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1.
J. Svetek V. Furtula M. Nemec E. A. Nothnagel M. Schara 《The Journal of membrane biology》1995,143(1):19-28
Translational diffusion of a fluorescent sterol probe was measured in the plasma membranes of protoplasts isolated from cortical cells of the primary root of maize seedlings. The apparent lateral diffusion coefficient was typically observed to be nearly insensitive to temperature, while the mobile fraction increased with increasing temperature. These fluorescence photobleaching recovery (FPR) measurements were compared with the electron paramagnetic resonance (EPR) spectra of the methyl ester of 13-doxyl palmitic acid in membranes of corn root tissue in situ. The complex spectra observed with this probe were analyzed as weighted sums of simpler spectra of various order parameters and rotational correlation times. The reconstituted spectra calculated from the model show that EPR also detects a mobile (less ordered, fluid) fraction, distinguished by the order parameter S=0.1 to 0.2, which becomes more abundant as temperature increases and is qualitatively comparable to the mobile fraction determined by the FPR method. The observed results on the mobile fractions and the diffusion rates for translational (FPR) as well as rotational (EPR) motions are interpreted in terms of membrane organization, thus providing information on the population and structural patterns of the coexisting domains with a special emphasis on the response of the membrane to temperature changes.This work was supported in part by grants from the Ministry of Science and Technology of the Republic of Slovenia and the International Research Program of the U.S. Department of Agriculture (USDA-JF 814-51) to M.S., and by grants from the Competitive Grants Program of the U.S. Department of Agriculture (88-37264-3807 and 90-37264-5471) to E.A.N. 相似文献
2.
Plasma-membrane dynamics in live protoplasts from maize (Zea mays L.) roots were characterized and examined for relationships as to the ability of the protoplasts to synthesize new cell walls and develop to cells capable of division. The lateral diffusion-coefficients and mobile fractions of fluorescence-labeled plasma-membrane proteins and lipids were measured by fluorescence photobleaching recovery. Small but significant effects on the diffusion of membrane proteins were observed after treatments with oryzalin or amiprophosmethyl, microtubule-disrupting drugs that increased the mobile fraction, and after treatments with cytochalasins B or D, microfilament-disrupting drugs that decreased the diffusion coefficient. A number of parameters were tested for correlative effects on membrane dynamics and protoplast performance in culture. Protoplasts isolated with a cellulase preparation from Trichoderma viride showed faster membrane-protein diffusion and a lower frequency of development to cells capable of division than did protoplasts isolated with a cellulase preparation from T. reesei. Membrane proteins in maize A632, a line less capable of plant regeneration from callus, diffused with a smaller diffusion coefficient but a greater mobile fraction than did membrane proteins in maize A634, a line with greater regeneration capacity. The plasma membranes of A632 and A634 protoplasts also differed with regard to lateral-diffusion characteristics of phospholipid and sterol probes, although the presence of both rapidly and slowly diffusing lipid components indicated the apparent existence of lipid domains in both A632 and A634. The protoplasts of the two lines did not differ significantly, however, in either wall regeneration or frequency of development to cells capable of division.Abbreviations and symbols D
lateral diffusion coefficient
- FITC
fluorescein-5-isothiocyanate
- FPR
fluorescence photobleaching recovery
- LY
Lucifer yellow
- LY-Chol
dilithium 4-amino-N-[(-(carbo(5-cholesten-3-yl)oxy)hydrazinocarbonyl)aminol]-1,8-naphthalimide-3,6-disulfonate
- LY-DC16:0PE
dilithium 4-amino-N-[3-(-(dipalmitoyl-sn-glycero-3-phosphoethanol-amino)ethylsulfonyl)phenyl]-1,8-naphthalimide-3,6-disulfonate 相似文献
3.
