Lipid-protein interactions: NMR study of melittin and its binding to lysophosphatidylcholine

Proton NMR of melittin differs according to the association state of the peptide in the monomer or tetramer. Melittin interacts with lysophosphatidylcholine micelles, whatever the association state of melittin; well resolved superimposed spectra from both components for all the lipid to peptide molar ratios are observed. Within the complexes, local mobility and fast exchange occurs. On binding concomitant shifts on Trp₁₉ indole lines and on the aliphatic CH₂ protons of the lipids are detected. The lipid perturbation is maximum for methylene groups in α and β of the ester bond, this could allow positionning of Trp₁₉ in the hydrophobic core of the lipids.     

ESR spectral changes induced by chlorpromazine in spin-labeled erythrocyte ghost membranes

chlorpromazine interacted preferentially with membrane proteins rather than membrane lipids in the initial incorporation into human erythrocyte ghosts, as demonstrated by means of the fluorescence quenching and a maleimide spin label. In this state the membrane fluidity increased. At higher concentrations of chlorpromazine, the membrane fluidity decreased and a motionally restricted signal from fatty acid spin labels appeared predominantly. However, no such signal appeared in protein-free vesicles. The temperature and pH dependences of the outer hyperfine splitting of this restricted signal were very similar to those of bovine serum albumin. On the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of chlorpromazine-treated and -untreated ghosts, it was found that there was no significant difference in membrane proteins between both samples except for the changes of a few bands which were not directly concerned with the occurrence of this restricted signal. These results suggest that the fatty acid spin labels bind preferably to membrane proteins as the lipid domain becomes packed with chlorpromazine.     

Comparison of cholesterol-phospholipid interaction in bilayers of liposomes and the red cell membrane

The energetics of interactions of cholesterol with phospholipid in simple liposome bilayers were compared with those in the bilayer of the human erythrocyte membrane, by measuring cholesterol distribution between erythrocytes and liposomes prepared from their whole phospholipid extract. With liposomes of a range of initial cholesterol contents, the equilibrium value for $\text{r}$, the ratio of cholesterol/phospholipid in the liposomes to that in the cells, is in the range 1.1–1.2. The closeness of this value to 1.0 indicates that overall cholesterol-phospholipid interaction in the cell membrane is similar to that in liposomes. However, while the deviation from 1.0 is small, and could arise from average cholesterol-phospholipid interactions in the membrane being only 0.06 to 0.1 kcal · mol^?1 weaker than in liposomes, it could also result from 10 to 20% of the cell membrane phospholipid being unavailable to mix with cholesterol.     

The involvement of the lipid phase transition in the plasma-induced dissolution of multilamellar phosphatidylcholine vesicles

Unsonicated liposomes prepared from dimyristoyl phosphatidylcholine were nearly completely dissolved during a 3 h incubation with rat plasma at or close to the phase-transition temperature of 24°C. At 37 or 15°C virtually no liposomal disintegration was observed even after 24 h of incubation. The liposomal solubilization, which was monitored by turbidity measurements or by determination of phospholipid sedimentability, was accompanied by the formation of a phospholipid-protein complex similar or identical to the one we previously reported to be formed from sonicated liposomes of egg phosphatidylcholine (Scherphof, G., Roerdink, F., Waite, M. and Parks, J. (1978) Biochim. Biophys. Acta 542, 296–307). Unsonicated multilamellar liposomes made of egg phosphatidylcholine were completely resistant to the dissolving potency of plasma when incubated at 37°C. Liposomes from equimolar mixtures of dimyristoyl and dipalmitoyl phosphatidylcholine were only degraded by plasma in the temperature range between 30 and 35°C at which temperature this cocrystallizing phospholipid mixture undergoes a phase transition. However, even at these temperatures the rate of dissolution of this mixture was significantly lower than of dimyristoyl phosphatidylcholine at 24°C. In the dissolving process of this mixture a slight preference for the lower-melting component was observed.The ability of cholesterol to completely abolish the susceptibility of dimyristoyl phosphatidylcholine liposomes to plasma at a 1:2 molar ratio of cholesterol to phospholipid substantiates the essential role of the phase transition in the process of liposome solubilization.When liposomes of the monotectic mixtures dimyristoyl and distearoyl phosphatidylcholine or dilauroyl and distearoyl phosphatidylcholine were incubated with plasma at temperatures in between those at which the constituent lipids undergo a phase change in the mixture, the liposomes were slowly disolved. Under those conditions a selective removal of the lipids in the liquid-crystalline phase was observed.It is concluded that for the plasma-induced dissolution of unsonicated liposomes, which is most probably achieved by interaction with (apo)lipoproteins, the presence of phase boundaries is required in much the same way as was first reported for phospholipases by Op den Kamp, J.A.F., de Gier, J. and Van Deenen, L.L.M. (1974) Biochim. Biophys. Acta 345, 253–256).     

