RESUMEN
In this last article of the trilogy, the unified biothermokinetic theory of ATP synthesis developed in the previous two papers is applied to a major problem in comparative physiology, biochemistry, and ecology-that of metabolic scaling as a function of body mass across species. A clear distinction is made between intraspecific and interspecific relationships in energy metabolism, clearing up confusion that had existed from the very beginning since Kleiber first proposed his mouse-to-elephant rule almost a century ago. It is shown that the overall mass exponent of basal/standard metabolic rate in the allometric relationship [Formula: see text] is composed of two parts, one emerging from the relative intraspecific constancy of the slope (b), and the other (b') arising from the interspecific variation of the mass coefficient, a(M) with body size. Quantitative analysis is shown to reveal the hidden underlying relationship followed by the interspecific mass coefficient, a(M)=P0M0.10, and a universal value of P0=3.23 watts, W is derived from empirical data on mammals from mouse to cattle. The above relationship is shown to be understood only within an evolutionary biological context, and provides a physiological explanation for Cope's rule. The analysis also helps in fundamentally understanding how variability and a diversity of scaling exponents arises in allometric relations in biology and ecology. Next, a molecular-level understanding of the scaling of metabolism across mammalian species is shown to be obtained by consideration of the thermodynamic efficiency of ATP synthesis η, taking mitochondrial proton leak as a major determinant of basal metabolic rate in biosystems. An iterative solution is obtained by solving the mathematical equations of the biothermokinetic ATP theory, and the key thermodynamic parameters, e.g. the degree of coupling q, the operative P/O ratio, and the metabolic efficiency of ATP synthesis η are quantitatively evaluated for mammals from rat to cattle. Increases in η (by â¼15%) over a 2000-fold body size range from rat to cattle, primarily arising from an â¼3-fold decrease in the mitochondrial H+ leak rate are quantified by the unified ATP theory. Biochemical and mechanistic consequences for the interpretation of basal metabolism, and the various molecular implications arising are discussed in detail. The results are extended to maximum metabolic rate, and interpreted mathematically as a limiting case of the general ATP theory. The limitations of the analysis are pointed out. In sum, a comprehensive quantitative analysis based on the unified biothermokinetic theory of ATP synthesis is shown to solve a central problem in biology, physiology, and ecology on the scaling of energy metabolism with body size.
Asunto(s)
Adenosina Trifosfato , Metabolismo Energético , Mamíferos , Mitocondrias , Termodinámica , Animales , Adenosina Trifosfato/metabolismo , Metabolismo Energético/fisiología , Mitocondrias/metabolismo , Mamíferos/metabolismo , Especificidad de la Especie , Ratones , Tamaño Corporal/fisiología , Modelos Biológicos , BovinosRESUMEN
The nonequilibrium coupled processes of oxidation and ATP synthesis in the fundamental process of oxidative phosphorylation (OXPHOS) are of vital importance in biosystems. These coupled chemical reaction and transport bioenergetic processes using the OXPHOS pathway meet >90% of the ATP demand in aerobic systems. On the basis of experimentally determined thermodynamic OXPHOS flux-force relationships and biochemical data for the ternary system of oxidation, ion transport, and ATP synthesis, the Onsager phenomenological coefficients have been computed, including an estimate of error. A new biothermokinetic theory of energy coupling has been formulated and on its basis the thermodynamic parameters, such as the overall degree of coupling, q and the phenomenological stoichiometry, Z of the coupled system have been evaluated. The amount of ATP produced per oxygen consumed, i.e. the actual, operating P/O ratio in the biosystem, the thermodynamic efficiency of the coupled reactions, η, and the Gibbs free energy dissipation, Φ have been calculated and shown to be in agreement with experimental data. At the concentration gradients of ADP and ATP prevailing under state 3 physiological conditions of OXPHOS that yield Vmax rates of ATP synthesis, a maximum in Φ of â¼0.5J(hmgprotein)-1, corresponding to a thermodynamic efficiency of â¼60% for oxidation on succinate, has been obtained. Novel mechanistic insights arising from the above have been discussed. This is the first report of a 3 × 3 system of coupled chemical reactions with transport in a biological context in which the phenomenological coefficients have been evaluated from experimental data.
