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
Mitochondria play an important role in the cell aging process. Changes in calcium homeostasis and/or increased reactive oxygen species (ROS) production lead to the opening of mitochondrial permeability transition pore (MPTP), depolarization of the inner mitochondrial membrane, and decrease of ATP production. Our work aimed to monitor age-related changes in the Ca2+ ion effect on MPTP and the ability of isolated rat liver mitochondria to accumulate calcium. The mitochondrial calcium retention capacity (CRC) was found to be significantly affected by the age of rats. Measurement of CRC values of the rat liver mitochondria showed two periods when 3 to 17-week old rats were tested. 3-week and 17-week old rats showed lower CRC values than 7-week old animals. Similar changes were observed while testing calcium-induced swelling of rat liver mitochondria. These findings indicate that the mitochondrial energy production system is more resistant to calcium-induced MPTP opening accompanied by the damaging effect of ROS in adult rats than in young and aged animals.
Assuntos
Envelhecimento/metabolismo , Mitocôndrias Hepáticas/metabolismo , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , Animais , Masculino , Ratos WistarRESUMO
Values of the calcium retention capacity (CRC) of rat liver mitochondria are highly dependent on the experimental conditions used. When increasing amounts of added calcium chloride are used (1.25-10 nmol), the values of the CRC increase 3-fold. When calcium is added in 75 s intervals, the CRC values increase by 30 % compared with 150 s interval additions. CRC values are not dependent on the calcium/protein ratio in the measured sample in our experimental design. We also show that a more detailed evaluation of the fluorescence curves can provide new information about mitochondrial permeability transition pore opening after calcium is added.
Assuntos
Cálcio/metabolismo , Fígado/metabolismo , Mitocôndrias Hepáticas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , Animais , Transporte Biológico , Masculino , Permeabilidade , Ratos , Projetos de PesquisaRESUMO
Novel strategies are needed that can stimulate endogenous signaling pathways to protect the heart from myocardial infarction. The present study tested the hypothesis that appropriate regimen of cold acclimation (CA) may provide a promising approach for improving myocardial resistance to ischemia/reperfusion (I/R) injury without negative side effects. We evaluated myocardial I/R injury, mitochondrial swelling, and ß-adrenergic receptor (ß-AR)-adenylyl cyclase-mediated signaling. Male Wistar rats were exposed to CA (8°C, 8 h/day for a week, followed by 4 wk at 8°C for 24 h/day), while the recovery group (CAR) was kept at 24°C for an additional 2 wk. The myocardial infarction induced by coronary occlusion for 20 min followed by 3-h reperfusion was reduced from 56% in controls to 30% and 23% after CA and CAR, respectively. In line, the rate of mitochondrial swelling at 200 µM Ca2+ was decreased in both groups. Acute administration of metoprolol decreased infarction in control group and did not affect the CA-elicited cardiprotection. Accordingly, neither ß1-AR-Gsα-adenylyl cyclase signaling, stimulated with specific ligands, nor p-PKA/PKA ratios were affected after CA or CAR. Importantly, Western blot and immunofluorescence analyses revealed ß2- and ß3-AR protein enrichment in membranes in both experimental groups. We conclude that gradual cold acclimation results in a persisting increase of myocardial resistance to I/R injury without hypertension and hypertrophy. The cardioprotective phenotype is associated with unaltered adenylyl cyclase signaling and increased mitochondrial resistance to Ca2+-overload. The potential role of upregulated ß2/ß3-AR pathways remains to be elucidated.NEW & NOTEWORTHY We present a new model of mild gradual cold acclimation increasing tolerance to myocardial ischemia/reperfusion injury without hypertension and hypertrophy. Cardioprotective phenotype is accompanied by unaltered adenylyl cyclase signaling and increased mitochondrial resistance to Ca2+-overload. The potential role of upregulated ß2/ß3-adrenoreceptor activation is considered. These findings may stimulate the development of novel preventive and therapeutic strategies against myocardial ischemia/reperfusion injury.