Lipid dependence of peptide-membrane interactions: Bilayer affinity and aggregation of the peptide alamethicin

Membrane incorporation and aggregation of the peptide alamethicin have been investigated as a function of lipid type. Head group and acyl chain regions both contribute to modulate alamethicin incorporation. Specifically, the peptide prefers thin membranes and saturated chains; incorporation is reduced by the presence of cholesterol. Aggregation of the peptide in the bilayer is virtually insensitive to changes in lipid composition. These findings show some analogies to results obtained with intrinsic membrane proteins and cast doubt on the use of global membrane parameters for interpreting lipid-peptide interactions.     

Raman spectroscopic studies on the interaction between counterion and polyion

The interaction between alkali metal ions and the polyacrylate ion was investigated by means of Raman spectroscopy. in comparison with the Raman spectra of propionate salts. The Raman bands due to the metal-oxygen bond were not apparent and no significant difference was observed among the Raman spectra of several univalent salts of polyacrylate. except in the case of the lithium salt. The apparent degree of dissociation of lithium polyacrylate, as determined from the relative intensity of a specific band characteristic of the carboxylate ion, was lower than those of the other alkali metal salts. It is concluded from the Raman data that the electrostatic interaction between counterions and a polyion is not specifically modified by forces of a nonionic nature. Moreover, it is pointed out that the local conformation of polyacrylate changes gradually with the degree of neutralization, but that the change is not like a conformational transition between globular and random coil forms.     

拉曼光谱检测胃癌变信号的研究

利用激光拉曼光谱对胃癌患者血清及非胃癌患者血清进行对比检测,并结合对体外培养胃癌细胞分泌物的检测,对胃癌特征拉曼峰作初步探讨。得出一种有重要临床应用价值的癌症辅助快速诊断方法。     

Lipid-associated chlorophyll Evidence from 13C-NMR of the photosynthetic spinach thylakoid membrane

The ¹³C-NMR spectrum at 90.5 MHz has been obtained for the photosynthetic thylakoid membrane of spinach. Specific lipid and chlorophyll resonances can be assigned in the high resolution spectrum, although protein resonances are not observed. It can be estimated from resonance intensities that at least 30% of the plant chlorophyll contributes to the high resolution ¹³C spectrum with the remainder broadened by incomplete motional averaging. The resonance linewidths of the observed chlorophyll phytol chains are approximately the same as those of the lipid hydrocarbon chains, indicating a similar motional state and suggesting that this particular pool of chlorophyll is lipidbound or at most only loosely associated with proteins.     

Lipid-protein interaction in the photolysis of octopus rhodopsin

Microvillar membranes of octopus photoreceptor cells were treated with phospholipase A₂, phospholipase C, hexane, or their combinations. By these means, various membrane preparations containing qualitatively and quantitatively different lipids were obtained. The lipid composition and phospholipid content of the membrane preparations obtained by the above methods were determined.Photochemical processes in the digitonin extract of the native and treated membranes have been studied by flash photometry. The results suggest that several different variations in the lipids can affect the rates of the photochemical transformations; these are: the content of phospholipid, the amount of unsaturated hydrocarbon chains and free fatty acids.     

Properties of the strongly immobilized signal observed in spin-labeled erythrocytes

A strongly immobilized signal from fatty acid spin labels was observed in human erythrocytes treated with oxidizing agents such as glutaraldehyde, hydrogen peroxide, phenylhydrazine and copper-ortho-phenanthroline. This signal was also observed in freshly prepared ghosts treated with potassium superoxide and in old erythrocyte ghosts. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of these samples demonstrated the diffuse, nondiscrete bands of high molecular weight due to the cross-linking of membrane proteins. The temperature and pH dependences of the outer hyperfine splitting of this signal were very similar to those of bovine serum albumin. We propose that the strongly immobilized signal reflects the interaction of the lipids with the cross-linked products of membrane proteins.     

