RESUMEN
The inflorescences of several members of the Arum lily family warm up during flowering and are able to maintain their temperature at a constant level, relatively independent of the ambient temperature. The heat is generated via a mitochondrial respiratory pathway that is distinct from the cytochrome chain and involves a cyanide-resistant alternative oxidase (AOX). In this paper we have used flux control analysis to investigate the influence of temperature on the rate of respiration through both cytochrome and alternative oxidases in mitochondria isolated from the appendices of intact thermogenic Arum maculatum inflorescences. Results are presented which indicate that at low temperatures, the dehydrogenases are almost in full control of respiration but as the temperature increases flux control shifts to the AOX. On the basis of these results a simple model of thermoregulation is presented that is applicable to all species of thermogenic plants. The model takes into account the temperature characteristics of the separate components of the plant mitochondrial respiratory chain and the control of each process. We propose that 1) in all aroid flowers AOX assumes almost complete control over respiration, 2) the temperature profile of AOX explains the reversed relationship between ambient temperature and respiration in thermoregulating Arum flowers, 3) the thermoregulation process is the same in all species and 4) variations in inflorescence temperatures can easily be explained by variations in AOX protein concentrations.
Asunto(s)
Araceae/enzimología , Flores/enzimología , Oxidorreductasas/metabolismo , Cinética , Mitocondrias/enzimología , Proteínas Mitocondriales , Proteínas de Plantas/metabolismo , TemperaturaRESUMEN
The diarylquinoline TMC207 kills Mycobacterium tuberculosis by specifically inhibiting ATP synthase. We show here that human mitochondrial ATP synthase (50% inhibitory concentration [IC(50)] of >200 microM) displayed more than 20,000-fold lower sensitivity for TMC207 compared to that of mycobacterial ATP synthase (IC(50) of 10 nM). Also, oxygen consumption in mouse liver and bovine heart mitochondria showed very low sensitivity for TMC207. These results suggest that TMC207 may not elicit ATP synthesis-related toxicity in mammalian cells. ATP synthase, although highly conserved between prokaryotes and eukaryotes, may still qualify as an attractive antibiotic target.
Asunto(s)
Antituberculosos/farmacología , Células Eucariotas/enzimología , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Mycobacterium tuberculosis/efectos de los fármacos , Quinolinas/farmacología , Animales , Bovinos , Línea Celular , Línea Celular Tumoral , Diarilquinolinas , Relación Dosis-Respuesta a Droga , Humanos , Concentración 50 Inhibidora , Ratones , Mitocondrias Cardíacas/metabolismo , Mitocondrias Hepáticas/metabolismo , ATPasas de Translocación de Protón Mitocondriales/genética , ATPasas de Translocación de Protón Mitocondriales/aislamiento & purificación , Consumo de Oxígeno/efectos de los fármacos , Sensibilidad y EspecificidadRESUMEN
We proposed that inhibition of mitochondrial adenine nucleotide translocator (ANT) by long chain acyl-CoA (LCAC) underlies the mechanism associating obesity and type 2 diabetes. Here we test that after long-term exposure to a high-fat diet (HFD): (i) there is no adaptation of the mitochondrial compartment that would hinder such ANT inhibition, and (ii) ANT has significant control of the relevant aspects of oxidative phosphorylation. After 7 weeks, HFD induced a 24+/-6% increase in hepatic LCAC concentration and accumulation of the oxidative stress marker N(epsilon)-(carboxymethyl)lysine. HFD did not significantly affect mitochondrial copy number, oxygen uptake, membrane potential (Deltapsi), ADP/O ratio, and the content of coenzyme Q(9), cytochromes b and a+a(3). Modular kinetic analysis showed that the kinetics of substrate oxidation, phosphorylation, proton leak, ATP-production and ATP-consumption were not influenced significantly. After HFD-feeding ANT exerted considerable control over oxygen uptake (control coefficient C=0.14) and phosphorylation fluxes (C=0.15), extra- (C=0.23) and intramitochondrial (C=-0.56) ATP/ADP ratios, and Deltapsi (C=-0.11). We conclude that although HFD induces accumulation of LCAC and N(epsilon)-(carboxymethyl)lysine, oxidative phosphorylation does not adapt to these metabolic challenges. Furthermore, ANT retains control of fluxes and intermediates, making inhibition of this enzyme a more probable link between obesity and type 2 diabetes.
