The assumption that nitric oxide inhibits mitochondrial ATP synthesis is correct

The assumption that reversible inhibition of mitochondrial respiration by nitric oxide (NO.) represents inhibition of ATP synthesis is unproven. NO. could theoretically inhibit the oxygen consumption with continued ATP synthesis, by acting as an electron acceptor from cytochrome c or as a terminal electron acceptor in stead of oxygen. We report here that NO. does reversibly inhibit brain mitochondrial ATP synthesis with a time course similar to its inhibition of respiration. Whilst such inhibition was largely reversible, there appeared to be a small irreversible component which may theoretically be due to peroxynitrite formation, i.e. as a result of the reaction between NO. and superoxide, generated by the mitochondrial respiratory chain.     

CK flux or direct ATP transfer: Versatility of energy transfer pathways evidenced by NMR in the perfused heart

How the myocardium is able to permanently coordinate its intracellular fluxes of ATP synthesis, transfer and utilization is difficult to investigate in the whole organ due to the cellular complexity. The adult myocardium represents a paradigm of an energetically compartmented cell since 50% of total CK activity is bound in the vicinity of other enzymes (myofibrillar sarcolemmal and sarcoplasmic reticulum ATPases as well as mitochondrial adenine nucleotide translocator, ANT). Such vicinity of enzymes is well known in vitro as well as in preparations of skinned fibers to influence the kinetic properties of these enzymes and thus the functioning of the subcellular organelles. Intracellular compartmentation has often been neglected in the NMR analysis of CK kinetics in the whole organ. It is indeed a methodological challenge to reveal subcellular kinetics in a working organ by a global approach such as NMR. To get insight in the energy transfer pathway in the perfused rat heart, we developed a combined analysis of several protocols of magnetization transfer associated with biochemical data and quantitatively evaluated which scheme of energetic exchange best describes the NMR data. This allows to show the kinetic compartmentation of subcellular CKs and to quantify their fluxes. Interestingly, we could show that the energy transfer pathway shifts from the phosphocreatine shuttle in the oxygenated perfused heart to a direct ATP diffusion from mitochondria to cytosol under moderate inhibition of ATP synthesis. Furthermore using NMR measured fluxes and the known kinetic properties of the enzymes, it is possible to model the system, estimate local ADP concentrations and propose hypothesis for the versatility of energy transfer pathway. In the normoxic heart, a 3-fold ADP gradient was found between mitochondrial intermembrane space, cytosol and ADP in the vicinity of ATPases. The shift from PCr to ATP transport observed when ATP synthesis decreases might result from a balance in the activity of two populations of ANT, either coupled or uncoupled to CK. We believe this NMR approach could be a valuable tool to reinvestigate the control of respiration by ADP in the whole heart reconciling the biochemical knowledge of mitochondrial obtained in vitro or in skinned fibers with data on the whole heart as well as to identify the implication of bioenergetics in the pathological heart.     

Mitochondrial nitric-oxide synthase: role in pathophysiology

The biochemistry of the mitochondrial production of nitric oxide is reviewed to gain insight into the basic role of this radical in mitochondrial and cellular oxidative metabolism. The mitochondrial production of nitric oxide is catalyzed by a nitric-oxide synthase (mtNOS). This enzyme has the same cofactor and substrate requirements as other constitutive nitric-oxide synthases. Its occurrence was demonstrated in various mitochondrial preparations from different organs and species using diverse approaches (oxidation of oxymyoglobin, electron paramagnetic resonance in conjunction with spin trap, radiolabeled L-arginine, immunohistochemistry, nitric-oxide electrode). MtNOS has been identified as the alpha isoform of nNOS, acylated at a Thr or Ser residue, and phosphorylated at the C-terminal end. Endogenous nitric oxide reversibly inhibits oxygen consumption and ATP synthesis by competitive inhibition of cytochrome oxidase. Nitric oxide is the first molecule that fulfills the requirement for a cytochrome oxidase activity modulator: it is a competitive inhibitor, produced endogenously at a fair rate near the target site, at concentrations high enough to exhibit an inhibitory effect on cytochrome oxidase. The role of the mitochondrial nitric oxide production is discussed in terms of the physiological (modulating oxygen gradients into tissues) and pathological (abrogation of oxygen gradient modification, apoptosis, protein nitrative/oxidative stress) implications.     

