RESUMO
Mitoplasts isolated from rat liver mitochondria were treated with dimethyladipimidate, a bifunctional alkylating agent. This agent causes, concurrently with modification of amino groups, loss of osmotic response. It was found that after cross-linking, the movement effector, succinate, was unable to induce the aspartate aminotransferase release from mitoplasts. In contrast, dimethyladipimidate-treated mitoplasts were still able to internalize 125I-labeled aspartate aminotransferase upon removal of exogenous succinate. The possible involvement of membrane asymmetry in the mechanism of translocation of porteins through the inner mitochondrial membrane is discussed.
Assuntos
Aspartato Aminotransferases/metabolismo , Membranas Intracelulares/metabolismo , Mitocôndrias Hepáticas/enzimologia , Animais , Reagentes de Ligações Cruzadas , Dimetil Adipimidato , Ratos , Succinatos/metabolismo , Sacarose/metabolismoRESUMO
We altered the cellular lipid composition of an insulin sensitive rat hepatoma cell line through supplementation of the culture medium with linoleic acid (18:2) or 25-hydroxycholesterol, and we studied the effects on insulin stimulation of aminoacid transport system A and glycogen synthesis. The basal rate of sodium-dependent aminoisobutyric acid uptake was slightly reduced in hydroxysterol-treated cells and increased in 18:2-enriched cells. Maximal insulin stimulation of transport was decreased by about 40% in both 18:2 and 25-hydroxycholesterol modified cells, as compared to control cells. In addition to reduced responsiveness, the hydroxysterol-treated cells also showed a diminished sensitivity to insulin, as revealed by a right-shift of the dose-response curve leading to a ED50 of 1.2 X 10(-8) M (P less than 0.02), as compared to 2.45 X 10(-9) M in control cells and 2.13 X 10(-9) M in 18:2 enriched cells. Concerning glycogen synthesis, the basal rate was unaffected by 25-hydroxycholesterol supplementation and slightly reduced in cells enriched in 18:2. Maximal insulin stimulation of glycogen synthesis was reduced by about 40% in both types of lipid modified cells. 25-Hydroxycholesterol again provoked a decrease in sensitivity to insulin: the ED50 was enhanced to 4.9 X 10(-9) M (P less than 0.05), as compared to 1.25 X 10(-9) M in control cells and 1.57 X 10(-9) M in 18:2-supplemented cells. Taken together with the previously reported changes of insulin binding to lipid modified hepatoma cells (Bruneau et al. (1987) Biochim. Biophys. Acta 928, 287-296) our results demonstrate an influence of alterations of the cellular lipid composition on both binding and biological actions of insulin, leading to an insulin-resistant state. Divergences between insulin binding and action were obtained and it was suggested that post-binding events may be responsible for the observed changes. Our findings may be relevant to experimental and clinical states of insulin resistance.
Assuntos
Aminoácidos/metabolismo , Resistência à Insulina , Glicogênio Hepático/biossíntese , Fígado/metabolismo , Lipídeos de Membrana/fisiologia , Receptor de Insulina/fisiologia , Animais , Transporte Biológico/efeitos dos fármacos , Hidroxicolesteróis/metabolismo , Insulina/farmacologia , Cinética , Ácido Linoleico , Ácidos Linoleicos/metabolismo , Neoplasias Hepáticas Experimentais , Fluidez de Membrana , Ratos , Sódio/fisiologiaRESUMO
Lipid composition was studied in liver and brain of normal (+/+), heterozygote (ob/+) and obese (ob/ob) mice. It was found that this genetic defect is expressed differently in the lipid composition of these organs. Cholesterol is increased in liver but strongly decreased in brain of obese animals. Phosphatide fatty acid composition is modified in liver and not in brain. In contrast, phospholipids and total ganglioside sialic acid are affected similarly in both organs. Although clinically normal, heterozygote (ob/+) mice already show an abnormal lipid composition in liver and brain. The potential importance of these results is presented.
