RESUMO
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
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
Electron paramagnetic resonance and fluorescence experiments have demonstrated that the lipid matrix of inner membrane of mitochondria was more fluid than the control membrane when incubated with succinate or with one of its non permeant and non metabolizable analog, phenylsuccinate, both of which induce a protein movement from the inner membrane towards the intermembrane space and the inner matrix. Besides, the increase of fluidity seemed more pronounced near the bilayer surface. Although the mechanisms involved in the protein movement are yet unknown, these results lead us to think that they are related to a membrane reorganization involving inter alia the lipid matrix.
Assuntos
Membranas Intracelulares/metabolismo , Bicamadas Lipídicas , Fluidez de Membrana/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Succinatos/farmacologia , Animais , Espectroscopia de Ressonância de Spin Eletrônica , Membranas Intracelulares/efeitos dos fármacos , Cinética , Ratos , Espectrometria de Fluorescência , Ácido SuccínicoRESUMO
Sodium valproate (VPA), the salt of a branched short-chain fatty acid, is a major antiepileptic whose mode of action, as yet unclear, may involve effects on the organization of membranes. VPA was either injected into rats whose liver and kidney mitochondria were then isolated, or was preincubated with isolated mitochondria. First, liver and kidney mitochondria were studied with paramagnetic probes. The electron paramagnetic resonance spectra of proteins of VPA-treated mitochondria spin-labeled with 4-maleimido-2,2,6,6-tetramethyl-1-pyrrolidinoxyl showed that the ratio of weakly immobilized to strongly immobilized SH groups was reduced with respect to control mitochondria, more so in liver than in kidney mitochondria of VPA-injected rats, and more so in kidney than in liver mitochondria for VPA-incubated mitochondria. Spectra of mitochondrial lipids spin-labeled with 5-doxyl stearic methyl ester showed that VPA had no significant effect on order parameters S. Second, the transmembrane movement of aspartate aminotransferase was studied by incubating liver mitochondria in a sucrose-succinate medium and then fractionating them. The translocation of aspartate aminotransferase from mitoplasts, vesicles formed of inner membrane and matrix, to the intermembrane fluid, was significantly higher in VPA-treated than in control mitochondria. Thus, VPA, at concentrations in the range of those used therapeutically, interacted with membranes by modifying the structural organization of the internal mitochondrial membrane, essentially the membrane protein conformation.