The temperature dependence of the yield of in vivo prompt and delayed chlorophyll fluorescence was investigated in maize and barley leaves. In the chilling-sensitive maize, delayed fluorescence at steady-state level showed a maximum near the temperature at which thylakoid membrane lipids undergo a phase transition as revealed by differential scanning calorimetry measurements. In the chilling-resistant barley, no phase transition was detected above 0°C and the delayed light emission varied in a monotonic fashion. It was shown that measurements of delayed luminescence intensity in vivo can provide a rapid and sensitive method for detecting the phase change of membrane lipids in intact leaves of chilling-sensitive plant species such as tomato, cotton, cucumber, castor bean or avocado. In contrast, the use of steady-state prompt chlorophyll fluorescence as an indicator of membrane fluidity change was not successful. 相似文献
4.
Temperature-induced Changes in Hill Activity of Chloroplasts Isolated from Chilling-sensitive and Chilling-resistant Plants 总被引:6,自引:3,他引:3
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The effect of temperature on Hill activity has been compared in chilling-sensitive and chilling-resistant plants. The Arrhenius activation energy (Ea) for the photoreduction of 2,6-dichlorophenolindophenol by chloroplasts isolated from two chilling-sensitive plants, mung bean (Vigna radiata L. var. Mungo) and maize (Zea mays L. cv. PX 616), increased at low temperatures, below 17 C for mung bean and below 11 C for maize. However, the Ea for this reaction in pea (Pisum sativum L. cv. Massay Gem), a chilling-resistant plant, likewise increased at temperatures below 14 C. A second change in Ea occurred at higher temperatures. The Ea decreased above about 28 C for mung bean, 30 C for maize, and 25 C for pea. At temperatures approaching 40 C, thermal inactivation of Hill activity occurred. These results, when taken together with previous results obtained with the chilling-resistant plant barley, indicate that chloroplasts from both chilling-sensitive and chilling-resistant plants can undergo a change in chloroplast membrane activity at low temperatures above freezing and that the presence of such a change in chloroplast membranes is not necessarily correlated with chilling sensitivity. 相似文献
5.
The temperature dependence of the local diffusion of fluorescent molecular probes of various polarities (alkane, long-chain fatty acid, short-chain alcohol and fatty acid), all labelled with 7-nitrobenz-2-oxa-1,3-diazol-4-yl in the cuticle of Clivia miniata Regel was studied by the technique of fluorescence recovery after photobleaching. The technique yields the coefficient of diffusion, D, in the plane of the cuticle over distances of some 10 m and the fraction, R, of mobile reporter molecules. The inner (more hydrophilic) and the outer (more hydrophobic) faces of the cuticle were studied separately by appropriate incubation. The value of D was found to depend sensitively on the polarity of the probe, the temperature and the position within the cuticle (outer hydrophobic or inner hydrophilic side). Depending on the type of probe, D increased (in the case of the alkane) or decreased (in the case of the alcohol) after removal of the (monomeric) waxes. The electron-spin-resonance (ESR) spectra of incorporated spin-labelled fatty-acid probes measured in the intact cuticle contained a major component similar to the spectrum recorded from the polymerized matrix from which waxes had been extracted, and a second component similar to the spectrum from the monomeric waxes. At low temperatures, the ESR spectra from labels at two different chain positions corresponded to chain motion which was slow on the ESR timescale. At high temperatures, the spectral component from the monomeric waxes indicated chain motions in the motional narrowing regime which were of an essentially isotropic nature.No evidence was found for a liquid-crystalline lipid phase such as found for the polar lipids in cell membranes, nor was there evidence for a sharp, thermotropic, lipid-phase transition either in the cuticle or in the waxes. Experiments with oriented samples did not demonstrate the presence of large domains with a uniform orientation of the lipid chains relative to the cuticular layers. The diffusion measurements and spin-label studies provide evidence for conformational changes of the cuticle extending over the whole temperature range studied (10° C to 70° C). These conformational changes are attributed to phase-separation processes within