渗透胁迫对小麦根质膜膜脂物理状态的影响

以两相法纯化的小麦(Triticum sativum L.)根质膜微囊为材料,研究了渗透胁迫下质膜物理状态的变化。结果表明,随着介质蔗糖浓度增加,质膜光散射值降低,二苯己三烯(DPH)荧光偏振值升高,MC540荧光强度增强,并且DPH长寿命组分的荧光寿命和平均寿命都缩短,暗示渗透胁迫使质膜微囊收缩变小,降低了质膜流动性和表面电荷密度,并且表明质膜的疏水性减弱。进一步实验发现,质膜内源色氨酸长寿命组分的寿命缩短,质膜H     

声波刺激对菊花愈伤组织膜脂物理状态和膜脂代谢的影响

以蔗糖梯度法纯化的菊花 (Dendranthemamorifolium (Ramat.)Tzvel.)愈伤组织质膜微囊为材料 ,研究了声波刺激下质膜膜脂代谢和物理状态的变化。结果表明 ,一定强度 (10 0dB)和频率 (10 0 0Hz)的声波刺激使质膜磷酸二脂含量和二苯己三烯 (DPH)荧光偏振值降低 ,质膜光散射值、MC5 40荧光强度和磷酸单脂含量增加。表明一定强度和频率的声波刺激使质膜变疏松 ,膜的流动性增加 ,膜表面电荷密度和疏水性降低 ,膜脂合成代谢增加 ,分解代谢减弱。由此可见 ,膜脂物理状态和膜脂代谢对声波刺激极为敏感。     

声波刺激对菊花愈伤组织膜脂物理状态和膜脂代谢的影响

以蔗糖梯度法纯化的菊花(Dendranthema morifolium (Ramat.) Tzvel.)愈伤组织质膜微囊为材料,研究了声波刺激下质膜膜脂代谢和物理状态的变化.结果表明, 一定强度(100 dB)和频率(1 000 Hz)的声波刺激使质膜磷酸二脂含量和二苯己三烯(DPH)荧光偏振值降低,质膜光散射值、MC540荧光强度和磷酸单脂含量增加.表明一定强度和频率的声波刺激使质膜变疏松,膜的流动性增加,膜表面电荷密度和疏水性降低,膜脂合成代谢增加,分解代谢减弱.由此可见,膜脂物理状态和膜脂代谢对声波刺激极为敏感.     

水稻幼苗根质膜氧化还原系统活性与抗冷性的关系

本试验以水稻幼苗为材料,研究冷胁迫和钙浸种、低温锻炼、低温锻炼结合钙浸种预处理分别对幼苗根质膜Ｆｅ（ＣＮ）３－６还原活性的影响。实验结果表明：冷胁迫降低了质膜Ｆｅ（ＣＮ）３－６还原活性;钙浸种、低温锻炼、低温锻炼结合钙浸种预处理均提高了质膜Ｆｅ（ＣＮ）３－６还源活性,尤其是削减了冷胁迫降低质膜Ｆｅ（ＣＮ）３－６还原活性的作用。根质膜Ｆｅ（ＣＮ）３－６还原活性与水稻幼苗抗冷力密切相关。     

水稻幼苗根系膜氧化还原系统活性与抗冷性的关系

本试验以水稻幼苗为材料,研究冷胁迫和钙浸种,低温锻炼,低温锻烧结合钙浸种预处理分别对幼苗根系膜Ｆｅ（ＣＮ）＾３－６还原性的影响。实验结果表明：冷胁迫降低了质膜Ｆｅ（ＣＮ）＾３－６还原活性;钙浸种,低温锻炼,低温锻炼结合钙浸种处理均提高了质膜Ｆｅ（ＣＮ）＾３－６还原活性、尤其是削减了冷胁迫降低质膜Ｆｅ（ＣＮ）＾３－６还原活性的作用。     

水分胁迫下钙螯合剂与CPZ抑制剂对棉花根和下胚轴两种ATPase的效应(简报)

水分胁迫下棉花根和下胚轴质膜（PM）H－ATPase和Ca2－ATPase活力、表观Km值以及Vmax降低。－0．3MPa和-1．1MPa胁迫下质膜AT－Pase活力随时间延长分别呈“V”字形变化和下降趋势。钙螯合剂（EGTA）、CaM抑制剂（CPZ）对棉花根和下胚轴质膜ATPase活性有明显的抑制效应,抑制程度为-1．1MPa大于-0．3MPa大于对照。     