Asunto(s)
Adenosina Trifosfato , Metabolismo Energético , Fosforilación Oxidativa , Termodinámica , Adenosina Trifosfato/metabolismo , Metabolismo Energético/fisiología , Oxidación-Reducción , Modelos Biológicos , Cinética , Adenosina Difosfato/metabolismo , HumanosRESUMEN
AIMS: To establish a FOF1-ATP synthase molecular motor biosensor to accurately identify colon cancer miRNAs. MAIN METHODS: The FOF1-ATP synthase molecular motor is extracted by fragmentation-centrifugation and connected to the colon cancer-specific miR-17 capture probe in the manner of the ε subunit-biotin-streptavidin-biotin system. Signal probes are designed for dual-signal characterization to increase detection accuracy. The FOF1-ATPase rotation rate decreases when the signaling and capture probes are combined with the target miRNA, resulting in a decrease in ATP synthesis. miR-17 concentrations are determined by changes in ATP-mediated chemiluminescence intensity and signal probe-mediated OD450nm. KEY FINDINGS: The chemiluminescence intensity and OD450nm show a good linear relationship with the miR-17 concentration in the range of 5 to 200 nmol L-1 (R2 = 0.9985, 0.9989). The colon cancer mouse model is established for the blood samples, and miR-17 in serum and RNA extracts is quantitatively determined using the constructed sensor. SIGNIFICANCE: The results are consistent with colon cancer progression, and the low concentration of miR-17 detecting accuracy is comparable to the PCR assay. In conclusion, the developed method is a direct, rapid, and promising method for miRNA detection of colon cancer.
Asunto(s)
Técnicas Biosensibles , Neoplasias del Colon , MicroARNs , Animales , Ratones , Adenosina Trifosfato , Biotina , Neoplasias del Colon/diagnóstico , Neoplasias del Colon/genética , MicroARNs/genética , Óxido Nítrico Sintasa , ATPasas de Translocación de ProtónRESUMEN
Tumor microenvironment (TME) plays an important role in the growth, invasion, and metastasis of tumor cells. The pH of TME is more acidic in solid tumors than in normal tissues. Although targeted delivery in TME has progressed, the complex and expensive construction of delivery systems has limited their application. FOF1-ATP synthase (FOF1-ATPase) is a rotation molecular motor found in bacteria, chloroplasts, and mitochondria. Here, FOF1-ATPase loaded chromatophores (chroma) isolated from thermophilic bacteria were extracted and utilized as a new delivery system targeting TME for the first time. Curcumin as model drug was successfully loaded by a filming-rehydration ultrasonic dispersion method to prepare a curcumin-loaded chroma delivery system (Cur-Chroma). The mobility and propensity distributions of Cur-Chroma reveal its specific pH-sensitive targeting driven by the transmembrane proton kinetic potential, demonstrating its distinct distribution in the TME and more favorable targeting delivery. Cellular uptake experiments indicated that Cur-Chroma entered cells through grid pathway-mediated endocytosis. In vivo studies have shown that Cur-Chroma can specifically target tumor tissue and effectively inhibit tumor growth with good safety. Curcumin's bioavailability and anti-tumor effects were significantly improved. These studies demonstrate that ATPase-loaded chromatophores are potentially ideal vehicles for anti-tumor drug delivery and have promising applications.
