蜂毒肽的溶血作用与红细胞膜上两种酶活性变化的关系

从蜂毒肽作用于红细胞膜上的Na^＋-K^＋-ATPase和葡萄糖-6-磷酸脱氢酶(G-6-PD)活性变化的角度,利用分光光度法测定酶活性,研究蜂毒肽与红细胞及膜作用过程中可能的靶点,讨论了蜂毒肽溶血过程与RBC膜上2种酶活性的变化．结果发现,蜂毒肽抑制RBC膜上酶活性的主要模式为附着/插入质膜与游离态并存模式,附着/插入质膜中的作用大于游离态的作用．Na^＋-K^＋-ATPase的K⁺结合位点是蜂毒肽的1个作用靶点．蜂毒肽插膜过程与其对此酶的作用随时间延长同步发生．蜂毒肽通过作用于葡萄糖-6-磷酸和NADP使G-6-PD的催化受到缓慢抑制,蜂毒肽形成四聚体的程度与酶活性密切相关．EDTA抑制蜂毒肽聚集,干扰蜂毒肽作用于G-6-P,蜂毒肽作用于底物G-6-P及辅酶NADP的生化机理相似,蜂毒肽抑制作用与G-6-PD的结构无关.     

跨膜Ca~(2+)梯度对肌质网Ca~3+-ATP酶调节的特异性

我们曾报道跨膜Ca~(2+)梯度可通过膜脂影响肌质网Ca~(2+)-ATP 酶的构象和活性。本文就跨膜Ca~(2+)梯度对肌质网Ca~(2+)-ATP 酶的调节是否具有特异性作进一步研究。结果表明这种特异性表现在两方面:一是跨膜Ca~(2+)梯度对肌质网Ca~(2+)-ATP 酶功能的调节不能归结于跨膜Ca~(2+)浓度梯度所导致的膜电位的作用,离子载体FCCP 可消除跨膜电位但并不影响肌质网Ca~(2+)-ATP 酶的活力;二是其它二价金属离子如Sr~(2+)的跨膜梯度对肌质网Ca~(2+)-ATP 酶活力基本无影响。荧光偏振系列探剂n-AS 测定的结果表明跨膜Ca~(2+)与Sr~(2+)梯度对嵌有Ca~(2+)-ATP 酶的脂酶体的中部流动性的影响有较大差异。而Ca~(2+)-ATP 酶的Ca~(2+)结合位点正处于脂双层中部,这进一步提示膜脂参与了跨膜Ca~(2+)梯度对Ca~(2+)-ATP 酶的调节作用。     

“正常”膜蛋白取向的细菌视紫红质脂质体制备及其瞬态光学响应

细菌视紫红质 (bR)是嗜盐菌质膜上的一种跨膜蛋白质 ,有其独特的光驱质子泵功能 ,可以被定向组装到磷脂脂质体膜上 ,并且表现出和细胞膜上相反的取向。通过细菌培养和细胞膜分离 ,获得了含bR蛋白质的紫膜悬浮体系 ,在pH =2 .5时将bR悬浮液和两亲性的DPPC磷脂混合、通过自组装的方式形成了含bR膜蛋白的磷脂脂质体 ,并通过瞬态光学响应测量考察了bR的取向和质子泵生物活性。结果表明 ,bR膜蛋白可以被整合到DPPC的脂质体膜上 ;蛋白质的质子泵运行规律的测量进一步验证了在酸性条件下所制备的脂质体上bR保持了不寻常的择优取向 ,与细胞膜上的“正常”取向一致 ,而与绝大多数文献报道的中性条件下制备的脂质体质子泵取向相反。     

跨膜Ca^2＋梯度对肌质网Ca^2＋—ATP酶调节的特异性

我们曾报道跨膜Ｃａ＾２＋梯度可通过膜脂影响肌质网Ｃａ＾２＋－ＡＴＰ酶的构象和活性。本文就跨膜Ｃａ＾２＋－ＡＴＰ酶的构象和活性。本文就跨膜Ｃａ＾２＋梯度对肌质网Ｃａ＾２＋－ＡＴＰ酶的调节是否具有特异性作进一步研究。结果表明这种特异性表现在两方面：一是跨膜Ｃａ＾２＋梯度对肌质网Ｃａ＾２＋－ＡＴＰ酶功能的调节不能归结于跨膜Ｃａ＾２＋深度梯度所导致的膜电位的作用,离子载体ＦＣＣＰ可消除跨膜电位但并不影响肌     