Assuntos
Adenilil Ciclases , Receptores Adrenérgicos beta , Aclimatação , Adrenérgicos , Animais , Masculino , Ratos , Ratos WistarRESUMO
The concentration-dependence of tert-butyl hydroperoxide (BHP) inhibitory effect on oxygen consumption in isolated rat liver mitochondria was measured in the presence of various respiratory substrates. Strong inhibitory effect at low concentrations of BHP (15-30 microM) was found for oxoglutarate and palmitoyl carnitine oxidation. Pyruvate and glutamate oxidation was inhibited at higher concentrations of BHP (100-200 microM). Succinate oxidation was not affected even at 3.3 mM BHP. Determination of mitochondrial membrane potential has shown that in the presence of NADH-dependent substrates the membrane potential was dissipated by BHP but was completely restored after addition of succinate. Our data thus indicate that beside peroxidative damage of complex I also various mitochondrial NADH-dependent dehydrogenases are inhibited, but to a different extent and with different kinetics. Our data also show that succinate could be an important nutritional substrate protecting hepatocytes during peroxidative damage.
Assuntos
Mitocôndrias Hepáticas/metabolismo , Estresse Oxidativo , Animais , Respiração Celular , Ácido Glutâmico/metabolismo , Ácidos Cetoglutáricos/metabolismo , Masculino , Potencial da Membrana Mitocondrial , Consumo de Oxigênio , Palmitoilcarnitina/metabolismo , Ácido Pirúvico/metabolismo , Ratos , Ratos Wistar , Ácido Succínico/metabolismo , terc-Butil Hidroperóxido/farmacologiaRESUMO
By determining the calcium retention capacity (CRC) of rat liver mitochondria, we confirmed and extended previous observations describing the activation of mitochondrial swelling by phosphate and tert-butyl hydroperoxide (t-BHP). Using CRC measurements, we showed that both phosphate and t-BHP decrease the extent of calcium accumulation required for the full mitochondrial permeability transition pore (MPTP) opening to 35 % of control values and to only 15 % when both phosphate and t-BHP are present in the medium. When changes in fluorescence were evaluated at higher resolution, we observed that in the presence of cyclosporine A fluorescence values return after each Ca(2+) addition to basal values obtained before the Ca(2+) addition. This indicates that the MPTP remains closed. However, in the absence of cyclosporine A, the basal fluorescence after each Ca(2+) addition continuously increased. This increase was potentiated both by phosphate and t-BHP until the moment when the concentration of intramitochondrial calcium required for the full opening of the MPTP was reached. We conclude that in the absence of cyclosporine A, the MPTP is slowly opened after each Ca(2+) addition and that this rate of opening can be modified by various factors such as the composition of the media and the experimental protocol used.
Assuntos
Cálcio/metabolismo , Mitocôndrias Hepáticas/metabolismo , Fosfatos/farmacologia , terc-Butil Hidroperóxido/farmacologia , Animais , Relação Dose-Resposta a Droga , Masculino , Mitocôndrias Hepáticas/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Ratos , Ratos WistarRESUMO
Mitochondria as an energy generating cell device are very sensitive to oxidative damage. Our previous findings obtained in hepatocytes demonstrated that Complex I of the respiratory chain is more sensitive to oxidative damage than other respiratory chain complexes. We present additional data on isolated mitochondria showing that palmityl carnitine oxidation is strongly depressed at a low (200 microM) tert-butyl hydroperoxide (tBHP) concentration, while oxidation of the flavoprotein-dependent substrate-succinate is not affected and neither is ATP synthesis inhibited by tBHP. In the presence of tBHP, the respiratory control index for palmityl carnitine oxidation is strongly depressed, but when succinate is oxidized the respiratory control index remains unaffected. Our findings thus indicate that flavoprotein-dependent substrates could be an important nutritional factor for the regeneration process in the necrotic liver damaged by oxidative stress.
Assuntos
Mitocôndrias/metabolismo , Oxidantes/farmacologia , Palmitoilcarnitina/metabolismo , Ácido Succínico/metabolismo , terc-Butil Hidroperóxido/farmacologia , Animais , Ácidos Graxos/metabolismo , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Fígado/citologia , Fígado/metabolismo , Masculino , Mitocôndrias/efeitos dos fármacos , NADP/efeitos dos fármacos , NADP/metabolismo , Estresse Oxidativo/fisiologia , Ratos , Ratos WistarRESUMO
Using high-resolution oxygraphy, we tested the changes of various parameters characterizing the mitochondrial energy provision system that were induced by peroxidative damage. In the presence of succinate as respiratory substrate, 3 mM t-butyl hydroperoxide increased respiration in the absence of ADP, which indicated partial uncoupling of oxidative phosphorylation. Low activity of coupled respiration was still maintained as indicated by the ADP-activated and oligomycin-inhibited respiration. However, during the incubation the phosphorylative capacity decreased as indicated by the continuous decrease of the mitochondrial membrane potential. Under these experimental conditions the maximum capacity of the succinate oxidase system was inhibited by 50% in comparison with values obtained in the absence of t-butyl hydroperoxide. Our data thus indicate that the oxygraphic evaluation of mitochondrial function represents a useful tool for evaluation of changes participating in peroxidative damage of cell energy metabolism.