Characterization and function of mitochondrial nitric-oxide synthase

The mitochondrial production of nitric oxide is catalyzed by a nitric-oxide synthase. This enzyme has the same cofactor and substrate requirements as other constitutive nitric-oxide synthases. Its occurrence was demonstrated in various mitochondrial preparations (intact, purified mitochondria, permeabilized mitochondria, mitoplasts, submitochondrial particles) from different organs (liver, heart) and species (rat, pig). Endogenous nitric oxide reversibly inhibits oxygen consumption and ATP synthesis by competitive inhibition of cytochrome oxidase. The increased K(m) of cytochrome oxidase for oxygen and the steady-state reduction of the electron chain carriers provided experimental evidence for the direct interaction of this oxidase with endogenous nitric oxide. The increase in hydrogen peroxide production by nitric oxide-producing mitochondria not accompanied by the full reduction of the respiratory chain components indicated that cytochrome c oxidase utilizes nitric oxide as an alternative substrate. Finally, effectors or modulators of cytochrome oxidase (the irreversible step in oxidative phosphorylation) had been proposed during the last 40 years. Nitric oxide is the first molecule that fulfills this role (it is a competitive inhibitor, produced at a fair rate near the target site) extending the oxygen gradient to tissues.     

Nature of enhanced mitochondrial oxidative metabolism by a calf blood extract

The effect of calf blood extract (Solcoseryl, SS) on mitochondrial oxidative function in various states was studied polarographically in vitro. 1) Mitochondrial respiration in all 4 conventional study states (Estabrook, 1967) was enhanced by the addition of SS, including states 1 and 2 (endogenous substrates only). 2) The effect of SS on mitochondrial oxygen consumption was concentration dependent, while ADP/O ratio remained constant. The effect of added respiratory substrates varied with the particular substrate at optimally active concentrations. With suboptimal substrate levels, ADP/O ratios were concentration dependent, in contrast to the SS effect. Under oligomycin ATPase inhibition, SS was no longer active, in contrast to DNP, which remained active. 3) In states 3 (added ADP) and 4 (ADP exhausted), oxygen consumption and oxidative phosphorylation were enhanced by SS in the presence or absence of citrate, glutamate, pyruvate, lactate, or ascorbate. However, in the presence of succinate, SS had no effect. 4) ADP/O ratio was decreased by SS in the presence of added substrate, suggesting that SS activation of H(+)-ATPase enhances ATP hydrolysis as well as oxidative phosphorylation and ATP synthesis. 5) The enhancing effect of SS on mitochondrial function is due to hydrophilic components of SS. The lipidic components obtained by Folch fraction of SS have no effect. It is concluded that the effects of SS respiratory substrates and uncouplers on mitochondrial function are essentially different. SS enhances both ATP synthesis and oxygen consumption by mitochondria.     

Role of calcium signaling in the activation of mitochondrial nitric oxide synthase and citric acid cycle

An apparent discrepancy arises about the role of calcium on the rates of oxygen consumption by mitochondria: mitochondrial calcium increases the rate of oxygen consumption because of the activation of calcium-activated dehydrogenases, and by activating mitochondrial nitric oxide synthase (mtNOS), decreases the rates of oxygen consumption because nitric oxide is a competitive inhibitor of cytochrome oxidase. To this end, the rates of oxygen consumption and nitric oxide production were followed in isolated rat liver mitochondria in the presence of either L-Arg (to sustain a mtNOS activity) or N(G)-monomethyl-L-Arg (NMMA, a competitive inhibitor of mtNOS) under State 3 conditions. In the presence of NMMA, the rates of State 3 oxygen consumption exhibited a K(0.5) of 0.16 microM intramitochondrial free calcium, agreeing with those required for the activation of the Krebs cycle. By plotting the difference between the rates of oxygen consumption in State 3 with L-Arg and with NMMA at various calcium concentrations, a K(0.5) of 1.2 microM intramitochondrial free calcium was obtained, similar to the K(0.5) (0.9 microM) of the dependence of the rate of nitric oxide production on calcium concentrations. The activation of dehydrogenases, followed by the activation of mtNOS, would lead to the modulation of the Krebs cycle activity by the modulation of nitric oxide on the respiratory rates. This would ensue in changes in the NADH/NAD and ATP/ADP ratios, which would influence the rate of the cycle and the oxygen diffusion.     

