Increase in lipid microviscosity of unilamellar vesicles upon the creation of transmembrane potential

Summary Diffusion potential of potassium ions was formed in unilamellar vesicles of phosphatidyl choline. The vesicles, which included potassium sulfate buffered with potassium phosphate, were diluted into an analogous salt solution made of sodium sulfate and sodium phosphate. The diffusion potential was created by the addition of the potassium-ionophore, valinomycin. The change in lipid microviscosity, ensuing the formation of membrane potential, was measured by the conventional method of fluorescence depolarization with 1,6-diphenyl-1,3,5-hexatriene as a probe. Lipid microviscosity was found to increase with membrane potential in a nonlinear manner, irrespective of the potential direction. Two tentative interpretations are proposed for this observation. The first assumes that the membrane potential imposes an energy barrier on the lipid flow which can be treated in terms of Boltzmann-distribution. The other interpretation assumes a decrease in lipid-free volume due to the pressure induced by the electrical potential. Since increase in lipid viscosity can reduce lateral and rotational motions, as well as increase exposure of functional membrane proteins, physiological effects induced by transmembrane potential could be associated with such dynamic changes.     

Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling

Cells respond to chemokine stimulation by losing their round shape in a process called polarization, and by altering the subcellular localization of many proteins. Classic imaging techniques have been used to study these phenomena. However, they required the manual acquisition of many cells followed by time consuming quantification of the morphology and the co-localization of the staining of tens of cells. Here, a rapid and powerful method is described to study these phenomena on samples consisting of several thousands of cells using an imaging flow cytometry technology that combines the advantages of a microscope with those of a cytometer. Using T lymphocytes stimulated with CCL19 and staining for MHC Class I molecules and filamentous actin, a gating strategy is presented to measure simultaneously the degree of shape alterations and the extent of co-localization of markers that are affected by CCL19 signaling. Moreover, this gating strategy allowed us to observe the segregation of filamentous actin (at the front) and phosphorylated Ezrin-Radixin-Moesin (phospho-ERM) proteins (at the rear) in polarized T cells after CXCL12 stimulation. This technique was also useful to observe the blocking effect on polarization of two different elements: inhibition of actin polymerization by a pharmacological inhibitor and expression of mutants of the Par6/atypical PKC signaling pathway. Thus, evidence is shown that this technique is useful to analyze both morphological alterations and protein redistributions.     

Comparison of the electron spin polarized spectrum found in plant photosystem I and in iron-depleted bacterial reaction centers with time-resolved K-band EPR; evidence that the photosystem I acceptor A1 is a quinone

The suggestion that the electron acceptor A₁ in plant photosystem I (PSI) is a quinone molecule is tested by comparisons with the bacterial photosystem. The electron spin polarized (ESP) EPR signal due to the oxidized donor and reduced quinone acceptor (P ₈₇₀ ⁺ Q^-) in iron-depleted bacterial reaction centers has similar spectral characteristics as the ESP EPR signal in PSI which is believed to be due to P ₇₀₀ ⁺ A ₁ ^- , the oxidized PSI donor and reduced A₁. This is also true for better resolved spectra obtained at K-band (24 GHz). These same spectral characteristics can be simulated using a powder spectrum based on the known g-anisotropy of reduced quinones and with the same parameter set for Q^- and A₁ ^-. The best resolution of the ESP EPR signal has been obtained for deuterated PSI particles at K-band. Simulation of the A₁ ^- contribution based on g-anisotropy yields the same parameters as for bacterial Q^- (except for an overall shift in the anisotropic g-factors, which have previously been determined for Q^-). These results provide evidence that A₁ is a quinone molecule. The electron spin polarized signal of P₇₀₀ ⁺ is part of the better resolved spectrum from the deuterated PSI particles. The nature of the P₇₀₀ ⁺ ESP is not clear; however, it appears that it does not exhibit the polarization pattern required by mechanisms which have been used so far to explain the ESP in PSI.Abbreviations hf hyperfine - A₀ A₀ acceptor of photosystem I - A₁ A₁ acceptor of photosystem I - Brij-58 polyoxyethylene 20 cetyl ether - CP1 photosystem I particles which lack ferridoxin acceptors - ESP electron spin polarized - EPR electron paramagnetic resonance - I intermediary electron acceptor, bacteriopheophytin - LDAO lauryldimethylamine - N-oxide, P₇₀₀ primary electron donor of photosystem I - PSI photosystem I - P₇₀₀ ^T triplet state of primary donor of photosystem I - P₈₇₀ primary donor in R. sphaeroides reaction center - Q quinore-acceptor in photosynthetic bacteria - RC reaction center     

Effects of membrane physical parameters on hematoporphyrin-derivative binding to liposomes: A spectroscopic study

Summary Physical parameters of membrane bilayers were studied for their effect on the binding of hematoporphyrin derivative (Hpd), which is used as a sensitizer in photodynamic therapy of cancerous tissues. The purpose of this study was to clarify which parameters were relevant, under physiological conditions, to the selectivity of Hpd binding to cancer cells. Fluorescence spectroscopy was used to measure the relative partitioning of the dye between the lipid and aqueous media. Increasing the microviscosity of the liposomes' membranes by various bilayer additives results in a strong reduction of Hpd binding, to an extent independent of the specific additive. The effect of temperature near the physiological value as well as the effect of cross membrane potential are small. Surface potential does not affect the binding constant, indicating that the binding species does not carry a net electric charge.     