Assuntos
Hepatócitos/metabolismo , Mitocôndrias Hepáticas/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , terc-Butil Hidroperóxido/farmacologia , Animais , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Separação Celular , Digitonina/farmacologia , Metabolismo Energético/efeitos dos fármacos , Técnicas In Vitro , Masculino , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Membranas Mitocondriais/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Cloreto de Potássio/farmacologia , Ratos , Ratos Wistar , Desacopladores/farmacologiaRESUMO
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
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
The changes in mitochondrial membrane potential (Deltapsi(m)) were used as an indicator for evaluating the mitochondrial permeability transition pore (MPTP) function. We found that in situ mitochondria in digitonin-permeabilized hepatocytes were coupled and responded to the addition of substrates, inhibitors and uncouplers. Ca(2+)-induced Deltapsi(m) dissipation was caused by MPTP opening because this process was inhibited by cyclosporin A. MPTP opening was enhanced by the pro-oxidant tert-butyl hydroperoxide.
Assuntos
Hepatócitos/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Animais , Cálcio/farmacologia , Eletrodos , Hepatócitos/efeitos dos fármacos , Técnicas In Vitro , Indicadores e Reagentes , Potencial da Membrana Mitocondrial , Poro de Transição de Permeabilidade Mitocondrial , Oniocompostos , Compostos Organofosforados , Consumo de Oxigênio , Ratos , terc-Butil Hidroperóxido/farmacologiaRESUMO
Using a novel method for evaluating mitochondrial swelling (Drahota et al. 2012a) we studied the effect of calcium (Ca(2+)), phosphate (P(i)), and triiodothyronine (T(3)) on the opening of mitochondrial membrane permeability transition pore and how they interact in the activation of swelling process. We found that 0.1 mM P(i), 50 microM Ca(2+) and 25 microM T(3) when added separately increase the swelling rate to about 10 % of maximal values when all three factors are applied simultaneously. Our findings document that under experimental conditions in which Ca(2+) and P(i) are used as activating factors, the addition of T(3) doubled the rate of swelling. T(3) has also an activating effect on mitochondrial membrane potential. The T(3) activating effect was also found after in vivo application of T(3). Our data thus demonstrate that T(3) has an important role in opening the mitochondrial membrane permeability pore and activates the function of the two key physiological swelling inducers, calcium and phosphate ions.
Assuntos
Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Tri-Iodotironina/metabolismo , Tri-Iodotironina/farmacologia , Animais , Cálcio/metabolismo , Cálcio/farmacologia , Relação Dose-Resposta a Droga , Masculino , Poro de Transição de Permeabilidade Mitocondrial , Ratos , Ratos WistarRESUMO
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
The influence of lysophosphatidylcholine (LPC) on H(+)-ATPase, cytochrome oxidase (COX), glycerolphosphate dehydrogenase (GPDH) and malate dehydrogenase (MDH) was followed. The activities of H(+)-ATPase and COX increased with increasing LPC concentration up to 0.5 mg/mg protein when maxima were achieved. This activatory effect is LPC-specific, because Lubrol-treated or frozen-thawed mitochondria showed lower activities of these enzymes. H(+)-ATPase was not influenced by higher concentration of LPC, while COX activity decreased with increasing amount of LPC. The activity of GPDH decreased at very low concentration of LPC and was not further modified at higher LPC concentration. In an attempt to find the concentration of LPC necessary for a complete permeabilization of inner mitochondrial membrane we followed the influence of lysolipid on the release of MDH activity from the mitochondrial matrix. The full activity of this enzyme was obtained with a concentration 0.75 mg LPC/mg protein indicating that mitochondria were completely broken. Our data indicate that LPC significantly affects activity of enzymes connected with mitochondrial membrane and can be useful for evaluation of the importance of phospholipid microenvironment for the enzyme function.
