没食子酸丙酯和没食子酸异丁酯对人红细胞膜结构与功能的影响

本文应用荧光探剂ANS(1—苯胺—8萘磺酸)、NPN(N—苯基—1—萘胺)和DPH(1.6—二苯基—1.3.5—已三烯)观察没食子酸丙醋和没食子酸异丁酯对人红细胞膜流动性和相变温度以及Na~ -K~ ATP酶活性的影响.实验结果指出该两种化合物均能:(1)降低与膜结合的荧光探剂强度但不改变探剂在水相与膜相的分配比例:(2)降低膜脂的相变温度,增加膜的流动性;(3)抑制红细胞膜Na~ -K~ ATP酶活性;(4)标记红细胞膜的DPH偏振度随化合物浓度的增加而降低,膜的流动性增加.在给定的浓度范围内,两种化合物的效应表现为明显的量效关系与构效关系.从上述结果推测该两种化合物可能是通过改变膜脂结构、膜蛋白的脂类环境而调节膜的功能,成为其治疗疾病的机理之一.     

丙二醛对脂质体和红细胞膜作用的激光Raman光谱研究

本文考察丙二醛处理前后的DPPC脂质体、DPPE脂质体和红细胞膜的Raman光谱的变化.我们发现丙二醛使DPPC脂质体相变温度上升10℃且流动性增加,以丙二醛处理DPPE脂质体后其预相变消失,相变温度上升,丙二醛可交联DPPE上的氨基形成Schiff碱.人血红细胞膜与丙二醛保温后膜磷脂trans构象减少,有序性下降;膜蛋白二级结构发生明显变化,α螺旋构象减少.     

血红蛋白对人红细胞膜流动性的影响

本文报道了pH7.5时血红蛋白和红细胞膜的结合效应.在10—45℃温度范围内观察到血红蛋白对膜脂质流动性的限制作用.看来这种限制作用不是脂质过氧化所致,而是血红蛋白和红细胞膜直接作用的结果.对流动性大的膜,血红蛋白的效应也随之增大.高铁血红蛋白及红细胞膜去胆固醇皆能修饰血红蛋白和膜的相互作用.     

冷冻前后人红细胞膜蛋白聚丙烯酰胺凝胶电泳分析

冷冻保存红细胞在血液保存工作中是一项新技术。以甘油为冷冻保护剂,加入新鲜红细胞中,在-80℃冷冻保存,可达到长期保存的目的,为了检验在血液保存过程中红细胞的质量,国外也曾对血库内保存的枸椽酸、枸椽酸钠、葡萄糖(简称ACD)全血中红细胞膜上蛋白质组分的改变情况进行研究。例如1969年布莱纳(Brener)等曾用淀粉凝胶电泳分析新鲜红细胞膜蛋白与保存一定时间后红细胞膜蛋白;1971年穆尔(Moore)对常规方法保存的全血,红细胞膜上蛋白质改变情况进行分析。本文使用硫酸十二醋钠盐(SDS)聚丙烯酰胺凝膜电泳分析冷冻前后红细胞膜蛋白,通过了解红细胞膜改变情况,进一步证明冷冻保存后红细胞结构完整,并肯定了其保存价值。     

不同温度及时间对液状保存血液质量的影响

目的：研究不同温度对血液液状保存时保存损害机制的影响,并探讨相应的防范措施。方法：取10名健康献血员静脉血,置于CP2D－A保存液中,于0℃和4℃环境条件下,分别于设定的时间（第1周末、第3周和第6周末）内取样检测GSH－Px、LPO、TSH、红细胞膜收缩蛋白、膜脂流动性等指标。结果：固定温度条件下随时间的延长血液过氧化反应增强,保存损害作用增加;同一时期内保存损害作用随温度的降低而减轻,以4℃组血液老化明显。结论：血液过氧化反应随保存期的延长而增加,随保存温度的降低而改善,0℃组保存血液的质量优于4℃组。     

高胆固醇血症血液流变性的研究

对实验性高胆固醇血症家兔血液流变性、红细胞流变性、红细胞膜脂分析以及与血清胆固醇浓度之间关系的研究指出随血清胆固醇浓度升高,全血表观粘度和血浆粘度升高,红细胞(?)积和(?)降低.进一步分析表明,随血清胆固醇浓度升高,红细胞膜中胆固醇Ch含量增加,引起膜胆固醇和磷脂(P1)的克分子比(Ch,P1)升高,膜的荧光偏振度(P)增大,表示红细胞流动性降低,而对照组各项指标在实验过程中保持稳定.     

