用定点自旋标记的方法研究天青蛋白疏水区的结构及其与P53蛋白的相互作用

定点自旋标记技术结合电子顺磁共振(ESR)波谱技术已成为检测蛋白质结构的有力工具.本文使用该方法研究了天青蛋白疏水区的结构特征及其与p53蛋白的相互作用.围绕天青蛋白疏水区构建了6个突变体,并对其进行自旋标记.溶液中标记位点的自旋谱线表明,45及63位自旋探针的运动受到很大限制,而59及65位的标记探针是在一个宽松的环...     

自旋标记法研究不同因素对嗜盐菌紫膜类脂物性的影响

本文用自旋标记方法研究了不同的pH和盐介质对光照前后嗜盐菌(H.halobium)紫膜类脂的影响.在低盐介质中,紫膜类脂序参数S的大小依次为pH2.5>pH7.0>pH12.0:旋转相关时间τ均在10~(-a)sec范围之内.pH12.0的暗适应和光适应悬液的ESR波谱十分相似,其它的ESR波谱均为暗适应>光适应.悬浮于Triton X-100中的紫膜的序参数最小,τ_e值在10~(-9)--10~(-10)sec之间;但其暗适应和光适应的ESR波谱仍有差别.在高盐介质中,紫膜类脂序参数均大于低盐介质,结构最为刚硬.实验表明类脂的序态变化与bR分子的构象改变有密切关系.     

肾性贫血红细胞膜蛋白巯基结合位置性质的ESR研究

用四种不同链长的马来酰亚胺氮氧自由基标记物研究了慢性肾衰竭(CRF)贫血病人红细胞膜蛋白巯基结合位置的性质。从所得的ESR波谱计算了弱、强固定化成分之比(W/S)。结果表明,由M(Ⅰ)、M(Ⅱ)和M(Ⅲ)标记病人红细胞膜所得的W/S值都比标记正常人红细胞膜得到的W/S位高(P<0.05),这说明慢性肾衰竭贫血病人红细胞膜蛋白的巯基结合位置的构象发生了变化;用M(Ⅳ)标记正常人红细胞和病人红细胞所得到的ESR波谱都只含有弱固定化波谱,没有强固定化波谱。得出的这个结果将为探讨CRF贫血的发病机理提供理论证据。     

超氧化物歧化酶的自旋标记研究

本文应用对-SH基特异性结合的自旋标记物对Cu_2Zn_2-SOD进行自旋标记研究,进一步观察了电离辐射对-SH基部位的影响,实验结果:(1)自旋标记后的Cu_2Zn_2-SOD表现出典型的弱固定化的ESR波谱信号特征。标记对Cu_2Zn_2-SOD的UV谱无明显影响。这表明该游离-SH基是位于酶蛋白分子的相对表层而不是包埋在疏水内部。(2)标记保温后立即取样及保温后17小时取样测酶活力,发现与未标记酶的活力相同。这表明该-SH基与酶的催化活性无直接关系。(3)在10—100Krad γ-射线照射下,酶活性及ESR信号幅度均随照射剂量增加而下降,两者之间有一定的相关性。     

Mn~(2+)和Cu~(2+)与脂质体和脂酶体相互作用的ESR研究

用ESR实验研究了Mn~(2 )、Cu~(2 )与DOPC,DPPC,SPL,DOPA,DPPA脂质体及其与H~ -ATP酶复合体重组的脂酶体的相互作用.通过Mn~(2 )—ESR谱线强度以及Cu~(2 )—ESR谱g因子的测量得出,磷脂分子头部不同的化学组成及其脂酰链的不同状态决定了Mn~(2 )、Cu~(2 )与膜脂结合的强弱程度,通过脂质体和脂酶体中自旋标记物5NS—ESR谱的测量进一步得出Mn~(2 )的结合增大了膜脂排列的序参数,而酶复合体的嵌入都导致与膜脂结合的Mn~(2 )比例减小.因而,当Mn~(2 )与脂酶体相互作用时,膜脂的排列最终达到一个平衡状态.在中性磷脂脂酶体的膜与Mn~(2 )之间,这种相互作用不明显.     