Asunto(s)
Antineoplásicos , Cromatóforos , Curcumina , Nanopartículas , Neoplasias , Humanos , Curcumina/química , Portadores de Fármacos/química , Microambiente Tumoral , Antineoplásicos/química , Neoplasias/tratamiento farmacológico , ATPasas de Translocación de Protón , Sistemas de Liberación de Medicamentos/métodos , Nanopartículas/químicaRESUMEN
The mitochondrial respiratory chain is an essential pathway in most studied eukaryotes due to its roles in respiration and other pathways that depend on mitochondrial membrane potential. Apicomplexans are unicellular eukaryotes whose members have an impact on global health. The respiratory chain is a drug target for some members of this group, notably the malaria-causing Plasmodium spp. This has motivated studies of the respiratory chain in apicomplexan parasites, primarily Toxoplasma gondii and Plasmodium spp. for which experimental tools are most advanced. Studies of the respiratory complexes in these organisms revealed numerous novel features, including expansion of complex size. The divergence of apicomplexan mitochondria from commonly studied models highlights the diversity of mitochondrial form and function across eukaryotic life.
Asunto(s)
Apicomplexa , Malaria , Plasmodium , Toxoplasma , Humanos , Transporte de Electrón , Mitocondrias/metabolismo , Plasmodium/metabolismo , Malaria/parasitología , Apicomplexa/metabolismoRESUMEN
Mechanisms coupling the chemical reactions of oxidation and ATP synthesis in cellular metabolism by the fundamental biological process of oxidative phosphorylation (OX PHOS) in mitochondria provide > 90% of the energy requirements in living organisms. Mathematical graph theory methods have been extensively used to characterize various metabolic, regulatory, and disease networks in biology. However, networks of energy coupling mechanisms in OX PHOS have not been represented and analyzed previously by these approaches. Here, the problem of biological energy coupling is translated into a graph-theoretical framework, and all possible coupling schemes between oxidation and ATP synthesis are represented as graphs connecting these processes by various intermediates or states. The problem is shown to be transformed into the hard problem of finding a Hamiltonian tour in the networks of possible constituent mechanisms, given the constraints of a cyclical nature of operation of enzymes and biological molecular machines. Accessible mathematical proofs of three theorems that guarantee sufficient conditions for the existence of a Hamiltonian cycle in simple graphs are provided. The results of the general theorems are applied to the set of possible coupling mechanisms in OX PHOS and shown to (1) unequivocally differentiate between the major theories and mechanisms of energy coupling, (2) greatly reduce the possibilities for detailed consideration, and (3) deduce the biologically selected mechanism using additional constraints from the cumulative experimental record. Finally, an algorithm is constructed to implement the graph-theoretical procedure. In summary, the enormous power and generality of mathematical theorems and approaches in graph theory are shown to help solve a fundamental problem in biology.
Asunto(s)
Adenosina Trifosfato , Fosforilación Oxidativa , Adenosina Trifosfato/química , Metabolismo Energético , Mitocondrias/metabolismo , Fenómenos Físicos , TermodinámicaRESUMEN
Proton translocation through the Fo fraction of FoF1-ATP synthase is one of the crucial processes in the catalytic cycle of the enzyme. However, the exact trace of protons movement has not been finally established yet because the location and structure of the half-channels are still the subject of investigation. We described the possible network of polar amino acids residues and water molecules that can favor the preferential proton pathway using molecular dynamics simulation of the membrane part of the E. coli ATP synthase embedded in the lipid bilayer and water environment. The inlet half-channel was a complex structure with two entrances in the form of aqueous lacunae and a highly conservative proton transfer chain near Asp61 of c-subunit including amino acids residues and three structural water molecules (W1-W3), while the outlet half-channel was just a water cavity through which a proton can easily move into the cytoplasm. Moreover, the side chains of Asn214 and Gln252 of a-subunit had the stable spatial positions (SP1-SP3). аAsn214 in position SP3 and аGln252 in SP1, SP2 were oriented towards cAsp61 and could presumably protonate it via W1. Herewith aAsn214 in SP1, SP2 was oriented to aHis245. Thus, the proton transfer chain is always unclosed, and switching between positions SP1/SP2 and SP3 of aAsn214 determines the time of proton transport and the movement in this region is the rate-limiting step. In addition, we found another rare position SP3, in which aGln252 is oriented to aAsn116 and aSer144, located outside of the "main H+ route" and being a dead end. The new findings would help to evaluate the whole process of the proton translocation through FoF1-ATP synthase.