用马来酰亚胺自旋标记研究库存血红细胞膜蛋白质构象

用两种马来酰亚胺自旋标记物—马来酰亚胺Ⅰ和马来酰亚胺Ⅴ研究了红细胞膜蛋白质构象及巯基结合位点性质在ACD-B方库存血保存期间的动态变化。结果发现,在35天的血液保存期间,马来酰亚胺Ⅰ所标记红细胞膜的S/w值很快下降到一低水平,而马来酰亚胺Ⅴ所标记红细胞膜的旋转相关时间则呈现迅速下降后缓慢升高的双相性变化。作者结合膜蛋白构象及其周围微观环境进行了讨论。     

用马来酰亚胺自旋标记研究库存血红细胞膜蛋白质构象

用两种马来酰亚胺自旋标记物—马来酰亚胺Ⅰ和马来酰亚胺Ⅴ研究了红细胞膜蛋白质构象及巯基结合位点性质在ACD-B方库存血保存期间的动态变化。结果发现,在35天的血液保存期间,马来酰亚胺Ⅰ所标记红细胞膜的S/w值很快下降到一低水平,而马来酰亚胺Ⅴ所标记红细胞膜的旋转相关时间则呈现迅速下降后缓慢升高的双相性变化。作者结合膜蛋白构象及其周围微观环境进行了讨论。     

萎缩性肌强直(MYD)患者红细胞膜生物物理特性的研究

作者用荧光方法测定了MYD患者红细胞和正常人红细胞在不同细胞外液K~+浓度下的膜电位,结果MYD患者红细胞膜电位比正常人高;荧光偏振法研究结果表明,MYD患者红细胞膜流动性比正常人高;细胞电泳方法发现MYD患者红细胞电泳迁移率亦较正常人高.这些生物物理特性的改变是相互联系的、反映了MYD患者红细胞膜结构的变化并将直接影响膜的代谢和功能.     

吗啡对大鼠红细胞膜生物物理特性的影响

为研究吗啡对生物体血液系统的影响,以大鼠为研究对象,分别培养了三周成瘾的吗啡依赖模型、体外吗啡作用和吗啡急性注射的模型,研究了吗啡对大鼠红细胞膜的生物物理特性的改变。作者采用新型激光衍射法测量了上述血样的弹性模量Ｅ和膜微粘度（μｍ）,同时通过ＤＰＨ标记的荧光偏振法测定了这些红细胞膜的流动性,还采用傅立叶变换的红外技术（ＦＴ－ＩＲ）测量了红细胞膜蛋白构象的动态变化,并对上述红细胞膜结构改变所引起的微观流变特性的变化进行了初步探讨。结果表明,大鼠对吗啡形成依赖会导致其红细胞膜流动性的下降,弹性剪切模量的升高和膜蛋白质二级结构的变化。而吗啡的体外作用和体内急性作用对大鼠红细胞膜特性的影响与吗啡依赖的作用相类似。由此我们推测吗啡对大鼠红细胞膜特性的影响可能不需通过受体,而是直接发生作用的。     

Heterogeneity in the fluidity of intact erythrocyte membrane and its homogenization upon hemolysis.

Intact erythrocytes were spin-labeled with various classes of phospholipid label. The ESR spectrum for phosphatidylcholine spin label was distinctly different from those for phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidic acid spin labels. The overall splitting for the former (52.5 G) was markedly larger than those for the others (approx. 47 G), suggesting a more rigid phosphatidylcholine bilayer phase and more fluid phosphatidylethanolamine and phosphatidylserine phases in the erythrocyte membrane. Evidence for asymmetric distribution of phospholipids in the membrane was obtained. Spin-labeled phosphatidylcholine incorporated into erythrocytes was reduced immediately by cystein and Fe3+, while the reduction of spin-labeled phosphatidylserine was very slow. The present results therefore suggest asymmetric fluidity in erythrocyte membrane; a more rigid outer layer and a more fluid inner layer. The heterogeneity in the lipid structure was also manifested in the temperature dependence of the fluidity. The overall splitting for phosphatidylcholine spin label showed two inflection points at 18 and 33 degrees C, while that for phosphatidylserine spin label had only one transition at 30 degrees C. When the spin-labeled erythrocytes were hemolyzed, the marked difference in the ESR spectra disappeared, indicating homogenization of the heterogenous fluidity. Mg2+ or Mg2+ + ATP prevented the hemolysis-induced spectral changed. Ca2+ did not prevent the homogenization and acted antagonistically to Mg2+. The heterogeneity preservation by Mg2+ was nullified by trypsin, pronase or N-ethylmaleimide added inside the cell. Some inner proteins may therefore be involved in maintaining the heterogeneous structure. The protecting action of Mg2+ was dependent on hemolysis temperature, starting to decrease at 18 degrees C and vanishing at 40 degrees C. The present study suggests that the heterogeneity in the fluidity of intact erythrocyte membranes arises from interactions between lipids and proteins in the membrane and also from interactions between the membrane constituents and the inner proteins. Concentration of cholesterol in the outer layer may also partly contribute to the heterogeneity.     