蜂毒肽抑杀病原菌的超微结构观察

本研究比较了蜂毒肽对4种不同病原菌生长的影响及其抑杀菌作用的差异,并利用电子显微镜观察了蜂毒肽抑杀菌过程中超微结构的变化.结果表明,蜂毒肽对白菜软腐病菌的杀菌作用最强,而且速效;对酵母菌和番茄叶霉病菌孢子同样具有很强的杀灭作用,但作用时间延长;对金黄葡萄球菌为低浓度抑菌,高浓度杀菌作用.实验中观察到,蜂毒肽在抑杀菌过程中超微结构的变化有3种方式致死细胞：一是膜孔洞式,即蜂毒肽作用后在细胞膜区形成小空泡,随之由内向外扩展穿透细胞壁形成孔洞,引起菌体内容物泄漏死亡,显示了膜孔洞的形成过程;二是质壁分离式,小空泡以横向扩展使细胞产生类似质壁分离样空腔,最后细胞裂解死亡;三是细胞质空腔式,蜂毒肽进入细胞后与细胞质中的类膜系统的膜结合引起解体,细胞质固缩,出现空腔,细胞死亡.值得注意的是蜂毒肽抑杀菌作用的强弱及速效性与其不同杀菌方式相关.综上结果,蜂毒肽各种抑杀菌方式的共同点是蜂毒肽与膜磷脂分子结合相互作用所致.     

蜂毒肽片段的合成及其与钙调蛋白的相互作用

采用固相法设计合成了４个蜂毒肽片段，Ｍｅｌ １２，Ｍｅｌ １３，Ｍｅｌ １４，Ｍｅｌ １５。应用电泳技术，抑制钙依赖性的磷赖性的磷酸二酯酶酶活方法和荧光技术研究了这些多肽与钙蛋白的相互作用，结果表明这些多肽与钙调蛋白均形成１：１复合物，抑制钙依赖性的磷酸二酯酶的活性，其中Ｍｅｌ １４和Ｍｅｌ １５对钙调蛋白的结合活性与完整的蜂毒肽比较接近。     

带电荷磷脂在脂质体内外不对称分布的理论解释

本文提出一模型可解释带电荷磷脂在脂质体脂双层内外的不对称分布。从此模型所得出的结论与大多数实验结果一致,同时,对理论与实验结果所存在的一些偏离也进行了讨论。从这一模型还可预期:由于膜中的带电荷磷脂,存在一跨膜电位。     

艾氏腹水癌细胞膜质子跨膜外转运与脂质体融合:Ca2+及脂质体膜脂成份的作用

研究了Ca^2 及脂质体膜脂成分对艾氏腹水癌细胞质膜质子跨膜转运驱动的脂质体融合中的作用。结果 表明Ca^2 促进质子跨膜转运驱动的质子跨膜转运驱动艾氏腹水癌细胞与脂质体间的融合,膜融合程度与膜表面电荷密度的相关曲线显示,在下述条件膜融合与膜表面电荷密度呈正相关：（1）介质Ca^2 浓度小于6mmol/L,脂质体磷脂组成为PE：PC：CL＝6：2：2;（2）介质Ca^2 浓度为6mmol/L,脂质体鳞脂组成为PE：PC：CL＝6：2：2;（3）无Ca^2 介质,脂质体磷脂组成为PE：CL＝8：2;（4）介质Ca^2 浓度10mmol/L,脂质体磷脂组成为PE：CL＝8：2。脂质体PE／PC含量对膜融合的影响表明,当PE含量减少PC含量增加时,膜融合程度不断下降,提示影响膜融合的另一因素可能是生物膜结构形成“柄”融合中介体的能力。     

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.     