Assuntos
Química Encefálica , Colesterol/análise , Gangliosídeos/análise , Fígado/análise , Fosfolipídeos/análise , Animais , Ácidos Graxos/análise , Heterozigoto , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Ácidos Siálicos/análise , Especificidade da EspécieRESUMO
Intramitochondrial large amplitude protein movements were studied by polyacrylamide gel electrophoresis on the intermembranal fluids of mitochondria which were incubated in presence of movement effectors. The specific and reversible property of this phenomenon was confirmed by this method. Its bilateral nature was observed. The eventuality of a partial inner membrane damage provoked by the synergic action of digitonine and movement effector, was discussed, evaluated and dismissed.
Assuntos
Mitocôndrias Hepáticas/metabolismo , Proteínas/metabolismo , Animais , Transporte Biológico , Ácidos Carboxílicos , Digitonina , Eletroforese em Gel de Poliacrilamida , Proteínas de Membrana/metabolismo , Membranas/metabolismo , Peso Molecular , RatosRESUMO
Acetylcholinesterase (Acetylcholine acetylhydrolase E.C. 3.1.1.7.) released from a rat brain synaptosomal fraction was shown to rebind upon incubation in the presence of 3 mM acetylcholine. This action was shown to be reversible. Glutamate, aspartate and gamma-aminobutyrate antagonise this effect. Solubilization of both bulk protein and acetylcholinesterase by Lubrol WX and triton X 100 after acetylcholine incubation of synaptosomal fractions is much lower than in the non incubated preparation. Local production of protons due to the hydrolysis of acetylcholine by the enzyme could partially explain the reassociation of the enzyme. We suggest that the observed phenomenon may play some physiological role in the function of acetylcholinesterase.
Assuntos
Acetilcolina/farmacologia , Acetilcolinesterase/metabolismo , Sinaptossomos/metabolismo , Acetilcolina/antagonistas & inibidores , Animais , Centrifugação com Gradiente de Concentração , Precipitação Química , Inibidores da Colinesterase , Concentração de Íons de Hidrogênio , Mitocôndrias/metabolismo , Octoxinol , Polietilenoglicóis , Ratos , Solubilidade , Sacarose/farmacologia , Sinaptossomos/efeitos dos fármacosRESUMO
The antiepileptic sodium valproate (VPA) systematically induces an asymptomatic hyperammonemia of renal origin in fasting normal human volunteers and in fasting rats, accompanied by an increased renal glutamine uptake. Fasting rats were injected with VPA and their mitochondria isolated, or isolated mitochondria of fasting rats were incubated with VPA. Transmembranal mitochondrial glutamine uptake and activities for five mitochondrial and three cytosolic enzymes involved in ammoniagenesis were measured. In VPA-incubated mitochondria, glutamine transport increased for VPA concentrations between 10(-3) and 10(-5) M; enzyme activities did not change. In mitochondria of VPA-treated rats, Km and Vmax were unaffected. These findings reflect membrane effects of VPA observed in other experimental settings.
Assuntos
Amônia/sangue , Glutamina/metabolismo , Rim/metabolismo , Mitocôndrias/metabolismo , Ácido Valproico/farmacologia , Amônia/metabolismo , Animais , Ligação Competitiva , Transporte Biológico/efeitos dos fármacos , Rim/efeitos dos fármacos , Rim/ultraestrutura , Cinética , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Ratos , Ratos EndogâmicosRESUMO
Plasma membrane "fluidity" of peripheral blood T lymphocytes from untreated patients with Hodgkin's disease (HD) and healthy controls was studied using the fluorescent probes 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4(trimethylamino)phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH). In 13 consecutive patients a significant increase of T lymphocyte plasma membrane microviscosity was observed with both DPH and TMA-DPH. These alterations seemed unrelated to the cholesterol (Chol) and phospholipid (PL) content of HD T lymphocytes since the Chol/PL ratio was comparable in both HD and control cells. Since prostaglandin E2 (PGE2) from monocytic origin has been claimed to be responsible for the impairment of cell-mediated immunity (CMI) associated with HD, we studied the effect of exogeneously added PGE2 (0.1 microM) on control subjects T lymphocyte membrane "fluidity". Using the fluorescent probe DPH and the spin labelled fatty acid probe 16 NMS for electron paramagnetic resonance study, we observed a PGE2-induced fluidization of control T lymphocyte membranes which is specifically located in the inner part of the plasma membrane, whereas the plasma membrane surface seemed unaffected by PGE2 as judged by the TMA-DPH probe. Thus, PGE2 does not appear to be responsible for the alterations of T lymphocyte membranes observed in HD. Intrinsic alterations and/or other mediators might be involved.