the cuticle. The phase separation in extracted waxes extended over a similar broad temperature range. This indicates that the transitions in the cuticle are largely determined by these components. At higher temperatures, however, the chain mobility in the regions of monomeric wax was considerably greater than that in the polymerized matrix. The experimental results strongly indicate that all three layers of the Clivia cuticle exhibit a multilamellar structure of alternatingly stacked, highly hydrophobic layers of welldefined thickness (5±0.5 nm) and more disordered layers of variable (4 to 15 nm) thickness. The lamellae are wellordered and extend over the whole leaf in the cuticle proper but are split-up into small domains in the inner and the external cuticular layer. Furthermore, changes of the molecular transport properties caused by the influence of ozone exerted during the growth of the plant were studied. We found that the diffusion coefficient increased both in the outer and the inner layer of the cuticle. A particularly large increase, by about a factor of three, was found for alkane diffusion in the hydrophobic outer face, pointing to defects in the polymerized matrix.Abbreviations MX-membrane
polymer matrix membrane (or monomeric wax-depleted cuticle)
- ESR
electron-spin resonance
-
n-SASL
n-(4,4-dimethyl-N-oxy-2-oxazolidinyl)-stearic acid
- NBD
7-nitrobenz-2-oxa-1,3-diazol-4-yl
The present work was made possible by a grant from the Bayerische Umweltministerium. Additional support by the Fonds der Chemischen Industrie is gratefully acknowledged. We are most grateful for very helpful discussions with Professor H. Ziegler, Professor J. Schönherr and Dr. M. Riederer from the Institut für Botanik, Technische Universität München, FRG. 相似文献
6.
Summary Lateral diffusion measurements have been made on lipids and proteins in the plasma membrane of live protoplasts derived from rose (Rosa sp. Paul's Scarlet) suspension-cultured cells. Two different fluorescent lipid probes exhibited markedly different diffusion rates, indicating possible heterogeneity in the lipid domain of the membrane. Membrane proteins were labeled directly with covalently-reactive fluorophores, and factors that might perturb the lateral diffusion of these labeled proteins were investigated. Treatment of the protoplasts with various cytoskeleton-disrupting drugs generally had little effect on protein diffusion, although treatment with oryzalin, a microtubule-disrupting drug, did slightly reduce the mobile fraction of membrane proteins. Elevation of the CaCl2 concentration in the medium from 1 mM to 10 mM significantly reduced the mobile fraction of membrane proteins and also increased the fraction of protoplasts that were able to regenerate cell walls and divide in culture. These results are discussed in relation to reported evidence of lipid domains in the plasma membranes of other cells and protoplasts. The relative importance of lipid domains and membrane-cytoskeleton interaction in governing protein diffusion is considered.Abbreviations D
lateral diffusion coefficient
- RCA
Ricinus communis agglutinin
- BPA
Bauhinia purpurea agglutinin
- DTAF
dichlorotriazinylaminofluorescein
- FTSC
fluorescein-5-thiosemicarbazide
- C18-Fl
5-(N-octadecanoyl)aminofluorescein
- LY-Chol
Lucifer yellow conjugate of cholesterol, i.e., dilithium 4-amino-N-[(-(carbo(5-cho-lesten-3-yl)oxy)hydrazinocarbonyl)amino]-1,8-naphthalimide-3,6-disulfonate
- APM
amiprophosmethyl
- DMSO
dimethylsulfoxide
- FPR
fluorescence photobleaching recovery
- sd
standard deviation
- FRAF
fluorescence redistribution after fusion
- M
mobile fraction 相似文献
7.
The temperature dependence of the yield of in vivo prompt and delayed chiorophyll fluorescence was investigated in maize and barley leaves. In the chilling-sensitive maize, delayed fluorescence at steady-state level showed a maximum near the temperature at which thylakoid membrane lipids undergo a phase transition as revealed by differential scanning calorimetry measurements. In the chilling-resistant barley, no phase transition was detected above 0°C and the delayed light emission varied in a monotonic fashion. It was shown that measurements of delayed luminescence intensity in vivo can provide a rapid and sensitive method for detecting the phase change of membrane lipids in intact leaves of chilling-sensitive plant species such as tomato, cotton, cucumber, castor bean or avocado. In contrast, the use of steady-state prompt chlorophyll fluorescence as an indicator of membrane fluidity change was not successful. 相似文献
8.