Metabolic and functional consequences of complete inhibition of creatine kinase by iodoacetamide in the perfused heart]

Treatment of perfused rat hearts with 0.5 mM iodoacetamide (IAAm) for 15 min at different workloads resulting in a nearly complete inhibition of creatine kinase (CK, 99%) was followed by a rapid decline of the phosphocreatine (PCr) level (30%) and a 2-fold increase of the P(i) level which then stabilized. Conversely, the ATP content started to drop monotonously at the beginning of the IAAm washout and reached 30% 90 min after the IAAm removal under medium load. Under low workload the ATP decay occurred at later periods. Neither the ADP-stimulated mitochondrial respiration in skinned fibers, nor the Ca(2+)-stimulated ATPase activity of myofibrils was affected by IAAm treatment. The sensitivity of the resting tension of skinned fibers to Ca2+ tended to a slight increase. The cardiac work index (PRP-pressure-rate product) decreased by 25%, while the end diastolic pressure (EDP) rose by 15 mm Hg when IAAm acted under medium load. In contrast, under low work these parameters were practically stable. The hearts poisoned with IAAm performed a two times lower maximal work and had reduced (by 35%) oxygen consumption rates. The efficiency of energy utilization for mechanical work decreased by 40%. The changes in PRP and EDP correlated with the cytosolic [ATP]/[ADP] ratio in such a way that the decrease in the latter was associated with a decrease in PRP and the elevation of EDP. These data suggest that the creatine kinase system is necessary for the effective translation of a high [ATP]/[ADP] ratio from the intermembrane space of mitochondria to the cytoplasm, myofibrils and ionic pumps. This provides a high level of mechanical work and good relaxation of the left ventricle and protects cytosolic adenine nucleotides from the breakdown.     

Analyzing the functional properties of the creatine kinase system with multiscale 'sloppy' modeling

In this study the function of the two isoforms of creatine kinase (CK; EC 2.7.3.2) in myocardium is investigated. The 'phosphocreatine shuttle' hypothesis states that mitochondrial and cytosolic CK plays a pivotal role in the transport of high-energy phosphate (HEP) groups from mitochondria to myofibrils in contracting muscle. Temporal buffering of changes in ATP and ADP is another potential role of CK. With a mathematical model, we analyzed energy transport and damping of high peaks of ATP hydrolysis during the cardiac cycle. The analysis was based on multiscale data measured at the level of isolated enzymes, isolated mitochondria and on dynamic response times of oxidative phosphorylation measured at the whole heart level. Using 'sloppy modeling' ensemble simulations, we derived confidence intervals for predictions of the contributions by phosphocreatine (PCr) and ATP to the transfer of HEP from mitochondria to sites of ATP hydrolysis. Our calculations indicate that only 15±8% (mean±SD) of transcytosolic energy transport is carried by PCr, contradicting the PCr shuttle hypothesis. We also predicted temporal buffering capabilities of the CK isoforms protecting against high peaks of ATP hydrolysis (3750 μM*s(-1)) in myofibrils. CK inhibition by 98% in silico leads to an increase in amplitude of mitochondrial ATP synthesis pulsation from 215±23 to 566±31 μM*s(-1), while amplitudes of oscillations in cytosolic ADP concentration double from 77±11 to 146±1 μM. Our findings indicate that CK acts as a large bandwidth high-capacity temporal energy buffer maintaining cellular ATP homeostasis and reducing oscillations in mitochondrial metabolism. However, the contribution of CK to the transport of high-energy phosphate groups appears limited. Mitochondrial CK activity lowers cytosolic inorganic phosphate levels while cytosolic CK has the opposite effect.     