Variations in Membrane Sensitivity of Brain Region Synaptosomes to the Effects of Ethanol In Vitro and Chronic In Vivo Treatment

The effects of chronic ethanol treatment on the membrane order of synaptosomes from the cerebral cortex, striatum, cerebellum, brainstem, and hippocampus of rats were determined by measuring the fluorescence polarization of diphenylhexatriene (DPH) that had been incorporated into the synaptosomal membranes. Fischer-344 rats either were fed a nutritionally complete ethanol-containing liquid diet for 5 months or pair-fed with a diet that contained sucrose substituted isocalorically for ethanol. Polarization values for synaptosomes from all the brain regions studied were similar except for those from cerebral cortical synaptosomal membranes, which were significantly less ordered. Ethanol in vitro (30-500 mM) decreased the polarization values in synaptosomes from sucrose-control rats for all brain regions, although the sensitivity of cerebellar synaptosomes to the membrane disordering effects of ethanol in vitro was significantly greater that of synaptosomes from other brain regions. Chronic ethanol treatment did not alter baseline polarization for any brain region. Cerebellar and brainstem synaptosomes from the ethanol-fed rats were significantly less susceptible to the membrane disordering effects of ethanol in vitro compared to their sucrose controls, suggesting that chronic ethanol administration results in tolerance to ethanol's membrane effects. Striatal synaptosomes exhibited intermediate tolerance, whereas the sensitivities of cortical and hippocampal synaptosomes to membrane disordering by ethanol in vitro were not significantly affected by the chronic ethanol treatment. These results suggest that synaptosomal membranes have different membrane order requirements depending on the brain region from which they are prepared. Variations in brain regional neuronal membrane sensitivity to ethanol and differential tolerance development may contribute to some of the acute and chronic behavioral effects of ethanol.     

The effect of erythrocyte associated light scattering on membrane fluorescence polarization

Summary The apparent membrane fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene has been reported to be lower in intact erythrocytes than in isolated erythrocyte membranes. Although this difference was once suggested to be caused by the fluidizing effect associated with the loss of erythrocyte proteins during membrane isolation, it is currently thought to be an artifact resulting from intense light scattering properties of intact erythrocytes which overwhelm extrapolation methods of correcting for light scattering. This study confirmed that, at erythrocyte concentrations greater than 10⁷ cells/ml, this difference was caused by intense light scattering; however, at erythrocyte concentrations less than 4.0 × 10⁶ cells/ml, the anisotropy values for erythrocytes and isolated membranes are identical, demonstrating that intense light scattering can be overcome with dilute suspensions of cells.     

荧光探针Bis-ANS和磷酸丙糖异构酶的相互作用

研究了极性荧光探针Bis-ANS和磷酸丙糖异构酶的相互作用。我们发现由磷酸丙糖异构酶(TIM)中Trp残基和结合在TIM分子上的Bis-ANS之间的能量传递引起的Trp残基荧光的淬灭呈双相性,表明Bis-ANS在TIM分子上可能有2个不相同的结合位点,其结合的解离平衡常数Kd分别为3.3μM和17.0μM。底物GDP引起已结合的Bis-ANS荧光强度进一步增强和荧光谱的蓝移说明GDP可影响Bis-ANS在TIM分子上结合部位的构象,使其疏水性增强。我们还观察到由于结合在同一TIM分子上的Bis-ANS之间的能量传递引起的退偏振,进一步证明Bis-ANS有2个结合部位在1—2800bar压力范围里,增高压力引起结合在TIM分子上的Bis-ANS荧光进一步增强和光谱蓝移,说明TIM在压力下解离成亚基的过程中发生了Weber提出的"conformationaldrift。     

脂肪族类两亲物对肌浆网膜脂区结构的影响

C18饱和脂肪酸和胺可增加DPH标记肌浆网(SR)的荧光偏振度,而C18单不饱和脂肪酸。胺和醇则使其偏振度下降。加入MgATP,可除去单不饱和脂肪胺引起的DPH标记的荧光偏振度下降,并使之高于未加脂肪胺的对照水平。饱和酸及相应胺可使标记于膜脂中层和深层的TAS和12AS的荧光偏振度上升,不饱和酸及相应胺和醇仅使12AS荧光偏振下降。说明脂肪族类两亲物对SR膜流动性的影响与脂肪链饱和程度有关。饱和者主要使膜中、深层流动性下降.不饱和者主要使膜深层流动性升高。     

Lipid fluidity and composition of the erythrocyte membrane from healthy dogs and labrador retrievers with hereditary muscular dystrophy

Erythrocyte membranes and their liposomes were prepared from clinically normal dogs and Labrador retrievers with hereditary muscular dystrophy. The static and dynamic components of fluidity of each membrane were then assessed by steady-state fluorescence polarization techniques using limiting hindered fluorescence anisotropy and order parameter values of 1,6-diphenyl-1,3,5-hexatriene (DPH) and fluorescence anisotropy values ofdl-2-(9-anthroyl)-stearic acid anddl-12-(9-anthroyl)-stearic acid, respectively. Membrane lipids were extracted and analyzed by thin-layer chromatography and gas chromatography. The results of these studies demonstrated that the lipid fluidity of erythrocyte membranes, and their liposomes, prepared from dystrophic dogs were found to possess significantly lower static and dynamic components of fluidity than control counterparts. Analysis of the composition of membranes from dystrophic dogs revealed a higher ratio of saturated fatty acyl chain/unsaturated chains (w/w) and lower double-bond index. Alterations in the fatty acid composition such as decrease in levels of linoleic (18:2) and arachidonic (20:4) acids and increase in palmitic (16:0) and stearic (18:0) acids were also observed in the membranes of dystrophic animals. These associated fatty acyl alterations could explain, at least in part, the differences in membrane fluidity between dystrophic and control dogs.