用自旋标记研究嗜盐菌紫膜的相变

本文用5′酮基软脂的甲酯的N′氧基4、4-二甲基(口恶)唑衍生物、TEMPO和4-(乙氧基氟代磷酸))-TEMPO′研究了嗜盐菌紫膜的相变.ESR测定表明:在醋酸钠缓冲溶液中(100mM、pH7.0)、30至70℃没有观察到相转变;在81℃附近获得了紫膜类脂的另一相变点,此转变点可能与紫膜类脂及蛋白质晶格的分离有关.紫膜蛋白质的热变性点则在100℃附近.当温度升至110℃时紫膜类脂烃链则趋“熔化”,紫膜蛋白质的热变性也呈不可逆的.文中还就不同的物理因素对紫模相变的影响进行了讨论.     

山莨菪碱对混合磷脂脂质体中酸性磷脂的选择性作用—激光拉曼光谱的研究

本文报道了用激光拉曼光谱研究山莨菪碱与二棕榈酰磷脂酸胆碱(DPPC)/ 二棕榈酰磷脂酸(DPPA)混合磷脂脂质体的相互作用.通过观察药物/磷脂体系相变过程中脂肪酸链结构的变化,发现山莨菪碱对酸性磷脂有明显的倾向性.参照天然神经突触膜上酸性磷脂成分配制的混合磷脂脂质体与药物作用结果表明仅有少量酸性磷脂存在,就使得药物对脂膜整体结构的影响发生了变化.并注意到山莨菪碱与酸性磷脂的这种选择性作用,没有在混合磷脂体系中引起分相.     

用自旋标记研究抗癌药物对中国地鼠肺正常细胞V_(79)和癌变细胞V_(79)-B_1膜流动性的影响

本文用脂肪酸自旋标记Ⅲ(10,3)在ESR波谱仪上研究了作用于不同环节的抗癌药物硫杂脯氨酸、阿糖胞苷、放线菌素D和5-氟-2′脱氧尿嘧啶对中国地鼠肺正常细胞V_(79)和癌变细胞V_(79)-B_1膜流动性的影响。从所得的ESR波谱计算了抗癌药物处理前后细胞膜脂肪酸链的序参数、平均涨落角度、旋转相关时间和微观粘度的变化。发现用抗癌药物处理以后,V_(79)和V_(79)-B_1细胞膜脂肪酸链的序参数增加,平均涨落角度变小,旋转相关时间和微观粘度增大,细胞膜流动性变小。这四种药物对细胞膜的疏水脂肪酸链作用类似,而硫杂脯氨酸对细胞膜表面作用最大,阿糖胞苷,放线菌素D和5-氟-2′脱氧尿嘧啶依次减小。做了序参数随药物处理时间的动力学曲线。发现除放线菌素D外,V_(79)-B_1细胞膜序参数对其他三种药物的反映比V_(79)细胞膜快。     

细菌视紫红质蛋白的赖氨酸和丝氨酸残基在其膜环境中的自旋标记研究

用化学修饰和自旋标记ESR技术研究了bR中丝氨酸和赖氨酸残基的构象,结果表明PH对bR分子构象的影响是很明显的,在酸性条件下带有自旋探针的丝氨酸(Ⅰ-bR)和赖氨酸(Ⅱ-bR)的ESR波谱参数迥然不同,这与丝氨酸和赖氨酸残基位点微环境中的电荷效应有关.顺磁增宽剂能猝灭膜表面的自由基,而剩余的强固定化ESR信号显示出bR分子内部的‘刚性’构象.用2%Triton X—100处理可大大增加bR分子的‘柔性’,其ESR波谱特征与bR分子失去部分α螺旋结构和改变了它的某些运动方式有关.     