Assuntos
Doença de Hodgkin/metabolismo , Fluidez de Membrana , Linfócitos T/metabolismo , Adulto , Colesterol/análise , Dinoprostona , Feminino , Polarização de Fluorescência , Humanos , Masculino , Fluidez de Membrana/efeitos dos fármacos , Pessoa de Meia-Idade , Fosfolipídeos/análise , Prostaglandinas E/farmacologia , Linfócitos T/análise , TemperaturaRESUMO
A rapid method for the isolation of acetylcholine receptor-rich membranes from Torpedo marmorata electric organ, using a Percoll density gradient, is presented. The preparation of purified membranes appeared on electron microscope examination as a homogeneous population of sealed vesicles, covered with the characteristic rosettes identified as acetylcholine receptor clusters. Biochemical characterization revealed an ?-bungarotoxin specific binding activity of 1.6-2.1 nmol/mg of protein, low acetylcholinesterase specific activity, a protein:lipid ratio (w/w) of 2.1 with high cholesterol content. Polyacrylamide gel electrophoresis under denaturing conditions showed the polypeptide bands characteristic of the receptor (?, ?, ? and ?), together with 43 kDa and ?100 kDa proteins (already described as ? and ?). The method is simple and rapid, and maintains constant osmotic conditions throughout. It thus represents an improvement over previous methods, and will be useful for routine preparation and specially for studying post-synaptic membrane components interactions.
RESUMO
Sodium valproate is an antiepileptic drug which may have side effects on different organs. Its mechanism of action, as yet unclear, may involve an effect on membranes. One possibility, an effect on mitochondrial membranes via an inhibition of oxidative phosphorylation, has been studied here by observing the transmembranal transport of a respiratory substrate, succinate, in rat kidney mitochondria incubated with valproate, or from rats injected with valproate. Succinate transport was inhibited in both conditions, which suggests that the effect was probably due to a direct effect of valproate rather than to an action of a valproate metabolite. For the valproate-incubated mitochondria, inhibition, described by a bell-shaped curve, started at a valproate concentration of 10(-7) M and was maximum at valproate 10(-5)M. Valproate's effect on mitochondrial transmembranal succinate transport can be compared to other evidence for membranal actions of valproate, actions which may clarify certain therapeutic or toxic properties of this drug.
Assuntos
Rim/metabolismo , Mitocôndrias/metabolismo , Succinatos/metabolismo , Ácido Valproico/farmacologia , Animais , Técnicas In Vitro , Rim/ultraestrutura , Cinética , Masculino , Mitocôndrias/efeitos dos fármacos , Ratos , Ratos EndogâmicosRESUMO
Rats were treated with intraperitoneal injections of sodium valproate (VPA), either acutely, one injection VPA 200 mg/kg, or chronically, VPA 600 mg/kg/day for 5 days, and the oxygen consumption, MO2, of isolated hepatic and cerebral mitochondria measured. For hepatic mitochondria, Stade IV MO2 decreased by more than 20%, and Stage III MO2 by more than 50%, in the presence of succinate or glutamate-malate substrates. A decoupling agent intensified this inhibition. With cerebral mitochondria, the effects were similar but weaker, for pyruvate-malate or glutamate-malate substrates. These findings suggest that VPA, a short-chain fatty acid, may affect the properties of the internal mitochondrial membrane, although an action on substrate carriers, or on indispensable mitochondrial metabolites, is not excluded. Inhibition of oxidative phosphorylation cannot, however, alone account for hepatotoxicities seen in VPA-treated subjects. These are rare, whereas inhibition of mitochondrial respiration by VPA is consistently observed.