Asunto(s)
Translocador 3 del Nucleótido Adenina/metabolismo , Grasas de la Dieta/administración & dosificación , Intolerancia a la Glucosa/etiología , Hígado/metabolismo , Mitocondrias Hepáticas/metabolismo , Fosforilación Oxidativa , Adenosina Trifosfato/metabolismo , Animales , Dieta , Intolerancia a la Glucosa/metabolismo , Hígado/química , Lisina/análogos & derivados , Lisina/análisis , Lisina/metabolismo , Estrés Oxidativo , RatasRESUMEN
OBJECTIVE: Reactive oxygen species (ROS) have been implicated in the progression of ventricular hypertrophy to congestive heart failure. However, the source of increased oxidative stress in cardiomyocytes remains unclear. METHODS: Here we examined NADPH oxidase and mitochondria as sources of ventricular ROS production in a rat model of right-ventricular (RV) failure (CHF) induced by pulmonary arterial hypertension (PAH). RESULTS: Western analysis showed increased expression of the catalytic subunit gp91(phox) of NADPH oxidase as well as its activator Rac1 in RV in CHF compared to non-failing myocardium (CON). In addition, analysis of mitochondrial respiratory chain complexes showed a selective increase in the expression of Complex II subunit B. Using lucigenin chemiluminescence, tissue homogenates showed increased NADPH oxidase and Complex II-dependent ROS production in failing RV, with no increase in the left ventricle. Functional analyses of isolated RV mitochondria showed an increase in Complex II activity as well as Complex II-associated ROS production in CHF vs CON. An increase in the reduction state of the mitochondrial Coenzyme Q in failing RV, together with increased expression of hypoxia-inducible factor 1 alpha, indicated conditions in CHF that strongly favor ROS production by mitochondria. Reduced ROS-scavenging capacity was indicated by decreased mRNA levels of superoxide dismutases. Oxidative stress in failing RV was indicated by a two-fold increase in the level of phospho-p38 mitogen-activated protein kinase and by immunohistochemical evidence of extensive protein nitration. CONCLUSIONS: These data show that the development of PAH-induced RV heart failure is associated with an increased capacity for ROS production by NADPH oxidase as well as mitochondria. The selective increase in expression and activity of mitochondrial Complex II may be particularly important for ventricular ROS production in heart failure.
Asunto(s)
Complejo II de Transporte de Electrones/metabolismo , Mitocondrias Cardíacas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Disfunción Ventricular Derecha/metabolismo , Animales , Biomarcadores/análisis , Fenómenos Biomecánicos , Ventrículos Cardíacos , Subunidad alfa del Factor 1 Inducible por Hipoxia/análisis , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Luminiscencia , Masculino , Glicoproteínas de Membrana/análisis , Microscopía Fluorescente , Mitocondrias Cardíacas/ultraestructura , Monocrotalina , Miocardio/metabolismo , Miocardio/ultraestructura , NADPH Oxidasa 2 , NADPH Oxidasas/análisis , NADPH Oxidasas/metabolismo , Estrés Oxidativo , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ubiquinona/metabolismo , Disfunción Ventricular Derecha/patología , Proteína de Unión al GTP rac1/análisisRESUMEN
Many organisms have several similar transporters with different affinities for the same substrate. Typically, high-affinity transporters are expressed when substrate is scarce and low-affinity ones when it is abundant. The benefit of using low instead of high-affinity transporters remains unclear, especially when additional nutrient sensors are present. Here, we investigate two hypotheses. It was previously hypothesized that there is a trade-off between the affinity and the catalytic efficiency of transporters, and we find some but no definitive support for it. Additionally, we propose that for uptake by facilitated diffusion, at saturating substrate concentrations, lowering the affinity enhances the net uptake rate by reducing substrate efflux. As a consequence, there exists an optimal, external-substrate-concentration dependent transporter affinity. A computational model of Saccharomyces cerevisiae glycolysis shows that using the low affinity HXT3 transporter instead of the high affinity HXT6 enhances the steady-state flux by 36%. We tried to test this hypothesis with yeast strains expressing a single glucose transporter modified to have either a high or a low affinity. However, due to the intimate link between glucose perception and metabolism, direct experimental proof for this hypothesis remained inconclusive. Still, our theoretical results provide a novel reason for the presence of low-affinity transport systems.
Asunto(s)
Proteínas de Transporte de Membrana/metabolismo , Transporte Biológico , Difusión , Cinética , Modelos Biológicos , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
To test whether long-chain fatty acyl-CoA esters link obesity with type 2 diabetes through inhibition of the mitochondrial adenine nucleotide translocator, we applied a system-biology approach, dual modular kinetic analysis, with mitochondrial membrane potential (Deltapsi) and the fraction of matrix ATP as intermediates. We found that 5 mumol/l palmitoyl-CoA inhibited adenine nucleotide translocator, without direct effect on other components of oxidative phosphorylation. Indirect effects depended on how oxidative phosphorylation was regulated. When the electron donor and phosphate acceptor were in excess, and the mitochondrial "work" flux was allowed to vary, palmitoyl-CoA decreased phosphorylation flux by 38% and the fraction of ATP in the medium by 39%. Deltapsi increased by 15 mV, and the fraction of matrix ATP increased by 46%. Palmitoyl-CoA had a stronger effect when the flux through the mitochondrial electron transfer chain was maintained constant: Deltapsi increased by 27 mV, and the fraction of matrix ATP increased 2.6 times. When oxidative phosphorylation flux was kept constant by adjusting the rate using hexokinase, Deltapsi and the fraction of ATP were not affected. Palmitoyl-CoA increased the extramitochondrial AMP concentration significantly. The effects of palmitoyl-CoA in our model system support the proposed mechanism linking obesity and type 2 diabetes through an effect on adenine nucleotide translocator.