细菌视紫红质蛋白的赖氨酸和丝氨酸残基在其膜环境中的自旋标记研究

用化学修饰和自旋标记ESR技术研究了bR中丝氨酸和赖氨酸残基的构象,结果表明PH对bR分子构象的影响是很明显的,在酸性条件下带有自旋探针的丝氨酸(Ⅰ-bR)和赖氨酸(Ⅱ-bR)的ESR波谱参数迥然不同,这与丝氨酸和赖氨酸残基位点微环境中的电荷效应有关.顺磁增宽剂能猝灭膜表面的自由基,而剩余的强固定化ESR信号显示出bR分子内部的‘刚性’构象.用2%Triton X—100处理可大大增加bR分子的‘柔性’,其ESR波谱特征与bR分子失去部分α螺旋结构和改变了它的某些运动方式有关.     

Use of spin labels to determine the percentage of interdigitated lipid in complexes with polymyxin B and polymyxin B nonapeptide

Long chain spin labels with the nitroxide group located near the terminal methyl of the chain were used to determine the percentage interdigitated lipid in complexes of polymyxin B (PMB) and polymyxin B nonapeptide (PMBN) with the acidic lipids dipalmitoylphosphatidylglycerol (DPPG) and dipalmitoylphosphatidic acid (DPPA) at varying mole ratios of drug to lipid and at different pH values. These spin labels are more motionally restricted in the interdigitated than in the non-interdigitated gel phase bilayer. This allows determination of the percentage interdigitated lipid by resolution of the spectrum into motionally restricted and more mobile components. At nonsaturating concentrations of PMB, significantly more DPPG than that which can be maximally PMB-bound, becomes interdigitated. As the temperature approaches the gel to liquid crystalline phase transition temperature, the bilayer becomes progressively non-interdigitated. The ESR spectrum indicates that PMB also causes interdigitation of DPPA. However, in contrast to DPPG, the amount of DPPA which is interdigitated at pH 6, is less than the amount which is expected to be PMB-bound. This is attributed to the ability of DPPA to participate in lateral interlipid hydrogen bonding interactions. Such lateral interactions would be abolished in the interdigitated bilayer and thus they are expected to inhibit its formation. At pH 9, where the interlipid interactions of DPPA are weakened, PMB induces even more lipid than that which is PMB-bound to become interdigitated. Indeed, the percentage interdigitated lipid is even greater than found for DPPG. This may be partly a result of the greater negative charge of DPPA at this pH. A greater repulsive negative charge is expected to favor interdigitation. PMBN is less effective than PMB at inducing interdigitation of DPPG and causes little or no interdigitation of DPPA at pH 6, even at saturating concentrations. PMBN also does not lower the phase transition temperature of DPPA at pH 6 as much as PMB. At pH 9, the effect of PMBN on DPPA is more similar to the effect of PMB. However, even for DPPG, and DPPA at pH 9, PMBN does not maintain interdigitation of the lipids at higher temperatures as effectively as PMB. PMBN's smaller perturbing effect and greatly decreased ability to cause interdigitation of DPPA at pH values below 9 may be related to a decreased ability to cause lateral separation of the lipid molecules, which is necessary in order to weaken the interlipid interactions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of phosphatidic acid on the radiation-initiated peroxidation of phosphatidylcholine in liposomes

It was found that the incorporation of anionic dipalmitoyl phosphatidic acid (DPPA) into phosphatidylcholine (PC) liposomes up to 15 mol % was accompanied by the intensification of accumulation of diene conjugates (DC), which are primary products of lipid peroxidation (LPO), if the LPO was initiated by gamma-irradiation of a 137Cs source. Monoethyl ester of DPPA, phosphatidylethanol (DPPEt), exerted a lesser influence at the same concentrations. Ca2+ ions inhibited the DC production not only in liposomes consisting of lipid mixture but in lipid membranes of PC alone as well. It was assumed that the electrostatic repulsion of negatively charged DPPA and DPPEt resulted in the loosening of polarside region of membrane hydrophobic layer and in consequence the access of hydroxyl radicals to hydrocarbon chains of PC. This assumption is in good agreement with the results of osmotic behavior of liposomes in hypertonic urea solution.     