Asunto(s)
Adenosina Trifosfato/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismo , ATPasas de Translocación de Protón/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Escherichia coli/genética , Transporte Iónico , Membrana Dobles de Lípidos/metabolismo , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica , Protones , AguaRESUMEN
Charge transfer across membranes is an important problem in a wide variety of fundamental physicochemical and biological processes. Since Mitchell's concept of the ion well advanced in 1968, several models of ion translocation across biomembranes, for instance through the membrane-bound FO portion of ATP synthase have been proposed. None of these models has considered the large desolvation free energy penalty of ~500 meV incurred in transferring a protonic charge from the aqueous phase into the membrane that hinders such charge transfer processes. The difficulty has been pointed out repeatedly. However, the problem of how the adverse ∆Gdesolvation barrier is overcome in order to enable rapid ion translocation in biomembranes has not been satisfactorily resolved. Hence the fact that the self-energy of the charges has been overlooked can be regarded as a main source of confusion in the field of bioenergetics. Further, in order to consider charges of a finite size (and not just point charges), the free energy of transferring the ions from water into a membrane phase of lower dielectric εm needs to be evaluated. Here a solution to the longstanding conundrum has been proposed by including the bound anion - the second ion in Nath's two-ion theory of energy coupling and ATP synthesis - in the free energy calculations. The mechanistic importance of the H+ - A- charge pair in causing rotation and ATP synthesis by ion-protein interactions is highlighted. The ∆G calculations have been performed by using the Kirkwood-Tanford-Warshel (KTW) theory that takes into account the self-energies of the ions. The results show that the adverse ∆Gdesolvation can be almost exactly compensated by the sum of the electrostatic free energy of the charge-charge interactions and the dipole solvation energy for long-range ion pairs. Results of free energy compensation using the KTW theory have been compared with experimental data on the ∆G of ion pairs and shown to be in reasonable agreement. A general thermodynamic cycle for coupled ion transfer has been constructed to further elucidate facilitated ion permeation between water and membrane phases. Molecular interpretations of the results and their implications for various mechanisms of energy transduction have been discussed. We firmly believe that use of electrostatic theories such as the KTW theory that properly include the desolvation free energy penalty arising from the self-energy of the relevant ions are crucial for quantifying charge transfer processes in bioenergetics. Finally, the clear-cut implication is that proton-only and single-ion theories of ATP synthesis, such as the chemiosmotic theory, are grossly inadequate to comprehend energy storage and transduction in biological processes.
Asunto(s)
ATPasas de Translocación de Protón Mitocondriales , Concentración de Iones de Hidrógeno , Fosforilación Oxidativa , Electricidad Estática , TermodinámicaRESUMEN
In a recent paper entitled "Chemiosmotic misunderstandings", it is claimed that "enough shortcomings in Mitchell's chemiosmotic theory have not been found and that a novel paradigm that offers at least as much explanatory power as chemiosmosis is not ready." This view is refuted by a wealth of molecular-level experimental data and strong new theoretical and computational evidence. It is shown that the chemiosmotic theory was beset with a large number of major shortcomings ever since the time when it was first proposed in the 1960s. These multiple shortcomings and flaws of chemiosmosis were repeatedly pointed out in incisive critiques by biochemical authorities of the late 20th century. All the shortcomings and flaws have been shown to be rectified by a quantitative, unified molecular-level theory that leads to a deeper and far more accurate understanding of biological energy coupling and ATP synthesis. The new theory is shown to be consistent with pioneering X-ray and cryo-EM structures and validated by state-of-the-art single-molecule techniques. Several new biochemical experimental tests are proposed and constructive ways for providing a revitalizing conceptual background and theory for integration of the available experimental information are suggested.