A 2H NMR study on alteration of the membrane organization of mouse B16 melanoma cells treated with 12-O-tetradecanoylphorbol-13-acetate

In order to monitor the membrane fluidity of cells without perturbation by an introduced probe, we developed a method for large-scale preparation of 2H-labeled melanoma cells for a 2H NMR study by incubating melanoma cells with [18,18,18-2H3]stearic acid/phosphatidylcholine liposomes for 2 h at 37 degrees C. It turned out that this treatment did not significantly change the cell viability, lipid metabolism or membrane fluidity. The 2H from C-18 of stearic acid is dominantly located at the original position of the fatty acid in the 2H-labeled membrane vesicles, as studied by a tracer experiment with [1-14C]stearic acid. We found that three to four 2H-labeled species were present at 19 degrees C in 2H NMR spectra of the 2H-labeled membrane vesicles prepared from B16 melanoma cells. The extent of peak-splittings due to 2H-quadrupole interaction decreased as the temperature rose, and a definite point of phase transition was not observed. At elevated temperature, 2H-labeled lipids undergo fast exchange between the bilayer and an isotropic phase such as oil phase of triolein or inverted micelles in lipid polymorphs. We further analyzed the change of membrane organization in mouse B16 melanoma cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA), which strongly inhibited melanogenesis. The magnitude of the quadrupole splitting at 19 degrees C in membranes from TPA-treated cells was significantly less (40%) than in the untreated control. This is mainly explained by decreased molecular ordering (fluidity) due to the increased amount of unsaturated fatty acids in the membranes of TPA-treated cells.     

Sphingomyelin phase transition in the sheep erythrocyte membrane

The dependence of membrane dynamics on the mole ratio of lecithin to sphingomyelin (L/S) was examined by the fluorescence depolarization of the fluidity probe DPH in membranes isolated from sheep and human erythrocytes. In these membranes L/S is the main variable of lipid composition (0.02 and 1.7, respectively). The sheep erythrocyte membrane, which is rich in sphingomyelin, displays a higher lipid microviscosity than the human erythrocyte membrane in addition to a broad gel/liquid-crystal phase transition in the range of 26–35°C. Single-walled lipid vesicles of high sphingomyelin content, when studied by the same technique, exhibited dynamic characteristics similar to those found in the sheep erythrocyte membrane. Both the apparent microviscosity and the transition temperature decreased with increasing the L/S. Membrane proteins of human and sheep erythrocytes were fluorescently labeled with the sulfhydryl reagent N-dansylaziridine and the emission spectrum was recorded as a function of temperature. In the human erythrocyte membranes a gradual increase in the ratio of emission maxima at 520 and 490 nm was observed between 6 and 40°C. At this temperature range the ratio of the above emission maxima in sheep erythrocyte membranes displayed a break between 20 and 28°C, which partially overlapped the phase transition observed for the lipid core. The effect of the lipid phase transition on membrane proteins for the lipid core. The effect of the lipid phase transition on membrane proteins was further assessed by comparing the activity of the membrane bound phospholipase A2 in the intact and detergent-solubilized sheep erythrocyte membranes. Below 31°C the lipids suppress the enzyme activity by about 90%, whereas above this temperature this suppression is progressively abolished.     

Characterization of membrane lipids of a general fatty acid auxotrophic bacterium by electron spin resonance spectroscopy and differential scanning calorimetry

Lipids in the plasma membrane of the general fatty acid auxotroph Butyrivibrio S2 pack as a bilayer that is characterized by a high order and high motional anisotropy and a low membrane fluidity compared to mammalian plasma membranes. Lipid packing as determined by the electron spin resonance (ESR) order parameter and membrane fluidity as measured by ESR correlation times are, however, comparable to those of other bacterial membranes. Membranes of the organism grown with saturated fatty acids of well-defined hydrocarbon chain length undergo a broad reversible endothermic phase transition, the peak temperature of which is well below the growth temperature; the end-point temperature of this thermal transition approximately coincides with the minimum temperature supporting significant growth of the organism. The lipid phase transition is also reflected in the temperature dependence of various ESR parameters, whereby the transition temperature thus derived is higher than the peak temperature of the endothermic transition but still lower than the growth temperature. ESR and calorimetry evidence taken together suggest that the endothermic transition is a gel to liquid-crystal transition and that, at the growth temperature, the plasma membrane of Butyrivibrio S2 is in the liquid-crystalline state. Similar values were measured for the order parameter of cell membranes of Butyrivibrio S2 regardless of whether the organism was grown on myristic, palmitic, or stearic acid. Butyrivibrio S2 has a mechanism enabling it to maintain membrane packing and fluidity at a fairly constant level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ontogeny of proximal colon basolateral membrane lipid composition and fluidity in the rabbit.