pH-dependent bilayer destabilization and fusion of phospholipidic large unilamellar vesicles induced by diphtheria toxin and its fragments A and B

The passage by the low endosomal pH is believed to be an essential step of the diphtheria toxin (DT) intoxication process in vivo. Several studies have suggested that this low pH triggers the insertion of DT into the membrane. We demonstrate here that its insertion into large unilamellar vesicles (LUV) is accompanied by a strong destabilization of the vesicles at low pH. The destabilization has been studied by following the release of a fluorescent dye (calcein) encapsulated in the liposomes. The influence of the lipid composition upon this process has been examined. At a given pH, the calcein release is always faster for a negatively charged (asolectin) than for a zwitterionic (egg PC) system. Moreover, the transition pH, which is the pH at which the toxin-induced release becomes significant, is shifted upward for the asolectin LUV as compared to the egg PC LUV. No calcein release is observed for rigid phospholipid vesicles (DPPC and DPPC/DPPA 9/1 mol/mol) below their transition temperature whereas DT induces an important release of the dye in the temperature range corresponding to the phase transition. The transition pH associated to the calcein release from egg PC vesicles is identical with that corresponding to the exposure of the DT hydrophobic domains, as revealed here by the binding of a hydrophobic probe (ANS) to the toxin. This suggests the involvement of these domains in the destabilization process. Both A and B fragments destabilize asolectin and PC vesicles in a pH-dependent manner but to a lesser extent than the entire toxin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of myelin basic protein with different ionization states of phosphatidic acid and phosphatidylserine

Myelin basic protein (BP) has a perturbing effect on some lipids, causing, among other effects, a decrease in the temperature and enthalpy of the phase transition. This is believed to be a result of penetration of some hydrophobic residues of the protein partway into the lipid bilayer. Variations in the perturbing effect of BP on different acidic lipids has been attributed to the ability of the lipids to participate in intermolecular hydrogen bonding which inhibits penetration of the protein. Participation in intermolecular hydrogen bonding depends on the ionization state of the lipid as well as the type of lipid. In order to further test the dependence of the degree of penetration of BP on the intermolecular hydrogen bonding properties of lipids, the effect of BP on the phase transition of lipids in different ionization states was studied using differential scanning calorimetry. Dipalmitoylphosphatidic acid (DPPA) and dimyristoylphosphatidylserine (DMPS) were studied at different pH-values from 4 to 9.5. The results were compared to data obtained earlier with phosphatidylglycerol (PG), which is in the same ionization state at pH-values above 4, in order to distinguish the effects of pH on the protein from effects on the lipids. The perturbing effect of BP on PG increases with increase in pH. This is probably a result of the increasing hydrophobicity of the protein as the histidines become deprotonated, which allows greater penetration of the protein into the bilayer. In contrast, the effect on DPPA was greatest at low pH, where the state of ionization of the lipid is less than 1 and protein binding utilizes all of the hydrogen bond accepting sites (P-O-) on the lipid. BP had no perturbing effect on DPPA at higher pH where the state of ionization is between 1 and 1.5, and hydrogen bond accepting and donating sites (P-OH) are still available even after binding of the protein. Thus hydrogen bonding occurs at high pH and penetration of hydrophobic residues of the protein into DPPA is inhibited. BP had a large perturbing effect on DMPS at all pH values above 4 suggesting that lipid intermolecular hydrogen bonding does not occur in the presence of the protein and its hydrophobic residues consequently can penetrate into the bilayer. The protein may inhibit hydrogen bonding by binding electrostatically to the anionic hydrogen bond accepting group of PS.     

