RESUMO
Age and sex play an essential role in the cardiac tolerance to ischemia-reperfusion injury: cardiac resistance significantly decreases during postnatal maturation and the female heart is more tolerant than the male myocardium. It is widely accepted that mitochondrial dysfunction, and particularly mitochondrial permeability transition pore (MPTP) opening, plays a major role in determining the extent of cardiac ischemia-reperfusion injury. We have observed that the MPTP sensitivity to the calcium load differs in mitochondria isolated from neonatal and adult myocardium, as well as from adult male and female hearts. Neonatal and female mitochondria are more resistant both in the extent and in the rate of mitochondrial swelling induced by high calcium concentration. Our data further suggest that age- and sex-dependent specificity of the MPTP is not the result of different amounts of ATP synthase and cyclophilin D: neonatal and adult hearts, similarly as the male and female hearts, contain comparable amounts of MPTP and its regulatory protein cyclophilin D. We can speculate that the lower sensitivity of MPTP to the calcium-induced swelling may be related to the higher ischemic tolerance of both neonatal and female myocardium.
Assuntos
Coração , Mitocôndrias Cardíacas/patologia , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Caracteres Sexuais , Animais , Cálcio/metabolismo , Coração/fisiopatologia , Humanos , Mitocôndrias Cardíacas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Traumatismo por Reperfusão Miocárdica/metabolismoRESUMO
Life manifests as growth, movement or heat production that occurs thanks to the energy accepted from the outside environment. The basis of energy transduction attracted the Czech researchers since the beginning of the 20th century. It further accelerated after World War II, when the new Institute of Physiology was established in 1954. When it was found that energy is stored in the form of adenosine triphosphate (ATP) that can be used by numerous reactions as energy source and is produced in the process called oxidative phosphorylation localized in mitochondria, the investigation focused on this cellular organelle. Although the Czech scientists had to overcome various obstacles including Communist party leadership, driven by curiosity, boldness, and enthusiasm, they characterized broad spectrum of mitochondrial properties in different tissues in (patho)physiological conditions in collaboration with many world-known laboratories. The current review summarizes the contribution of the Czech scientists to the bioenergetic and mitochondrial research in the global context. Keywords: Mitochondria, Bioenergetics, Chemiosmotic coupling.
Assuntos
Metabolismo Energético , Mitocôndrias , Humanos , Animais , História do Século XX , República Tcheca , Mitocôndrias/metabolismo , História do Século XXI , Pesquisa Biomédica/história , Pesquisa Biomédica/tendênciasRESUMO
Disorders of ATP synthase, the key enzyme in mitochondrial energy supply, belong to the most severe metabolic diseases, manifesting as early-onset mitochondrial encephalo-cardiomyopathies. Since ATP synthase subunits are encoded by both mitochondrial and nuclear DNA, pathogenic variants can be found in either genome. In addition, the biogenesis of ATP synthase requires several assembly factors, some of which are also hotspots for pathogenic variants. While variants of MT-ATP6 and TMEM70 represent the most common cases of mitochondrial and nuclear DNA mutations respectively, the advent of next-generation sequencing has revealed new pathogenic variants in a number of structural genes and TMEM70, sometimes with truly peculiar genetics. Here we present a systematic review of the reported cases and discuss biochemical mechanisms, through which they are affecting ATP synthase. We explore how the knowledge of pathophysiology can improve our understanding of enzyme biogenesis and function. Keywords: Mitochondrial diseases o ATP synthase o Nuclear DNA o Mitochondrial DNA o TMEM70.