Basolateral membranes from rabbit proximal colon were prepared from isolated colonocytes throughout postnatal maturation, using a modification of published techniques. In suckling (14-20 day) and post-weaning/mature (35-49 day) animals, membranes were purified approx. 10-fold, based upon the enrichment of ouabain-sensitive, sodium-potassium dependent adenosine triphosphatase activity. Membrane lipid analyses demonstrated age-dependent increases in total cholesterol and the cholesterol/phospholipid molar ratio, as well as decreases in phosphatidylethanolamine content and the fatty acid unsaturation index. Fluidity of basolateral membranes and membrane liposomes, determined from fluorescence anisotropy measurements using the lipid probes 1,6-diphenyl-1,3,5-hexatriene and DL-12-(9-anthroyl)stearic acid, demonstrated significant, ontogenic decreases in fluidity; and, additional studies showed that fluidity changes occurred early in the weaning period (by day 24 postnatally). Arrhenius plots of liposome anisotropies suggested a bilayer lipid thermotropic transition temperature of 22 degrees C in sucklings 26 degrees C in mature rabbits. These findings demonstrate that ontogeny of colonic basolateral membranes is associated with significant modulations in lipid composition and fluidity.     

Depth dependence of the perturbing effect of placing a bulky group (oxazolidine ring spin labels) in the membrane on the membrane phase transition

Electron paramagnetic resonance (EPR) and differential scanning calorimetry (I)SO have been used to study the effect on the phase transition of dimyristoylphosphatidylcholine membranes of incorporating various stearic acid spin labels (SASL's) that contain the bulky oxazolidine ring at various positions along the stearyl chain. SASL's lowered the phase transition temperature and decreased the size of the cooperative unit, with the effects stronger in the order of 9-> 12-> 5-> 16-SASL > stearic acid (no label). Incorporation of stearic acid without the spin label slightly increases the phase transition temperature. Incorporation of 9-SASL (3 mol% of lipid) decreased the transition temperature by 1.8C and the cooperative unit to 115 of that without the spin label, while the effect of 16-SASL was slight. The effect on transition enthalpy was small. It is concluded that the perturbing effect of placing a bulky group on the alkyl chain on phase transition is through inducing packing defects in the gel-phase.     

In vitro effects of ozone on human erythrocyte membranes: an EPR study

The effects of ozone at different concentrations (10, 30, 45 g/m3) on fluidity and thermotropic properties of erythrocyte membranes were investigated by EPR using two spin probes: 5-doxylstearic acid (5-DSA) and 16-doxylstearic acid (16-DSA). The effect of ozone on the erythrocyte membrane fluidity was a dose-dependent process. The ozone at concentration of 10 g/m3 caused rigidization of the membrane while at concentration of 45 g/m3 increased fluidity both on the surface and in the deeper hydrocarbon region of the membrane. Temperature transitions close to the polar heads region (monitored by 5-DSA) were not sensitive to an increase in ozone concentration. In the case of 16-DSA, low temperature thermotropic transition (around 20 degrees C) gradually decreased with the increase of ozone concentration. High temperature transition (around 40 degrees C) significantly differed at the ozone concentration of 10 g/m3 and 45 g/m3, being higher and lower, respectively, as compared to untreated cells. For the ozone concentration of 45 g/m3 the disappearance of the low temperature break and the appearance of two breaks at 37 degrees C and 16 degrees C were observed.     