脂质体通透性与脂多型性关系的研究

本文以TPE和TPE/DOPE(1:1.mol:mol)制成包裹荧光分子calcein的脂质体,通过测量荧光强度随扫描温度的变化,探讨了脂质体通透性与脂多型性之间的关系.结果表明,在不发生双层相(L)变成六角形Ⅱ相(H)相转变时,脂质体悬液的荧光强度不增加;当发生该转变时,脂质体悬液的荧光强度开始增加;完成该相转变后,脂质体悬液的荧光强度仍继续增加.据此,我们认为:脂质体的通透性与脂的多型性密切相关,当发生L→HⅡ相转变时,脂质体的通透性增加.由于荧光强度的变化对相变非常敏感,我们建议用测量脂质体荧光强度随温度的变化来监测脂质体稀悬液中脂的多型性.     

Diphtheria toxin induces leakage of acidic liposomes

Diphtheria toxin (DT) induces the leakage of dipalmitoylphosphatidic acid (DPPA) membranes but not neutral dipalmitoylphosphatidylcholine (DPPC) membranes. Cholesterol incorporated into liposomes enhances the membrane leakage induced by DT in acidic DPPA membranes but not in neutral DPPC membranes. Membrane leakage was determined by assaying the release of TEMPOcholine, a cationic spin probe from the multilamellar vesicles by using electron spin resonance methods. The effect of DT on membrane leakage is noticeable at 3 micrograms/ml concentrations, and reaches a plateau of about 20% leakage at 20 micrograms/ml. This saturation phenomenon led to the postulation that DT binds to the first shell of DPPA membranes and induces the leakage of TEMPOcholine limited to this layer of DPPA multimellar vesicles.     

Physical properties of phosphatidylcholine-phosphatidylinositol liposomes in relation to a calcium effect

Physical properties of binary mixtures of dipalmitoylphosphatidylcholine and yeast phosphatidylinositol were studied by ESR analysis using TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) and lipid spin probes, freeze-fracture electronmicroscopy and particle microelectrophoresis, and they were compared with those of phosphatidylcholine/bovine brain phosphatidylserine mixtures. The phase diagram of the binary mixtures of dipalmitoylphosphatidylcholine and phosphatidylinositol was obtained from the thermal features of TEMPO spectral parameter in the lipid mixtures. The phase diagram provided evidence that these two phospholipids in various combinations were miscible in the crystalline state. The addition of 10 mM Ca2+ slightly shifted the phase diagram upward. TEMPO titration of the binary mixture of dipalmitoylphosphatidylcholine and bovine brain phosphatidylserine revealed that 10 mM Ca2+ caused the complete phase separation of this lipid mixture. Studies of phase separations using phosphatidylcholine spin probe manifested that 10 mM Ca2+ induced almost complete phase separation in egg yolk phosphatidylcholine/bovine brain phosphatidylserine mixtures but only slight phase separation in egg yolk phosphatidylcholine/yeast phosphatidylinositol mixtures. However, some phase changes around the fluidus and the solidus curves were visualized by the freeze-fracture electronmicroscopy. The molecular motion of lipid spin probe was decreased by the addition of Ca2+ in the liposomes containing phosphatidylinositol. The temperature dependence of electrophoretic mobility was also examined in the absence and presence of 1 mM Ca2+. Liposomes of dipalmitoylphosphatidylcholine-phosphatidylinositol (90 : 10, mol/mol) exhibited a clear transition in the thermal features of electrophoretic mobilities. Raising the phosphatidylinositol content up to 25 mol% rendered the transition broad and unclear. The addition of 1 mM Ca2+ decreased the electrophoretic mobility but did not change its general profile of the thermal dependence. These results suggest that the addition of calcium ions induced a small phase change in the binary mixture of phosphatidylcholine and phosphatidylinositol while Ca2+ causes a remarkable phase separation in phosphatidylcholine/phosphatidylserine mixture. The physical role of phosphatidylinositol is discussed related to the formation of diacylglycerol.     