K(ATP)(+) channels, nitric oxide, and adenosine are not required for local metabolic coronary vasodilation

The role of ATP-sensitive K(+) (K(ATP)(+)) channels, nitric oxide, and adenosine in coronary exercise hyperemia was investigated. Dogs (n = 10) were chronically instrumented with catheters in the aorta and coronary sinus and instrumented with a flow transducer on the circumflex coronary artery. Cardiac interstitial adenosine concentration was estimated from arterial and coronary venous plasma concentrations using a previously tested mathematical model. Experiments were conducted at rest and during graded treadmill exercise with and without combined inhibition of K(ATP)(+) channels (glibenclamide, 1 mg/kg iv), nitric oxide synthesis (N(omega)-nitro-L-arginine, 35 mg/kg iv), and adenosine receptors (8-phenyltheophylline, 3 mg/kg iv). During control exercise, myocardial oxygen consumption increased ~2.9-fold, coronary blood flow increased ~2.6-fold, and coronary venous oxygen tension decreased from 19.9 +/- 0.4 to 13.7 +/- 0.6 mmHg. Triple blockade did not significantly change the myocardial oxygen consumption or coronary blood flow response during exercise but lowered the resting coronary venous oxygen tension to 10.0 +/- 0.4 mmHg and during exercise to 6.2 +/- 0.5 mmHg. Cardiac adenosine levels did not increase sufficiently to overcome the adenosine receptor blockade. These results indicate that combined inhibition of K(ATP)(+) channels, nitric oxide synthesis, and adenosine receptors lowers the balance between total oxygen supply and consumption at rest but that these factors are not required for local metabolic coronary vasodilation during exercise.     

Effects of exogenous donor of nitric oxide-sodium nitroprusside on energy production of rat reticulocytes

The effects of the sodium nitroprusside (SNP), a nitric oxide (NO) donor clinically used in the treatment of hypertensive emergencies on the energy production of rat reticulocytes were investigated. Rat reticulocyte-rich red blood cell suspensions were aerobically incubated without (control) or in the presence of different concentrations of SNP (0.1, 0.25, 0.5, 1.0 mM). SNP decreased total and coupled, but increased uncoupled oxygen consumption. This was accompanied by the stimulation of glycolysis, as measured by increased glucose consumption and lactate accumulation. Levels of all glycolytic intermediates indicate stimulation of hexokinase-phosphofructo kinase (HK-PFK), glyceraldehyde 3-phosphate dehydrogenase (GAPD) and pyruvate kinase (PK) activities in the presence of SNP. Due to the decrease of coupled oxygen consumption in the presence of SNP, ATP production via oxidative phosphorylation was significantly diminished. Simultaneous increase of glycolytic ATP production was not enough to provide constant ATP production. In addition, SNP significantly decreased ATP level, which was accompanied with increased ADP and AMP levels. However, the level of total adenine nucleotides was significantly lower, which was the consequence of increased catabolism of adenine nucleotides (increased hypoxanthine level). ATP/ADP ratio and adenylate energy charge level were significantly decreased. In conclusion, SNP induced inhibition of oxidative phosphorylation, stimulation of glycolysis, but depletion of total energy production in rat reticulocytes. These alterations were accompanied with instability of energy status.     

New control of mitochondrial membrane potential and ROS formation--a hypothesis

A new control of mitochondrial membrane potential delta(psi)m and formation of reactive oxygen species (ROS) is presented, based on allosteric ATP-inhibition of cytochrome c oxidase at high intramitochondrial ATP/ADP ratios. Since the rate of ATP synthesis by the ATP synthase is already maximal at low membrane potentials (100-120 mV), the ATP/ADP ratio will also be maximal at this delta(psi)m (at constant rate of ATP consumption). Therefore the control of respiration by the ATP/ADP-ratio keeps delta(psi)m low. In contrast, the known 'respiratory control' leads to an inhibition of respiration only at high delta(psi)m values (150-200 mV) which cause ROS formation. ATP-inhibition of cytochrome c oxidase is switched on and off by reversible phosphorylation (via cAMP and calcium, respectively). We propose that 'stress hormones' which increase intracellular [Ca2+] also increase delta(psi)m and ROS formation, which promote degenerative diseases and accelerate aging.     