Vincent Schram Jean-François Tocanne André Lopez 《European biophysics journal : EBJ》1994,23(5):337-348
Fluorescence Recovery After Photobleaching experiments were simulated using a computer approach in which a membrane lipid leaflet was mimicked using a triangular lattice obstructed with randomly distributed immobile and non-overlapping circular obstacles. Influence of the radius r and area fraction c of these obstacles and of the radius R of the observation area on the relative diffusion coefficient D
* (Eq. (1)) and mobile fraction M was analyzed. A phenomenological equation relating D
* to r and c was established. Fitting this equation to the FRAP data we obtained with the probe NBD-PC embedded in bacteriorhodopsin/egg-PC multilayers suggests that this transmembrane protein rigidifies the surrounding lipid phase over a distance of about 18 Å (two lipid layers) from the protein surface. In contrast, analysis of published diffusion constants obtained for lipids in the presence of gramicidin suggests that in terms of lateral diffusion, this relatively small polypeptide does not significantly affect the surrounding lipid phase. With respect to the mobile fraction M, and for point obstacles above the percolation threshold, an increase in R led to a decrease in M which can be associated with the existence of closed domains whose average size and diffusion properties can be determined. Adaptation of this model to the re-interpretation of the FRAP data obtained by Yechiel and Edidin (J Cell Biol (1987) 115:755–760) for the plasma membrane of human fibroblasts consistently leads to the suggestion that the lateral organization of this membrane would be of the confined type, with closed lipid domains of 0.5 µm2 in area.Abbreviations and notations used BR
bacteriorhodopsin
- DMPC
dimyristoylphosphatidylcholine
- diOC18
dioctadecyloxatricarbocyanine
- egf-PC
egg-yolk phosphatidylcholine
- NBD-PC
1-acyl2-[t2-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine
- MOPS
3-[N-morpholino]propane sulfonic acid
- FRAP
Fluoresence Recovery After photobleaching
- D
observed diffusion coefficient
-
D0
diffusion coefficient in the absence of obstacles
-
D
*
relative diffusion constant (Eq. 1)
-
M
mobile fraction
-
c
obstacle area fraction
-
r
obstacle radius
-
R
observation area radius
-
r
d
diffusion area radius
Correspondence to: A. Lopez 相似文献
9.
H+ flux kinetics were measured in solution around the roots of chilling-tolerant pea (Pisum sativum) and bean (Vicia faba), chilling-sensitive cucumber (Cucumis sativus) and pumpkin (Cucurbita pepo), and intermediate corn (Zea mays) species using a microelectrode technique to measure net flux. As a root warmed to room temperature alter 90 min at 4°C, at which temperature the H+ flux was near zero, the flux rose (influx) and then fell. These changes occurred at two apparent critical temperatures, which were higher for the more chilling-sensitive species. The First, lower, apparent critical temperature may represent the start of passive inward H+ transport. The higher critical temperature may represent the start of active H+ extrusion. From these apparent critical temperatures we have calculated the real critical temperature and the time delay of the chilling signal transduction process. Passive and active H+ transporters appear to have the same real critical temperature of chilling sensitivity, about 9°C, but have, respectively, 4 min and 11 min time delays. Measurement of these apparent critical temperatures may provide quick and reliable screening for chilling sensitivity in plant breeding programmes. Future ion flux studies may show the cellular location of chilling stress perception and the signal transduction pathways. 相似文献
10.