跨膜Ca~(2+)梯差对大豆下胚轴质膜H~+-ATPase活力的影响

采用两相法得到高纯度封闭的大豆下胚轴质膜微囊,研究了跨膜Ｃａ２＋梯差对质膜Ｈ＋－ＡＴＰａｓｅ质子转运和ＡＴＰ水解活力的影响。结果表明,在１０００：０．１,１０００：０．５,１０００：１及１０００：１０（μｍｏｌ／Ｌ：μｍｏｌ／Ｌ）几种梯差下,随着跨膜钙梯差的减小,质膜Ｈ＋－ＡＴＰａｓｅ质子转运活力逐步降低。然而,上述几种梯差对Ｈ＋－ＡＴＰａｓｅ水解活力的影响却很小。进一步研究发现,１０００：０．１及１０００：１（μｍｏｌ／Ｌ：μｍｏｌ／Ｌ）两种梯差对Ｋｍ值没有影响,但Ｋ＋对Ｈ＋－ＡＴＰａｓｅ的激活作用在两种梯差下存在显著差别。ＭＣ５４０荧光、ＤＰＨ荧光偏振结果表明,跨膜钙梯差影响着膜脂的聚集状态和流动性。本文对跨膜Ｃａ２＋梯差对于大豆下胚轴质膜Ｈ＋－ＡＴＰａｓｅ水解与质子转运活力影响的可能机制进行了讨论。     

磷脂模型膜的时间分辨纳秒荧光光谱的研究

叙述了用时间分辨率纳秒荧光技术研究大豆磷脂或合成磷脂模型膜（脂质体）的物理状态变化与荧光寿命以及时间分辨各向异性测量参数变化相关性的实验结果。用荧光探剂ＭＣ５４０标记模型膜的结果表明,荧光寿命（τ）的变化与脂质／探剂比例、磷脂组成、磷脂的极性头部、脂肪酸酰链长度等有关。用ＤＰＨ（１,６－ｄｉｐｈｅｎｙｌ－１,３,５－ｈｅｘａｔｒｉｅｎｅ）标记磷脂模型膜的时间分辨各向异性分析结果表明,序参数（Ｓ）、     

不同生境下木麻黄脯氨酸含量和质膜ATPase活性的研究

采集生长于恶劣环境和中生环境的普通木麻黄(Casuarina)小枝,超速离心提取粗质膜制剂后,用两相系统法纯化得到质膜微囊,研究不同生境下木麻黄的质膜ATPase活性,并测定木麻黄小枝的游离脯氨酸含量。实验结果表明:同一生境中的木麻黄ATPase活性相对一致,而同一树种木麻黄不同生境下质膜微囊H~+-ATPase、Ca~(2+)-ATPase、K~+-ATPase活性有显著差异,表现出以渗透胁迫为主的恶劣环境下的木麻黄质膜微囊ATPase活性和木麻黄细胞内游离脯氨酸明显高于中生环境下生长的木麻黄。说明普通木麻黄在干旱和盐胁迫下能调整生理生化过程来提高其质膜ATPase活性和增加细胞内脯氨酸含量提高渗透调节能力以保证其在恶劣环境的正常生长。     

不同生境下木麻黄脯氨酸含量和质膜ATPase活性的研究

采集生长于恶劣环境和中生环境的普通木麻黄(Casuarina)小枝,超速离心提取粗质膜制剂后,用两相系统法纯化得到质膜微囊,研究不同生境下木麻黄的质膜ATPase活性,并测定木麻黄小枝的游离脯氨酸含量。实验结果表明：同一生境中的木麻黄ATPase活性相对一致,而同一树种木麻黄不同生境下质膜微囊H^ -ATPase、Ca^2 ATPase、K^ -ATPase活性有显著差异,表现出以渗透胁迫为主的恶劣环境下的木麻黄质膜微囊ATPase活性和木麻黄细胞内游离脯氨酸明显高于中生环境下生长的木麻黄。说明普通木麻黄在干旱和盐胁迫下能调整生理生化过程来提高其质膜ATPase活性和增加细胞内脯氨酸含量提高渗透调节能力以保证其在恶劣环境的正常生长。     