Asunto(s)
Mitocondrias Hepáticas/metabolismo , Translocasas Mitocondriales de ADP y ATP/fisiología , Fosforilación Oxidativa , Palmitoil Coenzima A/fisiología , Animales , Técnicas In Vitro , Cinética , Masculino , Potenciales de la Membrana/fisiología , RatasRESUMEN
Impaired mitochondrial function contributes to copper- and cadmium-induced cellular dysfunction. In this study, we used modular kinetic analysis and metabolic control analysis to assess how Cd(2+) and Cu(2+) ions affect the kinetics and control of oxidative phosphorylation in isolated rat liver mitochondria. For the analysis, the system was modularized in two ways: (a) respiratory chain, phosphorylation and proton leak; and (b) coenzyme Q reduction and oxidation, with the membrane potential (Delta psi) and fraction of reduced coenzyme Q as the connecting intermediate, respectively. Modular kinetic analysis results indicate that both Cd(2+) and Cu(2+) ions inhibited the respiratory chain downstream of coenzyme Q. Moreover, Cu(2+), but not Cd(2+) ions stimulated proton leak kinetics at high Delta psi values. Further analysis showed that this difference can be explained by Cu(2+) ion-induced production of reactive oxygen species and membrane lipid peroxidation. In agreement with modular kinetic analysis data, metabolic control analysis showed that Cd(2+) and Cu(2+) ions increased control of the respiratory and phosphorylation flux by the respiratory chain module (mainly because of an increase in the control exerted by cytochrome bc(1) and cytochrome c oxidase), decreased control by the phosphorylation module and increased negative control of the phosphorylation flux by the proton leak module. In summary, we showed that there is a subtle difference in the mode of action of Cd(2+) and Cu(2+) ions on the mitochondrial function, which is related to the ability of Cu(2+) ions to induce reactive oxygen species production and lipid peroxidation.
Asunto(s)
Cadmio/metabolismo , Cobre/metabolismo , Iones/metabolismo , Mitocondrias Hepáticas/metabolismo , Fosforilación Oxidativa , Animales , Cadmio/química , Cobre/química , Transporte de Electrón/fisiología , Peróxido de Hidrógeno/metabolismo , Iones/química , Peroxidación de Lípido , Masculino , Oxidantes/metabolismo , Oxidación-Reducción , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Succinato Deshidrogenasa/metabolismo , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , Ubiquinona/metabolismoRESUMEN
The response of mitochondrial oxygen consumption to ADP in saponin-skinned cardiac fibre bundles has an apparent Km an order of magnitude higher than that in isolated mitochondria. Here we report that incubating skinned cardiac fibre bundles from wild-type mice or double-knockout mice lacking both cytosolic and mitochondrial creatine kinase (CK) with CK and creatine or with yeast hexokinase and glucose as extramitochondrial ADP-producing systems decreases the apparent Km of the bundles for ADP severalfold. We conclude that the affinity of mitochondria for ADP in mouse heart is of the same order of magnitude as that of isolated mitochondria, while the high apparent Km of the bundles is caused by diffusion gradients outside the mitochondria.
Asunto(s)
Adenosina Difosfato/fisiología , Membranas Intracelulares/fisiología , Mitocondrias Cardíacas/metabolismo , Miocardio/metabolismo , Animales , Creatina Quinasa/deficiencia , Creatina Quinasa/genética , Forma Mitocondrial de la Creatina-Quinasa , Difusión , Relación Dosis-Respuesta a Droga , Membranas Intracelulares/enzimología , Membranas Intracelulares/metabolismo , Isoenzimas/deficiencia , Isoenzimas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mitocondrias Cardíacas/enzimología , Mitocondrias Cardíacas/genéticaRESUMEN
Mitochondria in saponin-skinned cardiac fiber bundles were reported to have an order of magnitude lower apparent affinity to ADP than isolated mitochondria. Although ADP was measured outside the bundles, it was thought that the low affinity was not caused by diffusion gradients because of relatively short diffusion distances. Here we test the hypothesis that considerable ADP diffusion gradients exist and can be diminished by increasing the intrafiber ADP production rate. We increased the ADP-producing activity in rat heart skinned fiber bundles by incubating with 100 IU/ml yeast hexokinase and glucose. Consequently, we observed a significant decrease of the apparent Michaelis constant (K(m)) to ADP of the respiration rate of bundles from 216 +/- 59 to 50 +/- 9 microM. Fitting the results with a mathematical model, we estimated the K(m) of mitochondria in the bundles to be 25 microM. We conclude that the affinity to ADP of in situ mitochondria in heart is of the same order of magnitude as that of isolated mitochondria.