The fluorescence anisotropy decay of four different probes in bilayers of dimyristoylphosphatidylcholine was measured. The probes are diphenylhexatriene, diphenyloctatetraene, trimethylaminodiphenylhexatriene, and trans-parinaric acid. The data for each probe were analyzed in terms of two orientational order parameters, the ordinary order parameter and a higher one, and two rotational diffusion coefficients. The order parameters are largely independent of probe size, but depend on the position of the probes along the membrane normal, thus reflecting the profile of lipid order. If a probe is located in the plateau region of lipid order, its order parameters are interpreted as representing the rigid-body order of lipids. According to this interpretation, the total lipid order in the plateau region originates about equally from rigid-body order and conformational order. The two order parameters obtained for each probe are used to derive approximate angular distributions of the probe molecules. The diffusion coefficient for rotation about the long molecular axis is found to be infinitely large, indicating unhindered rotation about this axis. The diffusion coefficient for rotation about the short molecular axes is evaluated for a viscosity which results as 0.2 poise. This viscosity for rotational diffusion is an order of magnitude smaller than the viscosity for lateral diffusion indicating that at least two viscosities are required to characterize the fluidity of a lipid membrane.Abbreviations FAD
fluorescence anisotropy decay
- DMR
deuterium magnetic resonance
- ESR
electron spin resonance
- DMPC
dimyristoylphosphatidylcholine
- DPPC
dipalmitoylphosphatidylcholine
- DPH
1,6-diphenyl-1,3,5-hexatriene
- DPO
1,6-diphenyl-1,3,5,7-octatetraene
- TMA-DPH
1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene
- tPnA
trans-parinaric acid
- NPN
N-phenyl-1-naphthylamine
- BBO
2,5-bis(4-biphenylyl)oxazole 相似文献
11.
Molecular dynamics (MD) simulations were performed to study the migration of 2,4,6-trinitrotoluene (TNT) in the fluorine rubber
binder of polymer-bonded explosives (PBX) over a wide range of temperatures. The diffusion coefficient (D) of TNT is determined via microcanonical (NVE) MD simulation using the COMPASS force field. The calculated diffusion coefficient
(D) was then used to compute the migration time of TNT based on Fick’s second law and the results agree well with the experimental
data. The relation between D of TNT and temperature was confirmed and the results confirm the temperature-dependence of diffusion coefficients of TNT
in the binder, but a break is seen about the melt point (the temperature at which the elastomeric state of the binder changes
to a viscosity state) in the Arrhenius plot of ln(D) versus 1/T. 相似文献
12.
Heat Inducible Expression of a Chimeric Maize hsp70CAT Gene in Maize Protoplasts 总被引:6,自引:2,他引:4
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The response of maize (Zea mays L.) protoplasts to high temperature stress was investigated. After isolation and electroporation, protoplasts were preincubated for 12 hours at 26°C then incubated for 6 hours at elevated temperatures. The pattern of polypeptides synthesized by these protoplasts during the last hour was monitored by in vivo labeling with 35S-methionine. Incubation at 40° and 42°C resulted in the synthesis of polypeptides not detectable at 26°C. Introduction of a chimeric maize heat shock protein 70 promoter-chloramphenicol acetyltransferase coding region gene into protoplasts via electroporation resulted in the temperature-dependent induction of chloramphenicol acetyltransferase activity with maximal activity at 40°C. In the same protoplasts, a second chimeric gene, in which the firefly luciferase coding region was under the control of the 35S promoter from cauliflower mosaic virus, did not show an increase in expression after incubation at higher temperatures. Maize protoplasts provide a system to study molecular responses to high temperature stress. 相似文献
13.
Abstract Small molecule diffusion into Iota-Carrageenan gel was studied by using steady-state fluorescence (SSF) technique. Pyranine, dissolved in water was used as fluorescence probe. Fluorescence emission intensity, Ip, and scattered light intensity, Isc, were monitored to study diffusion and swelling processes at various temperatures respectively. Fickian and Li-Tanaka models were elaborated to produce diffusion, D, and collective diffusion, D 0, coefficients. Diffusion and swelling activation energies were also obtained and found to be 20.5 kj mol?1 and 28.2 kj mol?1, respectively. 相似文献
14.