Assuntos
ATPases Mitocondriais Próton-Translocadoras , Fenótipo , Humanos , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Mutação , Doenças Mitocondriais/genética , Doenças Mitocondriais/enzimologia , DNA Mitocondrial/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Animais , Mitocôndrias/enzimologia , Mitocôndrias/genéticaRESUMO
Mitochondrial retrograde signaling is a pathway of communication from mitochondria to the nucleus. Recently, natural mitochondrial genome (mtDNA) polymorphisms (haplogroups) received increasing attention in the pathophysiology of human common diseases. However, retrograde effects of mtDNA variants on such traits are difficult to study in humans. The conplastic strains represent key animal models to elucidate regulatory roles of mtDNA haplogroups on defined nuclear genome background. To analyze the relationship between mtDNA variants and cardiometabolic traits, we derived a set of rat conplastic strains (SHR-mtBN, SHR-mtF344 and SHR-mtLEW), harboring all major mtDNA haplotypes present in common inbred strains on the nuclear background of the spontaneously hypertensive rat (SHR). The BN, F344 and LEW mtDNA differ from the SHR in multiple amino acid substitutions in protein coding genes and also in variants of tRNA and rRNA genes. Different mtDNA haplotypes were found to predispose to various sets of cardiometabolic phenotypes which provided evidence for significant retrograde effects of mtDNA in the SHR. In the future, these animals could be used to decipher individual biochemical components involved in the retrograde signaling.
Assuntos
Doenças Cardiovasculares , DNA Mitocondrial , Animais , Doenças Cardiovasculares/metabolismo , DNA Mitocondrial/genética , Mitocôndrias/metabolismo , Fenótipo , Ratos , Ratos Endogâmicos F344 , Ratos Endogâmicos SHRRESUMO
Mitochondrial disorders manifest enormous genetic and clinical heterogeneity - they can appear at any age, present with various phenotypes affecting any organ, and display any mode of inheritance. What mitochondrial diseases do have in common, is impairment of respiratory chain activity, which is responsible for more than 90% of energy production within cells. While diagnostics of mitochondrial disorders has been accelerated by introducing Next-Generation Sequencing techniques in recent years, the treatment options are still very limited. For many patients only a supportive or symptomatic therapy is available at the moment. However, decades of basic and preclinical research have uncovered potential target points and numerous compounds or interventions are now subjects of clinical trials. In this review, we focus on current and emerging therapeutic approaches towards the treatment of mitochondrial disorders. We focus on small compounds, metabolic interference, such as endurance training or ketogenic diet and also on genomic approaches.
Assuntos
Terapia Genética/métodos , Mitocôndrias/metabolismo , Doenças Mitocondriais/terapia , Animais , Transporte de Elétrons , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Mitocôndrias/patologia , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismoRESUMO
Cytochrome c oxidase (COX), the terminal enzyme of mitochondrial electron transport chain, couples electron transport to oxygen with generation of proton gradient indispensable for the production of vast majority of ATP molecules in mammalian cells. The review summarizes current knowledge of COX structure and function of nuclear-encoded COX subunits, which may modulate enzyme activity according to various conditions. Moreover, some nuclear-encoded subunits posess tissue-specific and development-specific isoforms, possibly enabling fine-tuning of COX function in individual tissues. The importance of nuclear-encoded subunits is emphasized by recently discovered pathogenic mutations in patients with severe mitopathies. In addition, proteins substoichiometrically associated with COX were found to contribute to COX activity regulation and stabilization of the respiratory supercomplexes. Based on the summarized data, a model of three levels of quaternary COX structure is postulated. Individual structural levels correspond to subunits of the i) catalytic center, ii) nuclear-encoded stoichiometric subunits and iii) associated proteins, which may constitute several forms of COX with varying composition and differentially regulated function.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/enzimologia , Doenças Mitocondriais/enzimologia , Animais , Núcleo Celular/enzimologia , Núcleo Celular/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Genoma , Humanos , Mitocôndrias/genética , Doenças Mitocondriais/patologia , Especificidade de Órgãos , Subunidades Proteicas , Transdução de SinaisRESUMO
Brown adipose tissue (BAT) plays an important role in lipid and glucose metabolism in rodents and possibly also in humans. Identification of genes responsible for BAT function would shed light on underlying pathophysiological mechanisms of metabolic disturbances. Recent linkage analysis in the BXH/HXB recombinant inbred (RI) strains, derived from Brown Norway (BN) and spontaneously hypertensive rats (SHR), identified two closely linked quantitative trait loci (QTL) associated with glucose oxidation and glucose incorporation into BAT lipids in the vicinity of Wars2 (tryptophanyl tRNA synthetase 2 (mitochondrial)) gene on chromosome 2. The SHR harbors L53F WARS2 protein variant that was associated with reduced angiogenesis and Wars2 thus represents a prominent positional candidate gene. In the current study, we validated this candidate as a quantitative trait gene (QTG) using transgenic rescue experiment. SHR-Wars2 transgenic rats with wild type Wars2 gene when compared to SHR, showed more efficient mitochondrial proteosynthesis and increased mitochondrial respiration, which was associated with increased glucose oxidation and incorporation into BAT lipids, and with reduced weight of visceral fat. Correlation analyses in RI strains showed that increased activity of BAT was associated with amelioration of insulin resistance in muscle and white adipose tissue. In summary, these results demonstrate important role of Wars2 gene in regulating BAT function and consequently lipid and glucose metabolism.