Asunto(s)
Metabolismo Energético , Ósmosis , Adenosina Trifosfato/metabolismo , Animales , Humanos , Modelos Biológicos , Fosforilación Oxidativa , Fotosíntesis , Electricidad EstáticaRESUMEN
F-type ATP synthases are extraordinary multisubunit proteins that operate as nanomotors. The Escherichia coli (E. coli) enzyme uses the proton motive force (pmf) across the bacterial plasma membrane to drive rotation of the central rotor subunits within a stator subunit complex. Through this mechanical rotation, the rotor coordinates three nucleotide binding sites that sequentially catalyze the synthesis of ATP. Moreover, the enzyme can hydrolyze ATP to turn the rotor in the opposite direction and generate pmf. The direction of net catalysis, i.e. synthesis or hydrolysis of ATP, depends on the cell's bioenergetic conditions. Different control mechanisms have been found for ATP synthases in mitochondria, chloroplasts and bacteria. This review discusses the auto-inhibitory behavior of subunit ε found in FOF1-ATP synthases of many bacteria. We focus on E. coli FOF1-ATP synthase, with insights into the regulatory mechanism of subunit ε arising from structural and biochemical studies complemented by single-molecule microscopy experiments.
Asunto(s)
Adenosina Trifosfato/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , ATPasas de Translocación de Protón/metabolismo , Metabolismo Energético , Subunidades de Proteína/metabolismoRESUMEN
F1 is a soluble part of FoF1-ATP synthase and performs a catalytic process of ATP hydrolysis and synthesis. The γ subunit, which is the rotary shaft of F1 motor, is composed of N-terminal and C-terminal helices domains, and a protruding Rossman-fold domain located between the two major helices parts. The N-terminal and C-terminal helices domains of γ assemble into an antiparallel coiled-coil structure, and are almost embedded into the stator ring composed of α3ß3 hexamer of the F1 molecule. Cyanobacterial and chloroplast γ subunits harbor an inserted sequence of 30 or 39 amino acids length within the Rossman-fold domain in comparison with bacterial or mitochondrial γ. To understand the structure-function relationship of the γ subunit, we prepared a mutant F1-ATP synthase of a thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1, in which the γ subunit is split into N-terminal α-helix along with the inserted sequence and the remaining C-terminal part. The obtained mutant showed higher ATP-hydrolysis activities than those containing the wild-type γ. Contrary to our expectation, the complexes containing the split γ subunits were mostly devoid of the C-terminal helix. We further investigated the effect of post-assembly cleavage of the γ subunit. We demonstrate that insertion of the nick between two helices of the γ subunit imparts resistance to ADP inhibition, and the C-terminal α-helix is dispensable for ATP-hydrolysis activity and plays a crucial role in the assembly of F1-ATP synthase.
Asunto(s)
Adenosina Trifosfato/química , Proteínas Bacterianas/química , Cianobacterias/enzimología , ATPasas de Translocación de Protón/química , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cianobacterias/genética , Dominios Proteicos , Estructura Secundaria de Proteína , ATPasas de Translocación de Protón/genética , ATPasas de Translocación de Protón/metabolismo , Eliminación de SecuenciaRESUMEN
The core of F1-ATPase consists of three catalytic (ß) and three noncatalytic (α) subunits, forming a hexameric ring in alternating positions. A wealth of experimental and theoretical data has provided a detailed picture of the complex role played by catalytic subunits. Although major conformational changes have only been seen in ß-subunits, it is clear that α-subunits have to respond to these changes in order to be able to transmit information during the rotary mechanism. However, the conformational behavior of α-subunits has not been explored in detail. Here, we have combined unbiased molecular dynamics (MD) simulations and calorimetrically measured thermodynamic signatures to investigate the conformational flexibility of isolated α-subunits, as a step toward deepening our understanding of its function inside the α3ß3 ring. The simulations indicate that the open-to-closed conformational transition of the α-subunit is essentially barrierless, which is ideal to accompany and transmit the movement of the catalytic subunits. Calorimetric measurements of the recombinant α-subunit from Geobacillus kaustophilus indicate that the isolated subunit undergoes no significant conformational changes upon nucleotide binding. Simulations confirm that the nucleotide-free and nucleotide-bound subunits show average conformations similar to that observed in the F1 crystal structure, but they reveal an increased conformational flexibility of the isolated α-subunit upon MgATP binding, which might explain the evolutionary conserved capacity of α-subunits to recognize nucleotides with considerable strength. Furthermore, we elucidate the different dependencies that α- and ß-subunits show on Mg(II) for recognizing ATP.