Osmotic perturbations induce differential movements in the core and periphery of proteins, membranes and micelles

Polymeric structures, namely, micelles, membranes and globular proteins share the property of two distinct regions: a hydrophobic core and a hydrophilic exterior. The dynamics of these regions of the polymeric structures were probed using selective fluorophores 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-anilinonaphthalene-8-sulfonate (ANS), respectively. Perturbation of the polymers by external osmotic pressure, ionic strength and temperature was monitored in the two regions using steady state measurements of fluorescence intensity and anisotropy. While the fluorescence lifetime of DPH and ANS did not change significantly, parallel change in steady state anisotropy values and the rotational correlation time indicated mobility in the probe/probe-domain. Osmotic perturbation of the polymers in electrolyte media led to decreased DPH mobility. Enhanced ellipticity at 222 nm in bovine serum albumin was observed in 1.5 M NaCl and sucrose media. ANS exhibited a decreased anisotropy with progressive dehydration in proteins in NaCl media, in dimyristoylphosphatidylcholine (DMPC) vesicles in sucrose media, and in neutral laurylmaltoside micelles in both NaCl and sucrose media. Thus, ANS showed responses opposite to that of DPH in these systems. A comparison with several domain selective probes indicated that DPH reported findings common to depth probes while ANS reported data common to interfacial probes used for voltage monitoring.     

Differential adaptation of membranes of two osmotolerant fungi,Aspergillus chevalieri and Penicillium expansum to high sucrose concentrations

Aspergillus chevalieri and Penicillium expansum were able to tolerate sucrose concentrations in the growth media up to 80% (w/v). At 50% sucrose the growth rate is approximately 1.4 and 1.2 times, respectively, higher than in the control. While at 80% sucrose it drops to 35% and 45% of the control level for both fungi. Lipids and proteins in plasma membranes increased with increasing sucrose concentrations in the growth medium. Phospholipid content in membranes of both organisms being also increased, phosphatidyl glycerol was the major detected phospholipid and represented the highest increase. The fatty acid composition of fraction enriched plasma membrane of both fungi changed when they were grown in high sucrose concentrations. Some fatty acids which had not been detected in control cultures were present and the proportions of other fatty acids changed. At 50% sucrose the unsaturation index of membranes decreased by 20-25% in both fungi, indicating that the plasma membrane is less fluid at this concentration. At 80% sucrose a similar trend was observed for P. expansum but for A. chevalieri the unsaturation index was little changed compared with the control. The fluorescence polarization values of 1,6-diphenyl 1,3,5-hexatriene (DPH) in membranes of both fungi grown at 80% sucrose increased, indicating a decrease in membrane fluidity. At 50% sucrose the increase in saturation of membrane fatty acids would tend to reduce membrane fluidity but in A. chevalieri at 80% sucrose fatty acids did not become more saturated. In this case the marked increase in sterols at this sucrose concentration may be responsible for low membrane fluidity.     

Large solutes induce structural perturbations in proteins and membranes.

Structural perturbations in biopolymers with hydrophobic interiors i.e. specific proteins and dimyristoylphosphatidylcholine (DMPC) vesicles were investigated as a function of solute concentrations in the medium. 1,6-diphenyl-1,3,5-hexatriene (DPH) was used as fluorescent probe. Response of DPH was comparable to that of intrinsic tryptophan in BSA in terms of steady state and time resolved fluorescence. The solutes induced a decrease in steady state anisotropy as well as rotational correlation time (computed from lifetime measurements) for DPH in both proteins and membranes. Enhanced access of the quencher potassium iodide to tryptophan in bovine serum albumin (BSA) and ovalbumin, and enhanced terbium leakage in DMPC vesicles induced by various solutes concomitant with decreased anisotropy/correlation time were consistent with structural perturbations of the nature of defects or voids in these polymers.     