Assuntos
Tecido Adiposo Marrom/enzimologia , Lisofosfatidilcolinas/farmacologia , Mitocôndrias/enzimologia , Tecido Adiposo/enzimologia , Animais , Cricetinae , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Glicerolfosfato Desidrogenase/metabolismo , Membranas Intracelulares/enzimologia , Malato Desidrogenase/metabolismo , Mesocricetus , Mitocôndrias Hepáticas/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Ratos , Ratos EndogâmicosRESUMO
1. Isolation of ATPase from rat liver submitochondrial particles by chloroform treatment requires the presence of ATP or ADP during enzyme solubilization. In the absence of adenine nucleotides the enzyme activity is very low although all protein components of F1-ATPase are released. The low concentrations of ATP or ADP required (5 microM) indicate that the high affinity nucleotide-binding sites are involved in enzyme stabilization. Other nucleotides tested (ITP, GTP, UTP, CTP) were found to be less effective. 2. Polyacrylamide gel electrophoresis and immunodiffusion in agar plates revealed that in the absence of adenine nucleotides a fraction of F1-ATPase released by chloroform treatment is split into fragments. The part of the dissociated enzyme molecule has a molecular weight identical with that of a beta-subunit of F1-ATPase. 3. Dissociation of the F1-ATPase molecule could also be prevented by aurovertin. 4. Crude F1-ATPase solubilized by chloroform treatment can be further purified by Sepharose 6B gel filtration. Specific ATPase activity of the purified enzyme was 90 mumol Pi/min per mg protein and the enzyme was composed of five protein subunits (alpha, beta, gamma, delta, epsilon) with molecular weights 58 000, 55 000, 28 000, 13 000 and 8000, respectively. 5. Chloroform-released F1-ATPase from rat liver mitochondria displayed immunochemical cross-reactivity with that isolated from beef heart mitochondria.
Assuntos
Adenosina Trifosfatases/metabolismo , Mitocôndrias Hepáticas/enzimologia , Fatores Acopladores da Fosforilação Oxidativa/metabolismo , Adenosina Trifosfatases/isolamento & purificação , Animais , Bovinos , Clorofórmio , Estabilidade de Medicamentos , Imunodifusão , Cinética , Mitocôndrias Cardíacas/enzimologia , Peso Molecular , Fatores Acopladores da Fosforilação Oxidativa/isolamento & purificação , Ratos , Solubilidade , Partículas Submitocôndricas/enzimologiaRESUMO
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
FAD-linked L-glycerol-3-phosphate dehydrogenase purified from liver mitochondria of hyperthyroid rats was incorporated into unilamellar phospholipid liposomes. The incorporation influenced both Vmax,app and Km,app values of the enzyme for its substrate, L-glycerol 3-phosphate. The Km,app for the electron acceptor remained unchanged with a simultaneous slight enhancement of the corresponding Vmax,app value. The steady-state fluorescence anisotropies of the fluorescein isothiocyanate and trimethylammoniumdiphenylhexatriene labels were affected by sodium oleate and calcium ions in the case of both solubilized and liposome-incorporated L-glycerol-3-phosphate dehydrogenase. These results indicate that calcium ions cause a significant alteration of the enzyme conformation. Sodium oleate (used as a model of free fatty acids), besides its direct action on the enzyme itself, affects the enzyme indirectly as well, via alteration of the physical properties of the membrane.
Assuntos
Cálcio/farmacologia , Glicerolfosfato Desidrogenase/metabolismo , Lipossomos/metabolismo , Mitocôndrias Hepáticas/enzimologia , Ácido Oleico , Ácidos Oleicos/farmacologia , Animais , Cardiolipinas/análise , Cardiolipinas/farmacologia , Difenilexatrieno/análogos & derivados , Fluoresceína-5-Isotiocianato , Fluoresceínas , Corantes Fluorescentes , Glicerofosfatos/metabolismo , Hipertireoidismo/enzimologia , Cinética , Lipossomos/análise , Masculino , Fosfolipídeos/análise , Conformação Proteica/efeitos dos fármacos , Ratos , Espectrometria de Fluorescência , TiocianatosRESUMO
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.