Assuntos
Encéfalo/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Ácido Valproico/toxicidade , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Técnicas de Cultura , Masculino , Ratos , Ratos EndogâmicosAssuntos
Membranas/metabolismo , Proteínas/metabolismo , Animais , Complexo Antígeno-Anticorpo , Aspartato Aminotransferases/metabolismo , Transporte Biológico , Cloroplastos/metabolismo , Imunoglobulina M/metabolismo , Membranas Intracelulares/metabolismo , Cinética , Substâncias Macromoleculares , Fluidez de Membrana , Lipídeos de Membrana/fisiologia , Partículas Submitocôndricas/metabolismo , Temperatura , Toxinas Biológicas/metabolismoAssuntos
Transporte Biológico Ativo , Mitocôndrias Hepáticas/metabolismo , Proteínas/metabolismo , Acetatos/farmacologia , Aminoácidos/farmacologia , Animais , Aspartato Aminotransferases/metabolismo , Transporte Biológico Ativo/efeitos dos fármacos , Ácidos Dicarboxílicos/farmacologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Eletroforese em Gel de Poliacrilamida , Cetoácidos/farmacologia , Malato Desidrogenase/metabolismo , Masculino , Consumo de Oxigênio , Permeabilidade , Fosfatos/farmacologia , Potássio/farmacologia , Ligação Proteica , Conformação Proteica , Compostos de Amônio Quaternário/farmacologia , Ratos , Sódio/farmacologia , Succinatos/farmacologia , SacaroseRESUMO
Mitochondrial aspartate aminotransferase and malate dehydrogenase were purified to homogeneity from rat liver by the use of aspartate-coupled Sepharose, ion exchange, and Blue Sepharose chromatography. This procedure permits rapid preparation of these enzymes. The pI of each enzyme was determined and anomalous electrophoretic properties of aspartate aminotransferase were described.
Assuntos
Aspartato Aminotransferases/isolamento & purificação , Malato Desidrogenase/isolamento & purificação , Mitocôndrias Hepáticas/enzimologia , Animais , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Focalização Isoelétrica , RatosRESUMO
Two fluorescent probes, 2-(9-anthroyloxy)stearate and 12-(9-anthroyloxy)stearate, were used to investigate the effects of the neutralization of membrane charges on the organization of synaptic plasma membrane. Steady state fluorescence anisotropy measurements showed that a pH decrease provoked a rigidification of the synaptic membrane surface, whereas the bilayer core remained unaffected. The same effect was observed with negatively charged lipid vesicles. The relative distribution of proteins and the probes was estimated by fluorescence energy transfer from protein tryptophans to fluorescent probes: a pH decrease provoked an increase of the energy transfer, which was most pronounced with the surface probe, indicating an average closer packing between proteins and the probes. The modifications induced by a pH decrease were temperature dependent and were most marked at low temperatures. The results suggest that neutralization of the membrane charges provoked a redistribution of both membrane lipids and proteins. These findings are discussed in terms of a heterogeneous distribution of these membrane components.
Assuntos
Membranas Sinápticas/fisiologia , Animais , Encéfalo/fisiologia , Transferência de Energia , Polarização de Fluorescência/métodos , Concentração de Íons de Hidrogênio , Lipossomos , Ratos , TermodinâmicaRESUMO
Release and binding of aspartate aminotransferase, malate dehydrogenase and protein from and to water-shocked vesicles obtained from rat liver mitochondria was shown to occur. The characteristics of this phenomenon were shown to depend on the effectors used. They were in some way different from those observed with whole mitochondria under similar conditions.