The Effect of Temperature on Cold and Heat Resistance of Growing Plants: I. CHILLING-SENSITIVE SPECIES 总被引:1,自引:0,他引:1
DROZDOV S. N.; TITOV A. F.; TALANOVA V. V.; KRITENKO S. P.; SHERUDILO E. G.; AKIMOVA T. V. 《Journal of experimental botany》1984,35(11):1595-1602
The effect of temperature between 0 °C and 51 °C oncold and heat resistance of chilling-sensitive plants has beenstudied in maize, cucumber and tomato growing in controlledgrowth chambers. It has been shown that the normal temperaturerange (the background range) specific for each species doesnot affect their thermo-resistance. Temperatures outside thisbackground range induced the development of cold and heat resistancein leaves (these are the temperature ranges of cold and heathardening). Temperatures below + °9C and above +49 °Cfor maize, +8 °C and + 40°C for cucumber and +6 °Cand +42°C for tomato resulted in a decrease in leaf thermo-resistanceand injury (these are the temperature ranges of cold and heatinjury). At gradually declining or rising temperatures the initialpoints of plant injury were shifted slightly towards more extremetemperatures. The time necessary for dehardening of plants dependedon the degree of hardening. Dehardening is complete only atthe background temperatures. It has been assumed that the responsesto temperature of chilling-sensitive and chilling-resistantgrowing species belong qualitatively to the same type. Key words: Temperature, Thermo-resistance, Plants 相似文献
15.
Protoplasts were isolated from tissue fragments (<1 mm2) of three Philippine cultivars of Kappaphycus alvarezii: the giant cultivar, cultivar L and Bohol wild type, by enzymatic dissolution of cell walls. Yields of viable protoplasts from young and old thalli (apical, middle, basal segments) were compared at various temperatures, duration of treatment and pH using eight combinations of commercial enzymes (abalone acetone powder and cellulase), and prepared extracts from fresh viscera of abalone (Haliotis asinina) and a terrestrial garden snail. Isolated protoplasts were grown in various culture media, temperatures, photoperiods and irradiance values to determine the conditions that favor germination and growth.Protoplast yields in tissues treated with commercial enzymes and the garden snail extract were lower than those obtained in tissues treated with fresh abalone extracts. Generally, the number of viable protoplasts increased with duration of enzyme treatment at 25 °C with a maximum yield of 8.2 × 103 g−1 tissue at 48 h. Yields were consistently higher in all cultivars at pH 6.1. The yields were also high from the middle segments of the giant cultivar (3.7 × 103 g−1 tissue) and Bohol wild type (4.5 × 103 g−1 tissue) treated with fresh abalone extract, and from basal segments of cultivar L and tissues treated with garden snail extract. The germination rate of protoplasts was highest (39.8%) at 25 °C and 20 μmol photon m−2 s−1, using a 12:12 light dark photoperiod. The filament was 3.7 mm long by Day 5. These findings are relevant to developing cultures from protoplasts for genetic or strain improvement of K. alvarezii cultivars. 相似文献
16.
Dharanidaran Jayachandran Peter Smith Mohammad Irfan Junhong Sun John M. Yarborough Yannick J. Bomble Eric Lam Shishir P. S. Chundawat 《Biotechnology and bioengineering》2023,120(8):2253-2268
Carbohydrate binding modules (CBMs) are noncatalytic domains that assist tethered catalytic domains in substrate targeting. CBMs have therefore been used to visualize distinct polysaccharides present in the cell wall of plant cells and tissues. However, most previous studies provide a qualitative analysis of CBM-polysaccharide interactions, with limited characterization of engineered tandem CBM designs for recognizing polysaccharides like cellulose and limited application of CBM-based probes to visualize cellulose fibrils synthesis in model plant protoplasts with regenerating cell walls. Here, we examine the dynamic interactions of engineered type-A CBMs from families 3a and 64 with crystalline cellulose-I and phosphoric acid swollen cellulose. We generated tandem CBM designs to determine various characteristic properties including binding reversibility toward cellulose-I using equilibrium binding assays. To compute the adsorption (nkon) and desorption (koff) rate constants of single versus tandem CBM designs toward nanocrystalline cellulose, we employed dynamic kinetic binding assays using quartz crystal microbalance with dissipation. Our results indicate that tandem CBM3a exhibited the highest adsorption rate to cellulose and displayed reversible binding to both crystalline/amorphous cellulose, unlike other CBM designs, making tandem CBM3a better suited for live plant cell wall biosynthesis imaging applications. We used several engineered CBMs to visualize Arabidopsis thaliana protoplasts with regenerated cell walls using confocal laser scanning microscopy and wide-field fluorescence microscopy. Lastly, we also demonstrated how CBMs as probe reagents can enable in situ visualization of cellulose fibrils during cell wall regeneration in Arabidopsis protoplasts. 相似文献
17.