Asunto(s)
ATPasas de Translocación de Protón/química , Calorimetría , Simulación de Dinámica Molecular , Conformación Proteica , Subunidades de Proteína/química , TermodinámicaRESUMEN
Over the past 30years the mitochondrial permeability transition - the permeabilization of the inner mitochondrial membrane due to the opening of a wide pore - has progressed from being considered a curious artifact induced in isolated mitochondria by Ca(2+) and phosphate to a key cell-death-inducing process in several major pathologies. Its relevance is by now universally acknowledged and a pharmacology targeting the phenomenon is being developed. The molecular nature of the pore remains to this day uncertain, but progress has recently been made with the identification of the FOF1 ATP synthase as the probable proteic substrate. Researchers sharing this conviction are however divided into two camps: these believing that only the ATP synthase dimers or oligomers can form the pore, presumably in the contact region between monomers, and those who consider that the ring-forming c subunits in the FO sector actually constitute the walls of the pore. The latest development is the emergence of a new candidate: Spastic Paraplegia 7 (SPG7), a mitochondrial AAA-type membrane protease which forms a 6-stave barrel. This review summarizes recent developments of research on the pathophysiological relevance and on the molecular nature of the mitochondrial permeability transition pore. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou.
Asunto(s)
Proteínas de Transporte de Membrana Mitocondrial/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfato/metabolismo , Animales , Peptidil-Prolil Isomerasa F , Ciclofilinas/metabolismo , Dimerización , Humanos , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/efectos de los fármacos , Poro de Transición de la Permeabilidad Mitocondrial , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Enfermedades Neurodegenerativas/metabolismo , ATPasas de Translocación de Protón/química , ATPasas de Translocación de Protón/metabolismo , Paraplejía Espástica Hereditaria/genética , Paraplejía Espástica Hereditaria/metabolismoRESUMEN
Exosomes are 40-100-nm vesicles released by most cell types after fusion of multivesicular endosomes with the plasma membrane. Exosomes, ubiquitary in body fluids including urines, contain proteins and RNA species specific of the tissue of origin. Exosomes from urine have been extensively studied as a promising reservoir for disease biomarkers. Here, we report the proteome analysis of urinary exosomes compared to urinoma, studied by Orbitrap mass spectrometry. A discovery approach was utilized on the sample. 3429 proteins were present, with minimal overlapping among exosome and urinoma. 959 proteins (28%) in exosome and 1478 proteins (43%) in urinoma were exclusively present in only one group. By cytoscape analysis, the biological process gene ontology was correlated to their probability (P ≤ 0.05) to be functional. This was never studied before and showed a significant clustering around metabolic functions, in particular to aerobic ATP production. Urinary exosomes carry out oxidative phosphorylation, being able to synthesize ATP and consume oxygen. A previously unsuspected function emerges for human urinary exosomes as bioactive vesicles that consume oxygen to aerobically synthesize ATP. Determination of normal human urine proteome can help generate the healthy urinary protein database for comparison, useful for various renal diseases. BIOLOGICAL SIGNIFICANCE: The findings reported represent a significant advance in the understanding of the healthy human urinary proteome. The methodology utilized to analyze the collection of proteomic data allowed the assessment of the unique composition of urinary exosomes with respect to urinoma and to elucidate the presence in the former of molecular pathways previously unknown. The paper has the potential to impact its field of research, due to the biological relevance of the metabolic capacity of urinary exosomes, which may represent their important general feature.