Lipid-phase structure in epithelial cell membranes: comparison of renal brush border and basolateral membranes

The lipid-phase structures of brush border membrane vesicles (BBMV) and basolateral membrane vesicles (BLMV) isolated from rabbit renal cortex were compared by steady-state and phase-modulation measurements of diphenylhexatriene (DPH) and trans- and cis-parinaric acid (tPnA and cPnA) fluorescence. A temperature-scanning system was used which gave reproducible temperature profiles of steady-state and dynamic fluorescence parameters with a resolution of 0.1 degrees C. Steady-state anisotropy of DPH showed a triphasic dependence on temperature with slope discontinuities at 22 +/- 4 and 47 +/- 3 degrees C (BBMV) and at 23 +/- 2 and 48 +/- 1 degrees C (BLMV). At all temperatures, DPH anisotropy in BBMV was greater than that in BLMV. Ground-state heterogeneity analysis of tPnA and cPnA fluorescence lifetime data demonstrated the presence of long (greater than 12 ns) and short (less than 5 ns) lifetime components, interpreted in terms of solid-phase and fluid-phase lipid domains. The fraction of solid-phase phospholipid decreased from 0.9 to 0.1 for BBMV and from 0.7 to 0.3 in BLMV with increasing temperature (10-50 degrees C). In both membranes, tryptophan-PnA fluorescence energy-transfer measurements showed that membrane proteins were surrounded by a fluidlike phospholipid phase. These results demonstrate the inadequacy of steady-state DPH anisotropy data in defining the structural characteristics of complex biological membranes. Results obtained with the phase-sensitive parinaric acid probes demonstrate major differences in the phase structure of the two opposing cell membranes in both the bulk lipid and the lipid microenvironment around membrane proteins.     

A fluorescence spectroscopy study on the interactions of the TAT-PTD peptide with model lipid membranes

Protein-transduction domains (PTDs) have been shown to translocate into and through the living cells in a rapid manner by an as yet unknown mechanism. Regardless of the mechanism of translocation, the first necessary step must be binding of the PTD peptide to the surface of the lipid membrane. We used fluorescence spectroscopy to study the interaction between PTD of the HIV-1 Tat protein (TAT-PTD; residues 47-60 of Tat, fluorescently labeled with tryptophan) and the lipid bilayer labeled with various fluorescence membrane probes. The TAT-PTD tryptophan exhibited a decrease in fluorescence intensity and an increase in anisotropy upon interaction with lipid bilayers. The fluorescence changes were linearly proportional to the density of negative charge in the membrane. Kinetic analysis of the interaction showed two apparent dissociation constants. The value of one dissociation constant (Kd1 = 2.6 +/- 0.6 microM), which accounted for 24% of the interaction, was found to be independent of the negative charge density, suggesting its nonelectrostatic nature. The value of the second dissociation constant (Kd2), which accounted for 76% of the interaction, decreased linearly from 610 +/- 150 to 130 +/- 30 microM with an increase in negative charge density from 0 to 25 mol %, suggesting this interaction is electrostatic in nature. Even though the binding was predominantly electrostatic, it could not be reversed by high salt, indicating the presence of a second, irreversible, step in the interaction with lipid. When TAT-PTD was bound to lipid vesicles labeled with 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), fluorescence resonance energy transfer between the tryptophan and the probe occurred at a distance of 3.4 nm. No change in fluorescence anisotropy of either TMA-DPH or DPH was observed upon the interaction with TAT-PTD, indicating no significant disruption or perturbation of the lipid bilayer by the peptide. TAT-PTD did not cause dissipation of membrane potential (165 mV, negative inside). Inclusion of 3% pyrene-labeled phosphatidylglycerol (pyrene-PG) in the membrane revealed that TAT-PTD preferentially bound to the membrane in the liquid state. We conclude that membrane fluidity is an important physicochemical parameter, which may regulate binding of TAT-PTD to the membrane.     

Ethinylestradiol administration selectively alters liver sinusoidal membrane lipid fluidity and protein composition