Assuntos
Tecido Adiposo Marrom/metabolismo , Metabolismo Energético , Gordura Intra-Abdominal/metabolismo , Mutação , Obesidade/genética , Triptofano-tRNA Ligase/genética , Tecido Adiposo Marrom/patologia , Animais , Células Cultivadas , Metabolismo Energético/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Glucose/metabolismo , Gordura Intra-Abdominal/fisiopatologia , Metabolismo dos Lipídeos , Masculino , Mitocôndrias/metabolismo , Obesidade/metabolismo , Obesidade/fisiopatologia , Fenótipo , Locos de Características Quantitativas , Ratos Endogâmicos SHRRESUMO
Thyroid hormones are important regulators of mitochondrial metabolism. Due to their complex mechanism of action, the timescale of different responses varies from minutes to days. In this work, we studied selective T3 induction of the inner mitochondrial membrane enzyme-glycerophosphate dehydrogenase (mGPDH) in liver of euthyroid rats. We correlated the kinetics of the T3 level in blood, the mRNA level in liver, the activity and amount of mGPDH in liver mitochondria after a single dose of T3. The T3 level reached maximum after 1 h (80 nmol/l) and subsequently rapidly decreased. mGPDH mRNA increased also relatively fast, reaching a maximum after 12 h and fell to the control level after 72 h. An increase of mGPDH activity could be already found after 6 h and reached a maximum after 24 h in accordance with the increase in mGPDH content (2.4-fold vs. 2.7-fold induction). After 72 h, the mGPDH activity showed a significant 30% decrease. When the rats received three subsequent doses of T3, the increase of mGPDH activity was 2-fold higher than after a single T3 dose. The results demonstrate that mGPDH displays rapid induction as well as decay upon disappearance of a hormonal stimulus, indicating a rather short half-life of this inner mitochondrial membrane enzyme.
Assuntos
Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glicerolfosfato Desidrogenase/biossíntese , Mitocôndrias Hepáticas/enzimologia , Tri-Iodotironina/administração & dosagem , Animais , Ativação Enzimática/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/fisiologia , Masculino , RNA Mensageiro/biossíntese , Ratos , Ratos Wistar , Tri-Iodotironina/sangueRESUMO
We present clinical and laboratory data from 14 cases with an isolated deficiency of the mitochondrial ATP synthase (7-30% of control) caused by nuclear genetic defects. A quantitative decrease of the ATP synthase complex was documented by Blue-Native electrophoresis and Western blotting and was supported by the diminished activity of oligomycin/aurovertin-sensitive ATP hydrolysis in fibroblasts (10 cases), muscle (6 of 7 cases), and liver (one case). All patients had neonatal onset and elevated plasma lactate levels. In 12 patients investigated 3-methyl-glutaconic aciduria was detected. Seven patients died, mostly within the first weeks of life and surviving patients showed psychomotor and various degrees of mental retardation. Eleven patients had hypertrophic cardiomyopathy; other clinical signs included hypotonia, hepatomegaly, facial dysmorphism and microcephaly. This phenotype markedly differs from the severe central nervous system changes of ATP synthase disorders caused by mitochondrial DNA mutations of the ATP6 gene presenting mostly as NARP and MILS.
