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1.
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. 相似文献
2.
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. 相似文献
3.
4.
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 相似文献
5.
Variations in Membrane Sensitivity of Brain Region Synaptosomes to the Effects of Ethanol In Vitro and Chronic In Vivo Treatment 总被引:1,自引:1,他引:0
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. 相似文献
6.
Jagjivan R. Mehta Kyle G. Braund Gerald A. Hegreberg Vijay Thukral 《Neurochemical research》1991,16(2):129-135
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. 相似文献
7.
Peter H. Barry 《The Journal of membrane biology》1984,82(3):221-239
Summary Many neurones are extremely invaginated and possess branching processes, axons and dendrites. In general, they are surrounded by a restricted diffusion space. Many of these cells exhibit large, slow potential changes during the passage of current across their membranes. Whenever currents cross membranes separating aqueous solutions, differences in transport numbers of the major permeant ions give rise to local concentration changes of these ions adjacent to the membranes, which will result in various electrical and osmotic effects. These transport number effects are expected to be enhanced by the presence of membrane invaginations. Dendrites are equivalent to reversed invaginations and there should be significant changes in concentrations of permeant ions within them. In general, the effects of such changes on the electrical response of a cell will be greater when the concentration of a major permeant ion is low. The effects have been modelled in terms of two nondimensional parameters: the invagination transport number parameter and the relative area occupied by the invaginations A. If these two parameters are known, the magnitudes and time course of the slow potential changes can immediately be estimated and the time course converted to real time, if the length of the invaginations (l) and ionic diffusion coefficient (D) within them are also known. Both analytical and numerical solutions have been given and predictions compared. It is shown that in the case of large currents and potentials the analytical solution predictions will underestimate the magnitudes and rates of onset of the voltage responses. The relative magnitude of the transport number effect within the invaginations (or dendrites) and other transport number contributions to slow potential changes have also been assessed and order-of-magnitude values of these are estimated for some biological data. 相似文献
8.
The rise time, of Signal IIf and the decay time of P-680+ have been measured kinetically as a function of pH by using EPR. The Photosystem II-enriched preparations which were used as samples were derived from spinach chloroplasts, and they evolved oxygen before Tris washing. The onset kinetics of Signal IIf are in agreement, within experimental error, with the fast component of the decay of an EPR signal attributable to P-680+. The signal IIf rise kinetics also show good agreement with published values of the pH dependence of the decay of P-680+ measured optically (Conjeaud, H. and Mathis, P. (1980) Biochim. Biophys. Acta 590, 353–359). These results are consistent with a model where the species Z (or D1) responsible for Signal IIf is the immediate electron donor to P-680+ in tris-washed Photosystem II fragments. 相似文献
9.
In order to find a suitable reagent for extracting the muscarinic receptor from rat brain membranes 14 different detergents
were tested. Only the plant glycoside digitonin efficiently solubilized the receptor protein in its native form. At the same
time microviscosity of detergent micelles was determined by measuring the fluorescence polarization of a hydrophobic fluorescent
probe diphenylhexatriene incorporated into the micelles. In the case of digitonin the polarization value was close to the
corresponding value obtained for rat brain membrane fragments, while for the other detergents studied it remained considerably
lower. The results obtained indicate that the fluidity of detergent micelles may play an important role in retaining the active
conformation of the solubilized muscarinic receptor. 相似文献
10.
The effect of Ca2+ and Mg2+ on relative fluidity of phosphatidylcholine liposomes was studied by measuring the degree of chlorophyll fluorescence polarization.
An increase in the degree of fluorescence polarization was observed on incubation of liposomes with different concentrations
of Ca2+ or Mg2+. The results have been interpreted on the basis of increase in the size of liposomes which could be brought about by calcium
or magnesium induced fusion of small unilamellar liposomes to form larger vesicles. Fusion of liposomes has also been confirmed
by the experiments on efficiency of energy transfer from chlorophyll b to chlorophyll a, and transmission electron microscopy
of liposomes before and after incubation with Ca2+ and Mg2+. 相似文献