Administration of high-dose ethinylestradiol to rats decreases bile flow, Na,K-ATPase specific activity, and liver plasma membrane fluidity. By use of highly purified sinusoidal and bile canalicular membrane fractions, the effect of ethinylestradiol administration on the protein and lipid composition and fluidity of plasma membrane fractions was examined. In sinusoidal fractions, ethinylestradiol (EE) administration decreased Na,K-ATPase activity (32%) and increased activities of alkaline phosphatase (254%), Mg2+-ATPase (155%), and a 160-kDa polypeptide (10-fold). Steady-state and dynamic fluorescence polarization was used to study membrane lipid structure. Steady-state polarization of diphenylhexatriene (DPH) was significantly higher in canalicular compared to sinusoidal membrane fractions. Ethinylestradiol (5 mg/kg per day for 5 days) selectively increased sinusoidal polarization values. Similar changes were demonstrated with the probes 2- and 12-anthroyloxystearate. Time-resolved fluorescence polarization measurements indicated that EE administration for 5 days did not change DPH lifetime but increased the order component (r infinity) and decreased the rotation rate (R). However, 1 and 3 days after EE administration and with low doses (10-100 micrograms/kg per day for 5 days) the Na,K-ATPase, bile flow, and order component were altered, but the rotation rate was unchanged. Vesicles prepared from total sinusoidal membrane lipids of EE-treated rats, as well as phospholipid vesicles, demonstrated increased DPH polarization, as did intact plasma membrane fractions. Liver plasma membrane fractions showed no change in free cholesterol or cholesterol/phospholipid molar ratio, while esterified cholesterol content was increased with high-dose but not low-dose ethinylestradiol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of proteins on fluorophore lifetime heterogeneity in lipid bilayers.

The effect of three different membrane proteins on the fluorescence lifetime heterogeneity of 1,6-diphenyl-1,3,5-hexatriene (DPH) in phospholipid vesicle systems was investigated. For large unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) at 37 degrees C, the fluorescence decay was essentially monoexponential (8.6 and 8.2 ns, respectively) except for a minor component typical of DPH. For gramicidin D reconstituted into DMPC vesicles at a protein/lipid molar ratio of 1/7, the most appropriate analysis of the data was found to be in the form of a bimodal Lorentzian distribution. Centers of the major lifetime components were almost identical with those recovered for vesicles without proteins, while broad distributional widths of some 4.0 ns were recovered. Variation of the protein/lipid molar ratio in sonicated POPC vesicles revealed an abrupt increase in distributional width at ratios approximating 1/15-1/20, which leveled off at about 2.5 ns. For bacteriorhodopsin in DMPC vesicles and cytochrome b5 in POPC, the most appropriate analysis of the data was again found to be in the form of a bimodal Lorentzian also with broad distributional widths in the major component. Lifetime centers were decreased for these proteins due to fluorescence energy transfer to the retinal of the bacteriorhodopsin and heme of the cytochrome b5. Fluorescence energy transfer is distance dependent, and since a range of donor-acceptor distances would be expected in a membrane, lifetime distributions should therefore be recovered independently of other effects for proteins possessing acceptor chromophores.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of protein hydration on receptor conformation: decreased levels of bound water promote metarhodopsin II formation.

Neutral solutes were used to investigate the effects of osmotic stress both on the ability of rhodopsin to undergo its activating conformation change and on acyl chain packing in the rod outer segment (ROS) disk membrane. The equilibrium concentration of metarhodopsin II (MII), the conformation of photoactivated rhodopsin, which binds and activates transducin, was increased by glycerol, sucrose, and stachyose in a manner which was linear with osmolality. Analysis of this shift in equilibrium in terms of the dependence of ln(Keq) on osmolality revealed that 20 +/- 1 water molecules are released during the MI-to-MII transition at 20 degrees C, and at 35 degrees C 13 +/- 1 waters are released. At 35 degrees C the average time constant for MII formation was increased from 1.20 +/- 0.09 ms to 1.63 +/- 0.09 ms by addition of 1 osmolal sucrose or glycerol. The effect of the neutral solutes on acyl chain packing in the ROS disk membrane was assessed via measurements of the fluorescence lifetime and anisotropy decay of 1,6-diphenyl-1,3,5-hexatriene (DPH). Analysis of the anisotropy decay of DPH in terms of the rotational diffusion model showed that the angular width of the equilibrium orientational distribution of DPH about the membrane normal was progressively narrowed by increased osmolality. The parameter fv, which is proportional to the overlap between the DPH orientational probability distribution and a random orientational distribution, was reduced by the osmolytes in a manner which was linear with osmolality. This study highlights the potentially opposing interplay between the effect of membrane surface hydration on both the lipid bilayer and integral membrane protein structure. Our results further demonstrate that the binding and release of water molecules play an important role in modulating functional conformational changes for integral membrane proteins, as well as for soluble globular proteins.