蜂毒溶血肽对鸡红细胞及膜的生化作用

本文采用荧光分光光度、薄层层析、原子吸收、荧光显微图像等多种生化技术,系统研究了蜂毒肽作用于鸡红细胞及膜的生化机理。结果表明：蜂毒肽影响红细胞膜上及胞内两种酶的功能。它抑制膜Na⁺-K⁺-ATPase活性,导致胞内外离子转运异常,K⁺浓度失衡;它也抑制细胞内葡萄糖-6-磷酸脱氢酶活性,其正电区域干扰胞内带负电小分子的作用,影响红细胞正常代谢。蜂毒肽干扰膜中阴离子通道的转运功能,使细胞渗透压改变,引起膨胀而溶血。蜂毒肽对有核红细胞核内DNA没有作用,与其他抗微生物多肽作用的靶向不同。据此认为,抗菌蛋白类抗生素对细菌作用的生化机理与传统抗生素不同,这是细菌对其不易产生耐药性的重要原因。     

Orientation of membrane-bound melittin studied by a combination of HPLC and liquid secondary ion mass spectrometry (LSIMS)

Combining two analytical techniques, HPLC and liquid secondary ion mass spectrometry, the orientation of liposomal membrane-bound melittin was analyzed through its trypsin-digested products. We found that trypsin can access all proteolytic sites of the membrane-bound melittin when the liposomes have no transmembrane potential, whereas the proteolytic site near the N terminus of melittin is blocked when the liposomes have a negative transmembrane potential. The results suggest that the negative transmembrane potential may induce the melittin molecules to insert into the membrane perpendicularly, whereas melittin lies flat on the membrane surface in the absence of a negative potential.     

Voltage-dependent orientation of membrane proteins

In order to study the influence of electrostatic forces on the disposition of proteins in membranes, we have examined the interaction of a receptor protein and of a membrane-active peptide with black lipid membranes. In the first study we show that the hepatic asialoglycoprotein receptor can insert spontaneously into lipid bilayers from the aqueous medium. Under the influence of a trans-positive membrane potential, the receptor, a negatively charged protein, appears to change its disposition with respect to the membrane. In the second study we consider melittin, an amphipathic peptide containing a generally hydrophobic stretch of 19 amino acids followed by a cluster of four positively charged residues at the carboxy terminus. The hydrophobic region contains two positively charged residues. In response to trans-negative electrical potential, melittin appears to assume a transbilayer position. These findings indicate that electrostatic forces can influence the disposition, and perhaps the orientation, of membrane proteins. Given the inside-negative potential of most or all cells, we would expect transmembrane proteins to have clusters of positively charged residues adjacent to the cytoplasmic ends of their hydrophobic transmembrane segments, and clusters of negatively charged residues just to the extracytoplasmic side. This expectation has been borne out by examination of the few transmembrane proteins for which there is sufficient information on both sequence and orientation. Surface and dipole potentials may similarly affect the orientation of membrane proteins.     

Topology and dynamics of melittin within the liposome revealed by a combination of mass spectrometry and chemical modification

The topology and dynamics of melittin within the liposome were investigated by a mass spectrometry coupled with acetylation. The MALDI-TOF MS and MALDI-QIT-TOF MS/MS analyses revealed that only N-terminal amine of melittin was dominantly acetylated in the presence of liposome although all of four primary amines were completely and rapidly acetylated in aqueous solution. This result indicates that melittin adopts the N-terminal-outside transmembrane topology within the liposome. The time course of acetylation followed the first-order kinetics at any examined temperatures (6-30 °C). The rate constant was less than that of the acetylation of melittin in aqueous solution. The activation energy for acetylation (74 kJ mol⁻¹) was comparable to that for dissociation of a lipid monomer from the membrane, suggesting a float-like longitudinal motion of melittin within the liposome. These results demonstrate that a mass spectrometry combined with chemical modification is very efficient way for clarifying the topology and dynamics of peptides bound to the membrane.     

The presence of PEG-lipids in liposomes does not reduce melittin binding but decreases melittin-induced leakage

Poly(ethyleneglycol) (PEG), anchored at the surface of liposomes via the conjugation to a lipid, is commonly used for increasing the liposome stability in the blood stream. In order to gain a better understanding of the protective properties of interfacial polymers, we have studied the binding of melittin to PEG-lipid-containing membranes as well as the melittin-induced efflux of a fluorescent marker from liposomes containing PEG-lipids. We examined the effect of the polymer size by using PEG with molecular weights of 2000 and 5000. In addition, we studied the role of the anchoring lipid by comparing PEG conjugated to phosphatidylethanolamine (PE) which results in a negatively charged PEG-PE, with PEG conjugated to ceramide (Cer) which provides the neutral PEG-Cer. Our results show that interfacial PEG does not prevent melittin adsorption onto the interface. In fact, PEG-PE promotes melittin binding, most likely because of attractive electrostatic interactions with the negative interfacial charge density of the PEG-PE-containing liposomes. However, PEG-lipids limit the lytic potential of melittin. The phenomenon is proposed to be associated with the change in the polymorphic tendencies of the liposome bilayers. The present findings reveal that the protective effect associated with interfacial hydrophilic polymers is not universal. Molecules like melittin can sense surface charges borne by PEG-lipids, and the influence of PEG-lipids on liposomal properties such as the polymorphic propensities may be involved in the so-called protective effect.     