Uncoupling protein-4 (UCP4) increases ATP supply by interacting with mitochondrial Complex II in neuroblastoma cells

Mitochondrial uncoupling protein-4 (UCP4) protects against Complex I deficiency as induced by 1-methyl-4-phenylpyridinium (MPP(+)), but how UCP4 affects mitochondrial function is unclear. Here we investigated how UCP4 affects mitochondrial bioenergetics in SH-SY5Y cells. Cells stably overexpressing UCP4 exhibited higher oxygen consumption (10.1%, p<0.01), with 20% greater proton leak than vector controls (p<0.01). Increased ATP supply was observed in UCP4-overexpressing cells compared to controls (p<0.05). Although state 4 and state 3 respiration rates of UCP4-overexpressing and control cells were similar, Complex II activity in UCP4-overexpressing cells was 30% higher (p<0.05), associated with protein binding between UCP4 and Complex II, but not that of either Complex I or IV. Mitochondrial ADP consumption by succinate-induced respiration was 26% higher in UCP4-overexpressing cells, with 20% higher ADP:O ratio (p<0.05). ADP/ATP exchange rate was not altered by UCP4 overexpression, as shown by unchanged mitochondrial ADP uptake activity. UCP4 overexpression retained normal mitochondrial morphology in situ, with similar mitochondrial membrane potential compared to controls. Our findings elucidate how UCP4 overexpression increases ATP synthesis by specifically interacting with Complex II. This highlights a unique role of UCP4 as a potential regulatory target to modulate mitochondrial Complex II and ATP output in preserving existing neurons against energy crisis.     

A transient inhibition of mitochondrial ATP synthesis by nitric oxide synthase activation triggered apoptosis in primary cortical neurons

In order to investigate the relationship between nitric oxide-mediated regulation of mitochondrial function and excitotoxicity, the role of mitochondrial ATP synthesis and intracellular redox status on the mode of neuronal cell death was studied. Brief (5 min) glutamate (100 microM) receptor stimulation in primary cortical neurons collapsed the mitochondrial membrane potential (psi(m)) and transiently (30 min) inhibited mitochondrial ATP synthesis, causing early (1 h) necrosis or delayed (24 h) apoptosis. The transient inhibition of ATP synthesis was paralleled to a loss of NADH, which was fully recovered shortly after the insult. In contrast, NADPH and the GSH/GSSG ratio were maintained, but progressively decreased thereafter. Twenty-four hours after glutamate treatment, ATP was depleted, a phenomenon associated with a persistent inhibition of mitochondrial succinate-cytochrome c reductase activity and delayed necrosis. Blockade of either nitric oxide synthase (NOS) activity or the mitochondrial permeability transition (MPT) pore prevented psi(m) collapse, the transient inhibition of mitochondrial ATP synthesis, early necrosis and delayed apoptosis. However, blockade of NOS activity, but not the MPT pore, prevented the inhibition of succinate-cytochrome c reductase activity and delayed ATP depletion and necrosis. From these results, we suggest that glutamate receptor-mediated NOS activation would trigger MPT pore opening and transient inhibition of ATP synthesis leading to apoptosis in a neuronal subpopulation, whereas other groups of neurons would undergo oxidative stress and persistent inhibition of ATP synthesis leading to necrosis.     

Intracellular compartmentation of cardiac fibres from rainbow trout and Atlantic cod--a general design of heart cells

In mammalian cardiomyocytes, mitochondria and adjacent ATPases with participation of creatine kinase (CK) constitute functional compartments with an exchange of ADP and ATP delimited from cytosolic bulk solution. The question arises if this extends to ectothermic vertebrates: their low body temperature and thinner cardiomyocytes with a lower density of membrane structures may reduce the need and structural basis for compartmentation. In saponin-skinned cardiac fibres from rainbow trout and Atlantic cod, we investigated mitochondrial respiration induced by endogenous ADP generated by ATPases and its competition for this ADP with pyruvate kinase (PK) in excess. At low Ca(2+) activity (pCa = 7.0), PK lowered ATP-induced respiration by 40% in trout and 26% in cod. At high Ca(2+) activity (pCa = 5.41), PK had no effect. Additionally, ADP release from the fibres was almost zero but increased drastically upon inhibition of respiration with 1 mM Na-azide. This suggests that fibres are compartmented. PK abolished creatine-stimulated respiration in trout suggesting a less tight coupling of CK to respiration than in mammals. In conclusion, intracellular compartmentation seems to be a general feature of vertebrate cardiomyocytes, whereas the role of CK is unclear, but it seems to be less important for energy transport in species with lower metabolism.     