Lipid-cell interactions: A novel mechanism of transfer of liposome-entrapped substances into cells

A new approach has been developed for studying the transfer of liposome-entrapped substances into cells. The cells are incubated with liposomes containing two markers that in the free (non-entrapped) state enter the cells at different rates. Comparison of the ratio of cell-associated markers applied either in free or in liposome-entrapped form permits the evaluation of different pathways of cellular uptake of the intraliposomal substances. When epithelial cell sheets were incubated with egg phosphatidylcholine liposomes containing two different sugars they became cell-associated at a ratio different from their initial ratio inside the liposomes. Since the cell-associated ratio was shifted towards the value observed when the cells were incubated with a mixture of the two sugars in the free state, it is suggested that the liposomes become permeable during incubation and that the liberated substances enter the cells in the free form. On the other hand, cell-liposome interaction was demonstrated by NMR measurement and gel-filtration experiments to result in transformation of small unilamellar liposomes into larger multilayered aggregates. This transformation depends on the contact of the liposomes with the cell sheet. It is supposed that interliposomal aggregation is the underlying mechanism of cell-induced leakage of liposomes.     

Structural analysis of denaturant-protein interactions: Comparison between the effects of bromoethanol and SDS on denaturation and renaturation of triclinic lysozyme

This paper summarizes our crystallographic studies of the interaction of denaturants with cross-linked triclinic lysozyme. Electron density maps of various bromoethanol-lysozyme complexes are analyzed and compared to those reported earlier for SDS-lysozyme complexes. Despite differences in the chemical nature and size of the two denaturants their mode of interaction with the protein is quite similar, suggesting the existence of a general mechanism for binding of hydrophobic-hydrophilic denaturants to proteins. Our results are consistent with the conclusion that lysozyme consists of two domains connected by a flexible segment and that this segment represents an internal degree of freedom of the protein.The work was carried out during the tenure of a fellowship from the European Molecular Biology OrganizationWe are grateful to Dr. Gerson Cohen for providing us with his data processing programs, to Drs. David Haas, Paul Sigler, Thomas Creighton and Micael James for helpful discussions, and to Mr. Samuel Getteno for his invaluable technical assistance.     

胃癌患者手术前后血清的激光拉曼光谱研究

为了探讨胃癌患者血清光谱在手术前后的变化及临床应用价值,用光谱法分别检测了30例胃癌细胞培养液、20例健康人血清和41例胃癌患者手术前、后血清的激光拉曼光谱。结果表明细胞培养液的拉曼光谱与空白对照有明显不同,且峰值与培养时间增成正相关;胃癌患者血清的拉曼光谱与健康人有显著差异;手术后胃癌患者血清的拉曼光谱峰值比术前明显降低。研究表明拉曼光谱可以作为胃癌细胞体外研究的检测手段,并有潜力成为胃癌筛查、预后监测及疗效判断的新方法。     

The structure of melittin in lipid bilayer membranes

0.15 M inorganic phosphate dramatically increased the α-helix content of melittin in aqueous solution.When melittin interacted with egg yolk phosphatidylcholine liposomes in the absence of inorganic phosphate, it was converted to an α-helix rich form, as postulated by Dawson et al. (Dawson, C.R., Drake, A.F. Helliwell, J. and Hider, R.C. (1978) Biochim. Biophys. Acta 510, 75–86).     

Lipid-protein interactions. A comparative study of the binding of cardiotoxins and neurotoxins to phospholipid vesicles

It has recently been shown that cardiotoxin II from Naja mossambica mossambica specifically interacts with negatively charged phospholipids (Dufourcq, J. and Faucon, J.F. (1978) Biochemistry 17, 1170–1176). In order to investigate whether or not short neurotoxins give rise to similar interactions, four techniques have been used, namely intrinsic fluorescence, fluorescence polarization of 1,6-diphenylhexatriene, turbidity measurements and release of 6-carboxyfluorescein trapped inside single shelled vesicles.Neurotoxin III from Naja mossambica mossambica and neurotoxin I from the venom of the scorpion Androctonus australis Hector, specifically interact with negatively charged phospholipids leading to changes in tryptophan fluorescence and to a decrease of the fluidity of the bilayer. Cardiotoxin II from the same snake venom gives similar results. On the other hand, it seems that either a very weak or no interaction at all occurs in the case of neurotoxin I from the same Naja venom.There are important differences in the behaviour of cardiotoxin and neurotoxins: (i) neurotoxins lead to only weak release of 6-carboxyfluorescein from lipid vesicles, whereas cardiotoxin II induces fast and quantitative escape of the dye and then a general breakdown of the vesicular structure; (ii) binding of neurotoxins can be easily reversed by 100–200 mM NaCl or less than 1 mM Ca²⁺ and so it is essentially electrostatic, whereas binding of cardiotoxin II seems to involve some hydrophobic contribution.The short neurotoxins and cardiotoxins from snake venom having a great homology in sequence, their differences on binding properties are discussed in terms of changes in a particular area of the sequence.     