Assuntos
Enzimas/metabolismo , Mitocôndrias Hepáticas/metabolismo , Proteínas/metabolismo , Animais , Aspartato Aminotransferases/metabolismo , Técnicas In Vitro , Malato Desidrogenase/metabolismo , Membranas/efeitos dos fármacos , Membranas/metabolismo , Membranas/fisiologia , Mitocôndrias Hepáticas/enzimologia , Mitocôndrias Hepáticas/ultraestrutura , Oxaloacetatos/farmacologia , Ligação Proteica , Ratos , Cloreto de Sódio/farmacologia , Relação Estrutura-Atividade , Succinatos/farmacologia , ÁguaRESUMO
Paramagnetic labels specific for sulfhydryl (SH) groups have been used to study the conformational changes of the inner mitochondrial membrane. The EPR spectra of the SH-groups spin-labeled with maleimide or iodoacetamide show the existence of two populations of sulfhydryl groups, differing in their mobility (one weakly, the other strongly immobilized). The incubation with succinate or phenylsuccinate decreased the binding of these labels of the weakly immobilized sites while the number of total SH groups was the same before and after the incubation. These results suggest that succinate or phenylsuccinate induce a reversible change in protein conformation or in protein arrangement within the inner mitochondrial membrane. This change is concomitant to the protein movement between inner membrane and perimembranal space induced by either of these two molecules.
Assuntos
Membranas Intracelulares/análise , Proteínas de Membrana/análise , Mitocôndrias Hepáticas/análise , Succinatos , Animais , Espectroscopia de Ressonância de Spin Eletrônica , Conformação Proteica , Ratos , Ácido Succínico , Compostos de Sulfidrila/análiseRESUMO
Both salt-soluble and detergent-soluble rat brain globular acetylcholinesterases (SS- and DS- AChE EC 3.1.1.7) are amphiphiles, as shown by detergent dependency of enzymatic activity and binding to liposomes. Proteinase K and papain treatment transformed SS-AChE and DS-AChE into forms that, in absence of detergent, no longer aggregated nor bound to liposomes. In contrast, phosphatidylinositol-specific phospholipase C had no effect on these properties. Labeling DS-AChE with 3-(trifluoromethyl)-3-(m-(125I)-iodophenyl) diazirine ([125I]TID) revealed, by polyacrylamide gel electrophoresis under reducing conditions, one single band of 69 kD apparent molecular mass. The same pattern was previously obtained with Bolton and Hunter reagent-labeled enzyme. Proteinase K treatment transformed the 11 S [125I]TID labeled AChE into a 4 S form which no longer showed 125I-radioactivity and was unable to bind to liposomes. These results are compatible with the existence of a hydrophobic segment present both on salt-soluble and detergent-soluble 11 S AChE as well as on the minor forms 4 S and 7 S. This segment is not linked to the catalytic subunits by disulfide bounds in contrast to the 20 kD non-catalytic subunit described by Inestrosa et al.
Assuntos
Acetilcolinesterase/metabolismo , Encéfalo/enzimologia , Membrana Celular/enzimologia , Acetilcolinesterase/química , Animais , Azirinas , Sítios de Ligação , Reagentes de Ligações Cruzadas , Detergentes , Dissulfetos/metabolismo , Eletroforese em Gel de Poliacrilamida , Endopeptidase K , Lipossomos/metabolismo , Peso Molecular , Concentração Osmolar , Oxirredução , Papaína/metabolismo , Ratos , Ratos Wistar , Serina Endopeptidases/metabolismo , Solubilidade , Temperatura , Fosfolipases Tipo C/metabolismoRESUMO
We describe an affinity chromatography method in which dimethylaminoethylbenzoic acid-Sepharose 4B is used, making it possible to separate in one step the molecular forms of globular acetylcholinesterase (AChE, EC 3.1.1.7) or butyrylcholinesterase (ChE, EC 3.1.1.8). A crude extract containing these enzymes was deposited onto the chromatography gel, washed, and eluted by a linear gradient of tetramethylammonium chloride (0-0.3 M). With rat brain AChE, two well-separated peaks were eluted in the presence of 1% Triton X-100; the first peak corresponded to 4 S forms and the second to 11 S forms. This separation was very efficient for salt-soluble activity and less efficient for the detergent-soluble AChE. In this case, the 4 S peak represented only 6.5% of total detergent-soluble activity and was cross-contaminated by the 11 S form. Rat serum ChE was efficiently separated into two peaks of 7 S and 11 S. This method could potentially be adapted to separate other multimeric proteins with varying numbers of affinity sites.