Protein-dependent lipid lateral phase separation as a mechanism of human erythrocyte ghost resealing

The hypothesis of a correlation between a 10°–20°C lipid phase transition and the resealing process of human erythrocyte membrane has been investigated. The conditions required to reseal human erythrocyte ghosts have been studied by measuring the amount of fluorescein-labeled dextran (FD) that is trapped into the membrane. Temperature per se was sufficient to induce membrane resealing: (1) at 5 mM sodium phosphate, pH 7.8 (5P8), resealing began at 12°C; (2) at salt concentrations above 8 mM sodium phosphate, it occurred at lower temperature; and (3) in isotonic saline was detected just above 5°C. The removal of peripheral membrane proteins from unsealed membranes by chymotrypsin at 0°C in 5P8 was followed by membrane resealing. This seems to imply that the presence of proteins is necessary to maintain the membrane unsealed. Protein-induced lateral phase separation of lipids may be a reasonable mechanism for the observed phenomena. In fact, the permeability of phosphatidylserine-phosphatidylcholine mixed liposomes to FD is modified by lipid lateral phase separation induced by pH or poly-L-lysine. Electron spin resonance studies of membrane fluidity by a spin labeled stearic acid showed a fluidity break around 11°C, which may be due to a gel–liquid phase transition. Fluidity changes are abolished by chymotrypsin treatment. It is suggested that a lateral phase separation is responsible for the permeability of open ghosts to FD. Accordingly, disruption of phase separation apparently produces membrane reconstitution. In this respect peripheral proteins and particularly the spectrin-actin network, may play a major role in membrane resealing.     

Heterogeneity in the fluidity of intact erythrocyte membrane and its homogenization upon hemolysis

Intact erythrocytes were spin-labeled with various classes of phospholipid label. The ESR spectrum for phosphatidylcholine spin label was distinctly different from those for phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidic acid spin labels. The overall splitting for the former (52.5 G) was markedly larger than those for the others (approx. 47 G), suggesting a more rigid phosphatidylcholine bilayer phase and more fluid phosphatidylethanolamine and phosphatidylserine phases in the erythrocyte membrane. Evidence for asymmetric distribution of phospholipids in the membrane was obtained. Spin-labeled phosphatidylcholine incorporated into erythrocytes was reduced immediately by cystein and Fe³⁺, while the reduction of spin-labeled phosphatidylserine was very slow. The present results therefore suggest asymmetric fluidity in erythrocyte membrane; a more rigid outer layer and a more fluid inner layer. The heterogeneity in the lipid structure was also manifested in the temperature dependence of the fluidity. The overall splitting for phosphatidylcholine spin label showed two inflection points at 18 and 33 °C, while that for phosphatidylserine spin label had only one transition at 30 °C.When the spin-labeled erythrocytes were hemolyzed, the marked difference in the ESR spectra disappeared, indicating homogenization of the heterogeneous fluidity. Mg²⁺ or $\text{Mg}{}^{\mathrm{2+}}\text{+}\text{ATP}$ prevented the hemolysis-induced spectral changes. Ca²⁺ did not prevent the homogenization and acted antagonistically to Mg²⁺. The heterogeneity preservation by Mg²⁺ was nullified by trypsin, pronase or $\text{N-}\text{ethylmaleimide}$ added inside the cell. Some inner proteins may therefore be involved in maintaining the heterogeneous structure. The protecting action of Mg²⁺ was dependent on hemolysis temperature, starting to decrease at 18 °C and vanishing at 40 °C. The present study suggests that the heterogeneity in the fluidity of intact erythrocyte membranes arises from interactions between lipids and proteins in the membrane and also from interactions between the membrane constituents and the inner proteins. Concentration of cholesterol in the outer layer may also partly contribute to the heterogeneity.     

Alterations of human erythrocyte membrane fluidity by oxygen-derived free radicals and calcium

Two possible reasons for the structural alterations of cell membranes caused by free radicals are lipid peroxidation and an increase in the intracellular calcium ion concentration. To characterize the alterations in membrane molecular dynamics caused by oxygen-derived free radicals and calcium, human erythrocytes were spin-labeled with 5-doxyl stearic acid, and alterations in membrane fluidity were quantified by electron spin resonance oxidase (0.07 U/mL) decreased membrane fluidity, and the addition of superoxide dismutase and catalase inhibited the effect on membrane fluidity of the hypoxanthine-xanthine oxidase system. Hydrogen peroxide (0.1 and 1 nM) also decreased membrane fluidity and caused alterations to erythrocyte morphology. In addition, a decrease in membrane fluidity was observed in erythrocytes incubated with 2.8 mM CaCl₂. On the other hand, incubation of erythrocytes with calcium-free solution decreased the changes in membrane fluidity caused by hydrogen peroxide.

These results suggest that changes in membrane fluidity are directly due to lipid peroxidation and are indirectly the result of increased intracellular calcium concentration. We support the hypothesis that alterations of the biophysical properties of membranes caused by free radicals play an important role in cell injury, and that the accumulation of calcium amplifies the damge to membranes weakened by free radicals.