Low oxygen sensing and balancing in plant seeds: a role for nitric oxide

Storage product accumulation in seeds of major crop species is limited by their low internal oxygen concentration. Adjustment of energy and storage metabolism to oxygen deficiency (hypoxia) in seeds is highly relevant for agriculture and biotechnology. However, the mechanisms of low-oxygen sensing and balancing remain a mystery. Here, it is shown that normal hypoxia in seeds of soybean (Glycine max) and pea (Pisum sativum) triggers a nitrite-dependent increase in endogenous nitric oxide (NO) concentrations. NO, in turn, reduces the oxygen consumption of seeds, generating a localized decrease in both ATP availability and biosynthetic activity. Increasing oxygen availability reduces endogenous NO concentrations, thereby abolishing mitochondrial and metabolic inhibition. This auto-regulatory and reversible oxygen balancing, via NO, avoids seed anoxia and suggests a key role for NO in regulating storage activity. This hypothesis is reinforced by changes in energy status (ATP:ADP ratio), steady-state metabolite concentrations and biosynthetic fluxes under NO treatment. The proposed mechanism of low-oxygen sensing and balancing in plants offers the prospect of a new field of study in crop biotechnology.     

Effect of ATP translocation on citrulline and oxaloacetate synthesis by isolated rat liver mitochondria.

The possibility that the availability of ATP may affect the rate of synthesis of carbamoyl phosphate (measured as citrulline) by carbamoyl phosphate synthase (ammonia) was studied using respiring isolated rat liver mitochondria incubated with added ADP, with hexokinase, glucose, and ATP, or with atractylate, in order to enhance or prevent the efflux of mitochondrial ATP. The effects of these agents were compared with those on oxaloacetate synthesis from pyruvate. Addition of hexokinase, glucose, and ATP to isolated mitochondria resulted in an inhibition of citrulline synthesis which was proportional to the amounts of glucose 6-phosphate formed; under these conditions, matrix ATP and ATP/ADP tended to decrease. The addition of increasing amounts of ADP also resulted in proportional inhibition of citrulline synthesis, but in this case the matrix content of ATP and ADP increased, and ATP/ADP decreased very slightly. In the presence of atractylate, citrulline synthesis was maximal despite a 30% decrease in matrix ATP and ATP/ADP. These effects were observed whether pyruvate, succinate, glutamate, or β-OH-butyrate was used as the respiratory substrate. ADP, the hexokinase system, and atractylate had qualitatively similar but much less pronounced effects on oxaloacetate synthesis from pyruvate. Within the limits of variation observed in these experiments, the rate of synthesis of citrulline appears not to be affected by the matrix content of total ATP, total ADP, or by ATP/ADP. It is affected, however, by the velocity of translocation of ATP into the extramitochondrial medium. These findings suggest that carbamoyl phosphate synthase (ammonia) may be loosely associated with the mitochondrial inner membrane, and may compete for ATP with the ATP-ADP translocator to an extent determined by the extramitochondrial demands for ATP.     