Charge clusters and the orientation of membrane proteins

Summary Although hydrophobic forces probably dominate in determining whether or not a protein will insert into a membrane, recent studies in our laboratory suggest that electrostatic forces may influence the final orientation of the inserted protein. A negatively charged hepatic receptor protein was found to respond totrans-positive membrane potentials as though electrophoresing into the bilayer. In the presence of ligand, the protein appeared to cross the membrane and expose binding sites on the opposite side. Similarly, a positively charged portion of the peptide melittin crosses a lipid membrane reversibly in response to atrans-negative potential. These findings, and others by Date and co-workers, have led us to postulate that transmembrane proteins would have hydrophobic transmembrane segments bracketed by positively charged residues on the cytoplasmic side and negatively charged residues on the extra-cytoplasmic side. In the thermodynamic sense, these asymmetrically placed charge clusters would create a compelling preference for correct orientation of the protein, given the inside-negative potential of most or all cells. This prediction is borne out by examination of the few transmembrane proteins (glycophorin, M13 coat protein, H-2K^b, HLA-A2, HLA-B7, and mouse Ig heavy chain) for which we have sufficient information on both sequence and orientation.In addition to the usual diffusion and pump potentials measurable with electrodes, the microscopic membrane potential reflects surface charge effects. Asymmetries in surface charge arising from either ionic or lipid asymmetries would be expected to enhance the bias for correct protein orientation, at least with respect to plasma membranes. We introduce a generalized form of Stern equation to assess surface charge and binding effects quantitatively. In the kinetic sense, dipole potentials within the membrane would tend to prevent positively charged residues from crossing the membrane to leave the cytoplasm. These considerations are consistent with the observed protein orientations. Finally, the electrostatic and hydrophobic factors noted here are combined in two hypothetical models of translocation, the first involving initial interaction of the presumptive transmembrane segment with the membrane; the second assuming initial interaction of a leader sequence.     

Osmotic and pH transmembrane gradients control the lytic power of melittin.

Transmembrane osmotic gradients applied on large unilamellar 1-palmitoyl-2-oleoyl-phosphatidylcholine vesicles were used to modulate the potency of melittin to induce leakage. Melittin, an amphipathic peptide, changes the permeability of vesicles, as studied using the release of entrapped calcein, a fluorescent marker. A promotion of the ability of melittin to induce leakage was observed when a hyposomotic gradient (i.e., internal salt concentration higher than the external one) was imposed on the vesicles. It is proposed that structural perturbations caused by the osmotic pressure loosen the compactness of the outer leaflet, which facilitates the melittin-induced change in membrane permeability. Additionally, we have shown that this phenomenon is not due to enhanced binding of melittin to the vesicles using intrinsic fluorescence of the melittin tryptophan. Furthermore, we investigated the possibility of using a transmembrane pH gradient to control the lytic activity of melittin. The potency of melittin in inducing release is known to be inhibited by increased negative surface charge density. A transmembrane pH gradient causing an asymmetric distribution of unprotonated palmitic acid in the bilayer is shown to be an efficient way to modulate the lytic activity of melittin, without changing the overall lipid composition of the membrane. We demonstrate that the protective effect of negatively charged lipids is preserved for asymmetric membranes.     

Proteinase sensitivity of bacteriophage lambda tail proteins gpJ and pH in complexes with the lambda receptor.

Previous studies have shown that bacteriophage lambda initially binds to liposomes bearing its receptor protein by the tip of the tail fiber (type 1 complex). It then associates more directly so that the hollow tail tube is in direct contact with the membrane (type 2 complex). DNA can be injected across the lipid bilayer into the liposome from type 2 complexes. We show here that gpJ, the tail fiber protein, becomes more sensitive to proteolytic degradation in type 2 complexes, indicating that the tail fiber does not pass into the liposome and that the tail fiber may undergo a conformational change in type 2 complexes. Another bacteriophage protein, pH, is sensitive to proteolytic degradation in free bacteriophage, type 1 complexes, or type 2 complexes formed with free receptor, but is resistant to proteinases in type 2 complexes formed with liposomes. This finding suggests that pH associates with the membrane. We suggest that this association is part of the mechanism by which a transmembrane hole for DNA entry is formed.     

