after addition of ATP. However, when mitochondria were preincubated with the uncoupler for four minutes or more before the addition of ATP, its stimulating effect on mitochondrial ATPase activities was greatly reduced in all tumor cells tested, but not in normal adult liver. Reduction of the stimulating effect of the uncoupler by preincubation with it was also observed with mitochondrial ATPase of brain and fetal liver. Thus this pattern of change in the effect of uncoupler on preincubation may be common to tumor mitochondria, but it is not specific to tumor mitochondria. The pattern of uncoupler stimulation observed in fetal liver changed rapidly to that of adult liver immediately after birth. Thus the difference between the two uncoupler stimulation patterns is probably not due to a difference in molecular species of mitochondrial ATPase.     

Selective and reversible inhibition of the ATPase of Micrococcus denitrificans by 7-chloro-4-nitrobenzo-2-oxa-1,3 diazole

The covalent inhibitor of the beef heart mitochondrial ATPase 7-chloro-4-nitrobenzo-2-oxa-1,3 diazole inhibits the ATPase of phosphorylating particles prepared from Micrococcus denitrificans. Inhibition of both ATP synthesis and ATP hydrolysis occurs at similar rates, with a similar pH dependence, and in each case the inhibition is relieved by treatment with dithiothreitol. These results are compared with those previously obtained with the mitochondrial ATPase.