Assuntos
Predisposição Genética para Doença/genética , Doenças Mitocondriais/enzimologia , Doenças Mitocondriais/genética , Encefalomiopatias Mitocondriais/enzimologia , Encefalomiopatias Mitocondriais/genética , ATPases Mitocondriais Próton-Translocadoras/deficiência , Trifosfato de Adenosina/metabolismo , Adolescente , Idade de Início , Cardiomiopatia Hipertrófica Familiar/enzimologia , Cardiomiopatia Hipertrófica Familiar/genética , Cardiomiopatia Hipertrófica Familiar/fisiopatologia , Núcleo Celular/genética , Criança , Pré-Escolar , Face/anormalidades , Feminino , Hepatomegalia/enzimologia , Hepatomegalia/genética , Hepatomegalia/fisiopatologia , Humanos , Lactente , Recém-Nascido , Ácido Láctico/sangue , Masculino , Microcefalia/enzimologia , Microcefalia/genética , Mitocôndrias/enzimologia , Mitocôndrias/genética , Doenças Mitocondriais/fisiopatologia , Encefalomiopatias Mitocondriais/fisiopatologia , ATPases Mitocondriais Próton-Translocadoras/genética , SíndromeRESUMO
Mitochondrial respiratory chain enzyme Complexes are present in placenta at proportion similar to other tissues with exception of glycerophosphate dehydrogenase (mGPDH) which is expressed at a very high rate. As shown by Western blot quantification and respiratory chain enzyme activity measurements, the specific content of mGPDH is similar to that of succinate dehydrogenase or NADH dehydrogenase. Using fluorometric probe dichlorodihydrofluorescein diacetate we found that placental mitochondria display high rate of glycerophosphate-dependent hydrogen peroxide production. This was confirmed by oxygraphic detection of glycerophosphate-induced, KCN- or antimycin A-insensitive oxygen uptake. Hydrogen peroxide production by mGPDH was highly activated by one-electron acceptor, potassium ferricyanide and it was depressed by inhibitors of mGPDH and by cytochrome c. Our results indicate that mGPDH should be considered as an additional source of reactive oxygen species participating in induction of oxidative stress in placenta.
Assuntos
Glicerolfosfato Desidrogenase/metabolismo , Peróxido de Hidrogênio/metabolismo , Mitocôndrias/enzimologia , Placenta/enzimologia , Animais , Cricetinae , Feminino , Humanos , Oxirredutases/metabolismo , Oxigênio/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismoRESUMO
l-asparaginase (ASNase), a key component in the treatment of childhood acute lymphoblastic leukemia (ALL), hydrolyzes plasma asparagine and glutamine and thereby disturbs metabolic homeostasis of leukemic cells. The efficacy of such therapeutic strategy will depend on the capacity of cancer cells to adapt to the metabolic challenge, which could relate to the activation of compensatory metabolic routes. Therefore, we studied the impact of ASNase on the main metabolic pathways in leukemic cells. Treating leukemic cells with ASNase increased fatty-acid oxidation (FAO) and cell respiration and inhibited glycolysis. FAO, together with the decrease in protein translation and pyrimidine synthesis, was positively regulated through inhibition of the RagB-mTORC1 pathway, whereas the effect on glycolysis was RagB-mTORC1 independent. As FAO has been suggested to have a pro-survival function in leukemic cells, we tested its contribution to cell survival following ASNase treatment. Pharmacological inhibition of FAO significantly increased the sensitivity of ALL cells to ASNase. Moreover, constitutive activation of the mammalian target of rapamycin pathway increased apoptosis in leukemic cells treated with ASNase, but did not increase FAO. Our study uncovers a novel therapeutic option based on the combination of ASNase and FAO inhibitors.
Assuntos
Asparaginase/uso terapêutico , Ácidos Graxos/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Proteínas Monoméricas de Ligação ao GTP/fisiologia , Complexos Multiproteicos/fisiologia , Oxirredução , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Pirimidinas/biossíntese , Serina-Treonina Quinases TOR/fisiologiaRESUMO
1. Oligomycin-insensitive ATPase (ATP phosphohydrolase, EC 3.6.1.3) was purified from brown adipose tissue mitochondria. It had a specific activity of 50 units/mg which could be increased up to 85 units/mg by KHCO3. The isolated enzyme represented less than 0.5% of the initial membrane proteins.2. The enzyme had a molecular weight equal to beef heart ATPase and was composed of five subunits with molecular weights of 56 200, 54 300, 33 500, 13 400 and 9500 respectively. 3. Isolated ATPase was labile while cold and was activated by the divalent cations Mn2+, Mg2+, Co2+ and Cd2+. The optimum ATP/Mg2+ ratio found was 1.58 and the enzyme had a maximum activity at pH 8.5; the Km was 220 micrometer. 4. The ATPase activity was 55% inhibited by aurovertin. The isolated enzyme enhanced the fluorescence of aurovertin, quenched by ATP and Mg2+ and enhanced by ADP. 5. Oligomycin sensitivity and cold stability of isolated ATPase was restored by its reconstitution with both brown adipose tissue and beef heart particles depleted of ATPase. 6. The results presented demonstrate that the low ATPase activity of brown adipose tissue mitochondria is due to a reduced content of ATPase.