Adrenocorticotropic Hormone (ACTH) Responses Require Actions of the Melanocortin-2 Receptor Accessory Protein on the Extracellular Surface of the Plasma Membrane

The melanocortin-2 (MC2) receptor is a G protein-coupled receptor that mediates responses to ACTH. The MC2 receptor acts in concert with the MC2 receptor accessory protein (MRAP) that is absolutely required for ACTH binding and signaling. MRAP has a single transmembrane domain and forms a highly unusual antiparallel homodimer that is stably associated with MC2 receptors at the plasma membrane. Despite the physiological importance of the interaction between the MC2 receptor and MRAP, there is little understanding of how the accessory protein works. The dual topology of MRAP has made it impossible to determine whether highly conserved and necessary regions of MRAP are required on the intracellular or extracellular face of the plasma membrane. The strategy used here was to fix the orientation of two antiparallel MRAP molecules and then introduce inactivating mutations on one side of the membrane or the other. This was achieved by engineering proteins containing tandem copies of MRAP fused to the amino terminus of the MC2 receptor. The data firmly establish that only the extracellular amino terminus (N_out) copy of MRAP, oriented with critical segments on the extracellular side of the membrane, is essential. The transmembrane domain of MRAP is also required in only the N_out orientation. Finally, activity of MRAP-MRAP-MC2-receptor fusion proteins with inactivating mutations in either MRAP or the receptor was rescued by co-expression of free wild-type MRAP or free wild-type receptor. These results show that the basic MRAP-MRAP-receptor signaling unit forms higher order complexes and that these multimers signal.     

The use of fluoresceinphosphatidylethanolamine (FPE) as a real-time probe for peptide-membrane interactions

The characterization of fluorescelnphosphatidylethanolamlne (FPE) as a real-time Indicator of the electrostatic nature of a membrane surface is described. The conditions appropriate for the labelling of membranes and the implementation of FPE as a tool to monitor the interactions of various peptides with model membranes are outlined. It is shown that of the membrane-active peptides studied, Naja naja kaouthla cardiotoxin and pyrularia thionin bind to certain model membranes without insertion. Whereas the leader sequence of the nuclear encoded subunit IV of mammalian cytochrome c oxidase (E.C. 1.9.3.1), known as p-25, and melittin appear to bind and then partially insert into the membrane. It seems evident also that melittin does not adopt a fully transmembrane configuration. Melittin is known to promote membrane lysis and by employing a rapid-kinetic technique it is shown that the time-course of such lysis does not appear to correlate with peptide binding, but following binding a significant proportion of melittin must become inserted into the membrane before lysis appears to commence.     

Orientation and dynamics of melittin in membranes of varying composition utilizing NBD fluorescence

Melittin is a cationic hemolytic peptide isolated from the European honey bee, Apis mellifera. The organization of membrane-bound melittin has earlier been shown to be dependent on the physical state and composition of membranes. In this study, we covalently labeled the N-terminal (Gly-1) and Lys-7 of melittin with an environment-sensitive fluorescent probe, the NBD group, to monitor the influence of negatively charged lipids and cholesterol on the organization and dynamics of membrane-bound melittin. Our results show that the NBD group of melittin labeled at its N-terminal end does not exhibit red edge excitation shift in DOPC and DOPC/DOPG membranes, whereas the NBD group of melittin labeled at Lys-7 exhibits REES of approximately 8 nm. This could be attributed to difference in membrane microenvironment experienced by the NBD groups in these analogs. Interestingly, the membrane environment of the NBD groups is sensitive to the presence of cholesterol, which is supported by time-resolved fluorescence measurements. Importantly, the orientation of melittin is found to be parallel to the membrane surface as determined by membrane penetration depth analysis using the parallax method in all cases. Our results constitute the first report to our knowledge describing the orientation of melittin in cholesterol-containing membranes. These results assume significance in the overall context of the role of membrane lipids in the orientation and function of membrane proteins and peptides.