Effect of low-molecular-weight proteins on protein (lysozyme) binding to isolated brush-border membranes of rat kidney

Filtered proteins including the low-molecular-weight protein lysozyme are reabsorbed by the proximal tubule via adsorptive endocytosis. This process starts with binding of the protein to the brush-border membrane. The binding of ¹²⁵I-labelled egg-white lysozyme (EC 3.2.1.17) to isolated brush-border membranes of rat kidney and the effect of several low-molecular weight proteins on that binding was determined. The Scatchard plot revealed a one-component binding type with a dissociation constant of 5.3 μM and 53.0 nmol/mg membrane protein for the number of binding sites. The binding of the cationic lysozyme was inhibited competitively by the addition of cationic cytochrome c to the incubation medium, while the neutral myoglobin had no effect. The anionic β-lactoglobulin A inhibited the lysozyme binding in a noncompetitive manner. These data suggest that the binding takes place between positively charged groups of the protein molecule and negative sites on the brush-border membrane, and, the competition between the cationic cytochrome c and the cationic lysozyme for the binding sites may be responsible for the inhibitory effect of cytochrome c on renal lysozyme reabsorption. The binding step at the brush-border membrane appears to be cation-selective.     

Divalent cation enhancement of the agglutinability by soyabean lectin of liposomes prepared from total lipid of erythrocytes and of erythrocyte membranes

CaCl₂ or MgCl₂ but not NaCl enhances the soyabean lectin-induced agglutination of liposomes prepared from total lipids of erythrocyte membranes. The addition of purified phosphatidylserine to the total lipids of erythrocyte membranes before the formation of liposomes inhibits lectin-induced agglutinability of the preparation in the absence of CaCl₂, but not in its presence. When preformed phosphatidylserine liposomes are added to liposomes of total lipids of erythrocyte ghosts, they do not inhibit agglutination, indicating that phosphatidylserine does not inhibit the lectin directly. CaCl₂ or MgCl₂ but not NaCl also stimulates the soyabean lectin-induced agglutination of human erythrocyte membranes.Electron micrographs indicate that the liposome preparations are multilamellar and separate even in the presence of CaCl₂. When such liposomes are treated with lectin with or without CaCl₂, the electron micrographs show significant agglutination without apparent fusion. The reversal of the agglutination of liposomes by specific sugars followed by turbidimetric and electron microscopic techniques supports the conclusion that CaCl₂ stimulated lectin-induced agglutination is unaccompanied by fusion.The stimulation by divalent cations of lectin-induced agglutination of erythrocyte ghosts or of our liposomes may be due to a decrease in apparent surface charge of these membrane systems.     

Membrane interactions of antimicrobial peptides from Australian frogs

The membrane interactions of four antimicrobial peptides, aurein 1.2, citropin 1.1, maculatin 1.1 and caerin 1.1, isolated from Australian tree frogs, are reviewed. All four peptides are amphipathic α-helices with a net positive charge and range in length from 13 to 25 residues. Despite several similar sequence characteristics, these peptides compromise the integrity of model membrane bilayers via different mechanisms; the shorter peptides exhibit a surface interaction mechanism while the longer peptides may form pores in membranes.     

Incorporation of the antimicrobial protein seminalplasmin into lipid bilayer membranes

The interaction between seminalplasmin, an antimicrobial protein from bull semen, and lipid bilayers has been investigated. The fluorescence of the single tryptophan residue of the protein was measured. In the presence of phosphatidylcholine or phosphatidic acid bilayer vesicles the fluoresence maximum was shifted to shorter wavelengths, indicating transfer of the tryptophan to a more apolar environment. Circular dichroism spectra show an increased -helical content for the protein in the presence of lipid. Quenching experiments clearly show the incorporation of the protein with the tryptophan localized near the bilayer surface. The shift of the tryptophan fluorescence emission was used to monitor the lipid phase transition in phosphatidylcholine membranes.Abbreviations TEMPOL 2,2,6,6-Tetramethyl-4-hydroxy-piperidine-1-oxyl - DMPC 1,2-Dimyristoylphosphatidylcholine - DMPA 1,2-Dimyristoylphosphatidic acid - SL 5 2-(3-Carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinoxyl - SL 12 2-(10-Carboxydecyl)-4,4-dimethyl-2-hexyl-3-oxazolinoxyl