Asunto(s)
Exosomas/metabolismo , Consumo de Oxígeno , Proteoma/metabolismo , Urinoma/orina , Adulto , Femenino , Humanos , MasculinoRESUMEN
The present study shows that in isolated mitochondria and myoblast cultures depletion of cAMP, induced by sAC inhibition, depresses both ATP synthesis and hydrolysis by the FOF1 ATP synthase (complex V) of the oxidative phosphorylation system (OXPHOS). These effects are accompanied by the decrease of the respiratory membrane potential, decreased level of FOF1 connecting subunits and depressed oligomerization of the complex. All these effects of sAC inhibition are prevented by the addition of the membrane-permeant 8-Br-cAMP. These results show, for the first time, that cAMP promotes ATP production by complex V and prevents, at the same time, its detour to a mitochondrial membrane leak conductance, which is involved in cell death.
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Adenosina Trifosfatasas/metabolismo , Proteínas Portadoras/metabolismo , AMP Cíclico/fisiología , Proteínas de la Membrana/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfato/biosíntesis , Adenilil Ciclasas/fisiología , Animales , Proteínas Portadoras/química , Células Cultivadas , Potencial de la Membrana Mitocondrial , Proteínas de la Membrana/química , ATPasas de Translocación de Protón Mitocondriales , Mioblastos/metabolismo , Fosforilación Oxidativa , RatasRESUMEN
F1-ATPase (F1), an important rotary motor protein, converts the chemical energy of ATP hydrolysis into mechanical energy using rotary motion with extremely high efficiency. The energy-conversion mechanism for this molecular motor has been extensively clarified by previous studies, which indicate that the interactions between the catalytic residues and the ß- and γ-phosphates of ATP are indispensable for efficient catalysis and torque generation. However, the role of α-phosphate is largely unknown. In this study, we observed the rotation of F1 fuelled with an ATP analogue, adenosine 5'-[α-thio]-triphosphate (ATPαS), in which the oxygen has been substituted with a sulfur ion to perturb the α-phosphate/F1 interactions. In doing so, we have revealed that ATPαS does not appear to have any impact on the kinetic properties of the motor or on torque generation compared to ATP. On the other hand, F1 was observed to lapse into the ADP-inhibited intermediate states when in the presence of ATPαS more severely than in the presence of ATP, suggesting that the α-phosphate group of ATP contributes to the avoidance of ADP-inhibited intermediate formation.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , ATPasas de Translocación de Protón/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Bovinos , Hidrólisis , Cinética , Modelos Moleculares , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/metabolismoRESUMEN
The time course of ATP synthesis, oxygen consumption, and change in the membrane potential in Paracoccus denitrificans inside-out plasma membrane vesicles was traced. ATP synthesis initiated by the addition of a limited amount of either ADP or inorganic phosphate proceeded up to very low residual concentrations of the limiting substrate. Accumulated ATP did not decrease the rate of its synthesis initiated by the addition of ADP. The amount of residual ADP determined at State 4 respiration was independent of ten-fold variation of Pi or the presence of ATP. The pH-dependence of Km for Pi could not be fitted to a simple phosphoric acid dissociation curve. Partial inhibition of respiration resulted in a decrease in the rate of ATP synthesis without affecting the ATP/ADP reached at State 4. At pH8.0, hydrolysis of ATP accumulated at State 4 was induced by a low concentration of an uncoupler, whereas complete uncoupling results in rapid inactivation of ATPase. At pH7.0, no reversal of the ATP synthase reaction by the uncoupler was seen. The data show that ATP/ADP×Pi ratio maintained at State 4 is not in equilibrium with respiratory-generated driving force. Possible mechanisms of kinetic control and unidirectional operation of the Fo·F1-ATP synthase are discussed.