In the plasma membrane of various eucaryotic cell types, in particular blood platelets and erythrocytes, it is known that phospholipids are asymmetrically distributed between the two leaflets of the lipid bilayer and that this transverse asymmetry is controlled by an aminophospholipid translocase activity. In this respect, it was of interest to check whether there are differential transbilayer movements between amino- and neutral phospholipids in the apical plasma membrane of vascular endothelial cells which form the inner nonthrombogenic lining of the large blood vessel. In the first step we compared the transbilayer localization and also the rate of lateral motion of two fluorescent analogs of phosphatidylcholine and phosphatidylethanolamine, namely C6-NBD-PC and C6-NBD-PE, inserted into the apical plasma membrane of bovine aortic endothelial cells, in vitro. By the use of back-exchange experiments we have found that C6-NBD-PC could be removed from the cell membrane toward the culture medium regardless of the incubation conditions used, i.e., just after cell labeling at 0°C or even after further cell incubation for 1 h at 0 or 20°C. In contrast, C6-NBD-PE could be removed only when the cells were maintained at 0°C. After incubation for 1 h at 20°C, 85% of the probe molecules remained nonexchangeable, indicating probe translocation from the outer to the inner leaflet of the lipid bilayer. This "flip" process, which occurred at 20°C, was abolished when the endothelial cells were preincubated with N-ethylmaleimide, diamide, vanadate (VO3-4) and vanadyl (VO2+) ions, a set of substances which inhibit aminophospholipid translocase activity in various systems, and with a combination of sodium azide and 2-deoxyglucose which led to nearly complete ATP depletion in the cells. Fluorescence recovery after photobleaching experiments were also carried out to specify more precisely the localization and dynamics of the probes in the two leaflets of the plasma membrane lipid bilayer. They produced lateral diffusion coefficients D of 1.2 ± 0.05 × 10-9 cm2/s for C6-NBD-PC and 2.8 ± 0.3 × 10-9 cm2/s for C6-NBD-PE, when the two probes were located in the outer leaflet of the plasma membrane, just after cell labeling at 0°C. After cell incubation for one hour at 20°C, i.e., when C6-NBD-PC was still in the outer leaflet whereas C6-NBD-PE was translocated in the inner leaflet, D was observed to slightly increase for C6-NBD-PC (D = 1.9 ± 0.06 × 10-9 cm2/s) and to greatly increase by at least a factor of 3 for C6-NBD-PE (D = 9.1 ± 0.9 × 10-9 cm2/s). These results show that the plasma membrane of bovine aortic endothelial cells is equipped with a protein-dependent and energy-mediated phosphatidylethanolamine translocase activity and that the lateral diffusion rate of this phospholipid is much faster in the inner than in the outer leaflet of the lipid bilayer, thus indicating large differences in the fluidity of the two halves of this membrane. 相似文献
18.