Interaction of Paclitaxel with Phospholipid Bilayers

Abstract

Paclitaxel is an effective anticancer drug. Recently, paclitaxel encapsulated in liposomes was promoted as a better tolerated pharmaceutical formulation than that currently in use. The data presented in this study show the effects of paclitaxel on phospholipid bilayers. Experiments involving the phospholipid head group probe CAT-16 show significant disordering of the interfacial region. The pretransition was abolished and the main phase transition temperature in paclitaxel loaded liposomes was reduced. 2T II values of 7-NSA and 16-NSA spin probes reporting from the middle and from the core of the phospholipid bilayer, respectively, show that the presence of paclitaxel eliminated the pretransition (from Lβ/ to Pβ/) while inducing a slight reduction in the main (Pβ/ to Lα) phase transition temperature; in the same temperature interval, the central resonance line width δ H O displayed a greater rate of spin label reorientation in paclitaxel loaded bilayers. Further data are presented clearly demonstrating that the presence of paclitaxel in liposomal membrane increases the solubility of hydrophobic compounds. Differential scanning calorimetry was used to confirm that the presence of paclitaxel stabilized the lamellar structure of the bilayer and increased the transition temperature from lamellar Lα phase to hexagonal H II phase of TPE liposomes. The encapsulation of paclitaxel in liposomes depends on phospholipid characteristics; more drug is contained in the bilayer of liposomes containing unsaturated fatty acid chains and phosphorylcholine headgroups, such as DEPC and egg PC.     

Permeability of bilayer lipid membranes in phase transition. The significance of intermolecular phosphate-phosphate hydrogen bonding

The effect of pH on the phase transition temperature of 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA) and 1,2-dipalmitoyl-sn-glycero-3-thionphosphate (thion-DPPA) has been investigated. The phase transition was detected using the jump like increase effect in the conductance of the planar bilayer membrane. It is shown that the steepness of pH-dependence of the phase transition temperature differs for these two kinds of lipids in the pH range of 3.5-8. This result is explained in terms of decreased intermolecular hydrogen bonding between the head groups of thion-DPPA. Calculations taking into account the ability of DPPA molecules to intermolecular phosphate-phosphate hydrogen bonding were made. The results of calculations are in good agreement with the experimental data obtained in this study.     

pH dependent changes in the reactivity of the primary electron acceptor of system II in spinach chloroplasts to external oxidant and reductant.]

Two pure phospholipids, dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine, have been studied using freeze-fracture electron microscopy and the partitioning of the spin label, TEMPO. It is found that the characteristic band pattern, corresponding to monoclinic symmetry in multilamellar liposomes, is observed only in freeze-fracture electron microphotographs when samples are quenched from temperatures intermediate between the chain melting transition temperature and the pretransition temperature of the membrane. Markings are also observed on fracture faces of samples quenched from below the pretransition, but these "bands" are few in number and are widely and irregularly spaced. The lipid membranes used for freeze-fracture were prepared using detergent dialysis and are thought to consist of one, two, or some small number of concentric bilayer shells. These observations are in excellent accord with the recent, prior studies of Janiak, M.J., Small, D.M. and Shirley, G.G., ((1976) Biochemistry 15, 4575--4580), who found monoclinic symmetry (Pbeta' structure) in multilamellar liposomes of these phospholipids only when the sample temperature was intermediate between the main, chain melting transition temperature, and the pretransition temperature. The significance of these results for relating freeze-fracture electron microphotographis to phase diagrams derived from spin label or calorimetric data is discussed briefly. 2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO) partitioning data show distinct differences between liposomal preparations of these lipids, and other preparations having fewer bilayers per vesicular structure, with respect to the position, width, and hysteresis of the pretransition.