Assuntos
Adenosina Trifosfatases/isolamento & purificação , Tecido Adiposo Marrom/enzimologia , Aclimatação , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/metabolismo , Animais , Cátions Bivalentes/farmacologia , Temperatura Baixa , Cricetinae , Estabilidade de Medicamentos , Ativação Enzimática , Cinética , Substâncias Macromoleculares , Mesocricetus , Mitocôndrias/enzimologia , Mitocôndrias Musculares/enzimologia , Peso Molecular , Oligomicinas/farmacologia , SolubilidadeRESUMO
A simple method for isolation of adenosine triphosphatase (EC 3.6.1.3) from mitochondria is described. The enzyme is released from mitochondrial Lubrol particles by drastic sonication and purified by gel filtration on Sepharose 6-B. The described procedure is effective in isolating adenosine triphosphatase from rat liver as it is from beef heart mitochondria. The enzyme isolated from beef heart has a specific activity of 120 mumol P/min per mg protein and enzyme isolated from rat liver has a specific activity of 70 mumol P/min per mg protein when measured as a release of inorganic phosphate.
Assuntos
Adenosina Trifosfatases/isolamento & purificação , Mitocôndrias Hepáticas/enzimologia , Mitocôndrias Musculares/enzimologia , Adenosina Trifosfatases/metabolismo , Animais , Bovinos , Cromatografia em Gel , Detergentes , Concentração de Íons de Hidrogênio , Cinética , Métodos , Miocárdio , Ratos , Especificidade da EspécieRESUMO
Embryonic development of mouse and rat brown adipose tissue was characterized by electron microscopy and by quantifying the mitochondrial oxidative, phosphorylating and thermogenic capacities immunochemically, using antibodies against cytochrome oxidase, F1-ATPase and uncoupling protein, respectively. Mitochondria and cytochrome oxidase were detected from the 15-16th day of pregnancy and their amounts continuously increased toward birth. F1-ATPase was also found on the 15th day but it reached a maximum level already on the 19th day when the uncoupling protein appeared and rapidly increased during further maturation of brown adipose tissue. It thus appears that mitochondria in early prenatal brown adipose tissue lack completely uncoupling protein and are nonthermogenic. They transform into typical thermogenic mitochondria abruptly only 2 days before birth.
Assuntos
Tecido Adiposo Marrom/fisiologia , Regulação da Temperatura Corporal , Proteínas de Transporte , Desenvolvimento Embrionário e Fetal , Proteínas de Membrana/fisiologia , Mitocôndrias/fisiologia , Tecido Adiposo Marrom/enzimologia , Tecido Adiposo Marrom/ultraestrutura , Animais , Diferenciação Celular , Complexo IV da Cadeia de Transporte de Elétrons/isolamento & purificação , Embrião de Mamíferos , Canais Iônicos , Proteínas de Membrana/isolamento & purificação , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/enzimologia , Proteínas Mitocondriais , ATPases Translocadoras de Prótons/isolamento & purificação , Ratos , Ratos Endogâmicos , Proteína Desacopladora 1RESUMO
1. The content of the membrane sector of the ATPase complex (Fo) in brown adipose tissue mitochondria was determined by means of specific [14C]-DCCD binding. 2. The specific DCCD binding to the F0 protein was distinguished from the nonspecific binding to the other membrane proteins and phospholipids by: (a) Scatchard plot analysis of the equilibrium binding data, (b) SDS-polyacrylamide gel electrophoresis of the 14C-labelled membrane proteins, (c) partial purification of the chloroform-methanol extractable DCCD-binding protein. It was found that the specific DCCD binding was present in three polypeptides of a relative molecular weight of 9000, 16 000 and 32 000. In brown adipose tissue mitochondria the specific binding was 10-times lower than in heart or liver mitochondria. The binding to the other membrane proteins and to phospholipids was quite similar in all mitochondrial preparations studied. 3. The decreased quantity of the specific binding sites in brown adipose tissue mitochondria demonstrated that the reduction of F0 parallels the reduction of the F1-ATPase and revealed that in these mitochondrial membranes the ratio between the respiratory chain enzymes and the ATPase complex is 10- to 20- times higher than in heart or liver mitochondria.