Measurements of the steady state polarization of fluorescence from perylene and 9-vinylanthracene embedded in bilayer membranes were performed as a function of temperature. Similar measurements were made when these probes were dissolved in hydrocarbons as model solvents. The effects of cholesterol and n-alkyl alcohol additions to bilayers and head group variation were also examined. Results were expressed in terms of the average rotation rates of the probes.At 25°C, the calculated rotation rate for perylene in egg phosphatidylcholine vesicles was 275 × 106 sec?1 as compared to 2400 × 106 sec?1 for perylene in n-hexadecane. However, the activation energies for probe rotation in both environments was about 7 kcal/mole suggesting similar rotational diffusion mechanisms. Membrane microviscosity evaluations were performed according to a recently published scheme and an assessment of this method of viscosity estimation was given. The presence of an approximately equimolar amount of cholesterol impeded probe rotation (90 × 106 sec?1 at 25°C) and reduced the activation energy (4.9 kcal/mole) for probe rotation. In contrast, addition of n-alkyl alcohols to the vesicle suspension acted to increase probe rotation rates, an indication of fluidization of the membranes. This is in accord with spin label and cation permeability data for similar membranes.It was concluded that this method of probing can adequately report changes in membrane dynamic structure when these changes occur uniformly over the membrane surface. The interpretation is less clear when structural changes occur only in patches or domains of the membrane thereby producing a non-uniform surface distribution of probes. 相似文献
19.
The influence of pH and temperature on the structural organization, fluidity and permeability of the hyperthermophilic archaeon
membrane was investigated in situ by a combination of electron paramagnetic resonance (EPR) and fluorescence emission spectroscopy. For EPR measurements, Aeropyrum pernix cells, after growing at different pHs, were spin-labeled with the doxyl derivative of palmitic acid methylester (MeFASL[10,3]).
From the EPR spectra maximal hyperfine splitting (2A
max) and empirical correlation time (τemp), which are related to mean membrane fluidity, were determined. The mean membrane fluidity increases with temperature and
depends on the pH of the growth medium. Computer simulation of the EPR spectra shows that membrane of A. pernix is heterogeneous and consists of the regions characterized with three different types of motional characteristics, which
define three types of membrane domains. Order parameter and proportion of the spin probes in the three types of domains define
mean membrane fluidity. The fluidity changes of the membrane with pH and temperature correlate well with the ratio between
the fluorescence emission intensity of the first and third bands in the vibronic spectra of pyrene, I1/I3. At pH 7.0 a decrease of I1/I3 from 2.0 to 1.2, due to the penetration of pyrene into the nonpolar membrane region, is achieved at temperatures above 65°C,
the lower temperature limit of A. pernix growth. 相似文献
20.
《生物化学与生物物理学报:生物膜》2015,1848(4):942-950
The naphthalene-based fluorescent probes Patman and Laurdan detect bilayer polarity at the level of the phospholipid glycerol backbone. This polarity increases with temperature in the liquid–crystalline phase of phosphatidylcholines and was observed even 90 °C above the melting temperature. This study explores mechanisms associated with this phenomenon. Measurements of probe anisotropy and experiments conducted at 1 M NaCl or KCl (to reduce water permittivity) revealed that this effect represents interactions of water molecules with the probes without proportional increases in probe mobility. Furthermore, comparison of emission spectra to Monte Carlo simulations indicated that the increased polarity represents elevation in probe access to water molecules rather than increased mobility of relevant bilayer waters. Equilibration of these probes with the membrane involves at least two steps which were distinguished by the membrane microenvironment reported by the probe. The difference in those microenvironments also changed with temperature in the liquid–crystalline phase in that the equilibrium state was less polar than the initial environment detected by Patman at temperatures near the melting point, more polar at higher temperatures, and again less polar as temperature was raised further. Laurdan also displayed this level of complexity during equilibration, although the relationship to temperature differed quantitatively from that experienced by Patman. This kinetic approach provides a novel way to study in molecular detail basic principles of what happens to the membrane environment around an individual amphipathic molecule as it penetrates the bilayer. Moreover, it provides evidence of unexpected and interesting membrane behaviors far from the phase transition. 相似文献