Similar mitochondrial activation kinetics in wild-type and creatine kinase-deficient fast-twitch muscle indicate significant Pi control of respiration

Past simulations of oxidative ATP metabolism in skeletal muscle have predicted that elimination of the creatine kinase (CK) reaction should result in dramatically faster oxygen consumption dynamics during transitions in ATP turnover rate. This hypothesis was investigated. Oxygen consumption of fast-twitch (FT) muscle isolated from wild-type (WT) and transgenic mice deficient in the myoplasmic (M) and mitochondrial (Mi) CK isoforms (MiM CK(-/-)) were measured at 20°C at rest and during electrical stimulation. MiM CK(-/-) muscle oxygen consumption activation kinetics during a step change in contraction rate were 30% faster than WT (time constant 53 ± 3 vs. 69 ± 4 s, respectively; mean ± SE, n = 8 and 6, respectively). MiM CK(-/-) muscle oxygen consumption deactivation kinetics were 380% faster than WT (time constant 74 ± 4 s vs. 264 ± 4 s, respectively). Next, the experiments were simulated using a computational model of the oxidative ATP metabolic network in FT muscle featuring ADP and Pi feedback control of mitochondrial respiration (J. A. L. Jeneson, J. P. Schmitz, N. A. van den Broek, N. A. van Riel, P. A. Hilbers, K. Nicolay, J. J. Prompers. Am J Physiol Endocrinol Metab 297: E774-E784, 2009) that was reparameterized for 20°C. Elimination of Pi control via clamping of the mitochondrial Pi concentration at 10 mM reproduced past simulation results of dramatically faster kinetics in CK(-/-) muscle, while inclusion of Pi control qualitatively explained the experimental observations. On this basis, it was concluded that previous studies of the CK-deficient FT muscle phenotype underestimated the contribution of Pi to mitochondrial respiratory control.     

5'-p-Fluorosulfonylbenzoyl adenosine inhibits progesterone synthesis in human placental mitochondria

The human placental mitochondria have an ATP-diphosphohydrolase (apyrase) activity. In this paper we characterized the effect of 5'-p-fluorosulfonylbenzoyl adenosine (FSBA) on placental apyrase, and its repercussion on progesterone synthesis and oxygen consumption. Apyrase activity was inhibited by FSBA. Nucleosides tri- and diphosphates protected against FSBA inactivation, but divalent cations did not, indicating that FSBA attaches itself to an ATP-binding site of apyrase. In mitochondria, the inactivation of apyrase by FSBA was associated with inhibition of progesterone synthesis. Also, the oxygen consumption induced by ATP but not by ADP, was inhibited, clearly showing that FSBA exclusively inactivated the apyrase in human placental mitochondria. It is concluded that the apyrase activity is closely related to progesterone synthesis, probably associated with the cholesterol transport between mitochondrial membranes.     

Energy conservation in Nitrobacter

Abstract The generation of ATP and NADH in total cells of Nitrobacter was measured under aerobic and anaerobic conditions. NADH synthesis was driven by an ATP independent reaction with nitrite or nitric oxide as electron donors. The rate of NADH formation was about 200 times higher, if nitric oxide instead of nitrite served as electron donor. Approximately 2 mol nitric oxide were needed for reduction of 1 mol NAD⁺. Nitrite caused an end-product inhibition of the nitric oxide induced NADH synthesis. ATP was synthesized by NADH oxidation with oxygen and nitrate as terminal electron acceptors.     

Increased activities of antioxidant enzymes and decreased ATP concentration in cultured myoblasts with the 3243A-->G mutation in mitochondrial DNA

The MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) is most commonly caused by the 3243A-->G mutation in mitochondrial DNA, resulting in impaired mitochondrial protein synthesis and decreased activities of the respiratory chain complexes. These defects may cause a reduced capacity for ATP synthesis and an increased rate of production of reactive oxygen species. Myoblasts cultured from controls and patients carrying the 3243A-->G mutation were used to measure ATP, ADP, catalase and superoxide dismutase, which was also measured from blood samples. ATP and ADP concentrations were decreased in myoblasts with the 3243A-->G mutation, but the ATP/ADP ratio remained constant, suggesting a decrease in the adenylate pool. The superoxide dismutase and catalase activities were higher than in control cells, and superoxide dismutase activity was slightly, but not significantly higher in the blood of patients with the mutation than in controls. We conclude that impairment of mitochondrial ATP production in myoblasts carrying the 3243A-->G mutation results in adenylate catabolism, causing a decrease in the total adenylate pool. The increase in superoxide dismutase and catalase activities could be an adaptive response to increased production of reactive oxygen species due to dysfunction of the mitochondrial respiratory chain.