Assuntos
Adenosina Trifosfatases/metabolismo , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo/metabolismo , Carbodi-Imidas/metabolismo , Proteínas de Transporte/metabolismo , Dicicloexilcarbodi-Imida/metabolismo , Animais , Radioisótopos de Carbono , Proteínas de Transporte/isolamento & purificação , Bovinos , Cinética , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Hepáticas/metabolismo , Peso Molecular , Especificidade de Órgãos , ATPases Translocadoras de Prótons , RatosRESUMO
Brown adipose tissue of developing hamster was characterized by western blotting, enzyme activity measurements and immunoelectron microscopy. During the first postnatal week the tissue contained significant amounts of differentiating mitochondria and comparable quantities of active cytochrome oxidase and ATP synthase. The uncoupling protein appeared on the 7/8th day and its specific content increased 80-times between day 8 and day 17. In parallel, the specific content and activity of cytochrome oxidase increased 3-times but ATP synthase decreased 2-times. The total content of uncoupling protein and of cytochrome oxidase in interscapular brown adipose tissue increased 360- and 11-times, respectively. Analysis of isolated mitochondria showed that the observed differences result mainly from changes of the enzymic equipment of the mitochondrial membrane. During the same interval, propylthiouracil-insensitive "type II' thyroxine 5'-deiodinase activity in brown adipose tissue increased 10-times. It was concluded that the thermogenic function of the hamster brown adipose tissue develops after the first postnatal week due to highly differentiated synthesis of mitochondrial proteins leading to replacement of preexisting, uncoupling protein-lacking nonthermogenic mitochondria by thermogenic ones, similarly as shown in brown adipose tissue of the embryonic mouse and rat (Houstek, J., et al. (1988) Biochim. Biophys. Acta 935, 19-25).
Assuntos
Tecido Adiposo Marrom/crescimento & desenvolvimento , Proteínas de Transporte , Proteínas de Membrana/fisiologia , Mitocôndrias/fisiologia , Desacopladores/análise , Tecido Adiposo Marrom/análise , Tecido Adiposo Marrom/ultraestrutura , Animais , Animais Recém-Nascidos , Peso Corporal , Cricetinae , Complexo IV da Cadeia de Transporte de Elétrons/análise , Metabolismo Energético , Imuno-Histoquímica , Iodeto Peroxidase/análise , Canais Iônicos , Proteínas de Membrana/análise , Mesocricetus , Microscopia Eletrônica/métodos , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais , ATPases Translocadoras de Prótons/análise , Proteína Desacopladora 1RESUMO
Differentiation and biogenesis of mitochondria in brown adipose tissue (BAT) was studied in situ and in cell culture by Western blotting, enzyme activity measurements, [35S]methionine incorporation and immunofluorescence microscopy. In different rodent species the perinatal development of BAT thermogenic function resulted from the formation of thermogenic mitochondria which replaced the preexisting nonthermogenic mitochondria. Their biogenesis was characterized by the sudden appearance and rapid increase of the uncoupling protein (UCP), increase of cytochrome oxidase (COX) and decrease of H(+)-ATPase. In primary cell culture, differentiation of precursor cells from mouse BAT to typical multilocular adipocytes was accompanied by increasing content of COX and H(+)-ATPase. A selective synthesis of UCP was induced by activation of beta-adrenergic receptors or by elevated levels of cellular cAMP. UCP was quantitatively incorporated into mitochondria and within 24 h after stimulation reached near physiological concentration. Both in situ and in cell culture, the conditions enabling the expression of UCP gene were accompanied by activation of intracellular thyroxine 5'-deiodinase.
Assuntos
Tecido Adiposo Marrom/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Desacopladores/metabolismo , Tecido Adiposo Marrom/citologia , Tecido Adiposo Marrom/enzimologia , Animais , Regulação da Temperatura Corporal , Diferenciação Celular , Células Cultivadas , Cricetinae , Iodeto Peroxidase/metabolismo , Mesocricetus , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Perinatologia , Ratos , Ratos EndogâmicosRESUMO
To study the assembly of mitochondrial F1F0 ATP synthase, cultured human cells were labeled with [35S]methionine in pulse-chase experiments. Next, two-dimensional electrophoresis and fluorography were used to analyze the assembly pattern. Two assembly intermediates could be demonstrated. First the F1 part appeared to be assembled, and next an intermediate product that contained F1 and subunit c. This product probably also contained subunits b, F6 and OSCP, but not the mitochondrially encoded subunits a and A6L. Both intermediate complexes accumulated when mitochondrial protein synthesis was inhibited, suggesting that mitochondrially encoded subunits are indispensable for the formation of a fully assembled ATP synthase complex, but not for the formation of the intermediate complexes. The results and methods described in this study offer an approach to study the effects of mutations in subunits of mitochondrial ATP synthase on the assembly of this complex. This might be of value for a better understanding of deficiencies of ATP synthase activity in mitochrondrial diseases.
Assuntos
Mitocôndrias/enzimologia , Miopatias Mitocondriais/enzimologia , ATPases Translocadoras de Prótons/biossíntese , Antibacterianos/farmacologia , Western Blotting , Doxiciclina/farmacologia , Eletroforese em Gel Bidimensional , Humanos , Cinética , Mitocôndrias/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
1. In isolated bovine heart mitochondria, the 14C-labelled dicyclohexylcarbodiimide (DCCD) induced inhibition of the ATPase activity is accompanied by labelling of three polypeptides of Mx 9000, 16 000 and 33 000. Of these, only the 9000 polypeptide reacts with [14C]DCCD proportionally to the inhibitory effect, being saturated when the enzyme is maximally inhibited. 2. The 9000 and 16 000 polypeptides are extracted by neutral chloroform/methanol (2 : 1 v/v) while the 33 000 polypeptide remains in the non-extractable residue. No disaggregation of the polypeptides takes place during the extraction. 3. In the ATPase complex immunoprecipitated with antibody against F1, the 9000 and 16 000 polypeptides are present, but the 33 000 polypeptide is absent. 4. The results obtained indicate that the 33 000 polypeptide is not a component of the ATPase complex. As far as F0 is concerned, two types of the binding sites for DCCD were demonstrated, corresponding to the 9000 and 16 000 polypeptides. Their existence is explained by a non-random arrangement among individual monomers of the DCCD-binding protein.
Assuntos
Adenosina Trifosfatases/metabolismo , Carbodi-Imidas/metabolismo , Proteínas de Transporte/metabolismo , Dicicloexilcarbodi-Imida/metabolismo , Mitocôndrias Cardíacas/metabolismo , Animais , Radioisótopos de Carbono , Proteínas de Transporte/isolamento & purificação , Bovinos , Cinética , Peso Molecular , Peptídeos/isolamento & purificaçãoRESUMO
Isolated, nucleotide-depleted bovine-heart F1-ATPase exhibits a break in Arrhenius plot with a 2.7-fold increase in activation energy of ATP hydrolysis below 18-19 degrees C. Analysis of intrinsic tyrosine fluorescence and of the circular dichroism of F1-ATPase showed an abrupt and reversible conformational change occurring at the break temperature, characteristic of a structural tightening at low temperature. Analysis of catalytic nucleotide binding sites using fluorescent ADP analog, 3'-O-(1-naphthoyl)adenosine diphosphate did not show any significant change in affinity of nucleotide binding around the transition temperature but the bound fluorophore exerted a more restricted motion and slower rotation at temperature below the break, indicating a change in the mobility of groups in the close neighbourhood. It is concluded that, as a result of temperature, two kinetically distinct states of F1-ATPase are induced, due to a change in enzyme conformation, which influences directly the properties of catalytic nucleotide binding sites.