Interaction of the toxic plant protein--ricin, with model membranes. A fluorescence method of study]

The fluorescence method has been used to investigate ricin and its isolated subunits interaction with some model membranes. Three liposome types were used as a model of biological membrane: 1) liposomes constructed from lecithin and cholesterol (9:1, M:M) 2) from ganglioside receptors GM1 and 3) from the mixture of GM1, lecithin and cholesterol (1:9:1). Interaction of the protein with liposome evokes changes in the parameters of both intrinsic protein fluorescence and fluorescence of the covalently bound dansyl. Binding constants were calculated from a decrease of the intrinsic fluorescence intensity as well as from the changes in the dansyl rotation anisotropy. Measurements were carried out at neutral and acidic pH. There was good correlation of the results obtained by different methods. It was shown that association constants were different for intact ricin and its subunits. The constants also depend on liposome composition and pH of the solution. The present study has demonstrated that interaction of ricin with liposome is accounted for not only by receptor centers but also by other hydrophobic regions of ricin that are inaccessible in the native toxin and may represent the region of the subunits interaction.     

Glutaryl phosphatidylcholine effects on pH and composition dependent behavior of liposomes studied by spin-labeling method

The influence of glutaryl phosphatidylcholine on the molecular organization of phosphatidylcholine liposomes was studied by spin-labeling technique. The ESR signals given by the 5-nitroxide stearic acid label showed that the presence of glutaryl lecithin (i) significantly increased the negative charge density of the polar liposome surface with increasing proton concentration depending on the bulk solution pH, and (ii) apparently decreased the packing (order) of the hydrophobic region close to the surface, essentially in the presence of saturated phospholipids. The spectral information--S (order parameter) and alpha N (isotropic nitrogen coupling constant)--resulted in the location of the probe near or in the polar zone of the membrane or in the hydrophobic region, depending on the protonation/deprotonation of the fatty acid carboxyl group of the probe. The microviscosity of the inner region of the membrane monitored by the 12- and 16-probes was not significantly altered by glutaryl lecithin. On the other hand, glutaryl lecithin has a lesser effect on liposomes containing anionic polar head groups, such as dipalmitoyl phosphatidylglycerol or phosphatidylinositol, the anionic charge of which already had the same effect on protonation of the polar surface. The temperature dependence of dipalmitoyl phosphatidylcholine liposome dynamic behavior indicates that the glutaryl lecithin effect is completely different above and below the gel-to-liquid crystalline phase transition point.     

Resistance of liposomes with different physico-chemical characteristics to the effect of plasma

The chemical composition, liquid content sign and value of charge as well as structure and size of lipid vesicles are studied for the effect they exert on the liposome permeability for 22Na+ in the presence of human blood plasma. The rate of the isotope outlet from the electroneutral lecithin liposomes is determined by the size of vesicles and the quantity of phospholipid bilayers in their membrane. The presence either of a negative or a positive charge on the surface of the liposome membrane has no essential effect on the outlet rate of the radioactive marker. Introduction of different amounts of cholesterol or sphingomyelin into the liposome composition decreases considerably the lipid vesicle permeability and an increase in the liquid content of their membranes due to the temperature elevation is accompanied by a sharp rise in the isotope outlet rate. A conclusion is drawn on the possibility to control the outlet rate of the liposome content in the presence of blood plasma.     

Polyene antibiotic action on lecithin liposomes: effect of cholesterol and fatty acyl chains

The effect of cholesterol incorporation upon amphotericin B and nystatin susceptibility of lecithin liposome systems containing various fatty acids has been studied. Cholesterol was shown to: 1) confer sensitivity to low concentrations of amphotericin B in liposomes derived from egg lecithin, and 2) suppress the amphotericin B and nystatin-induced response in liposomes derived from dipalmitoyl or distearoyl lecithins. This clear cut difference cannot be explained by mechanisms of drug action so far presented. They are discussed in connection with the possibility that susceptibility to these polyene antibiotics is related to the over-all state of the membrane organization, in particular to the over-all conformation of membrane components.     

Various properties of urease encapsulated into liposomes

The enzymic activity of plant urease encapsulated into liposomes from egg lecithin was studied. Liposomes contained 3-5% of the initial enzymic preparation. Incorporation of urease into liposomes increases the permeability of the lecithin membrane for urea. The liposome membrane provides protection of the incorporated material from the inhibitory action of heavy metal ions. Kinetics of the reactions catalyzed by the free enzyme and encapsulated one is different. Km for the encapsulated enzyme is 1 X 10(-3) M and for free urease--4 X 10(-4) M, that is related to limited substrate mass transfer rate and as a result of it due to inhomogeneity of the catalysis proceeding in liposomes.     

Effect of cholesterol on the interaction of the amphibian antimicrobial peptide DD K with liposomes

DD K is an antimicrobial peptide previously isolated from the skin of the amphibian Phyllomedusa distincta. The effect of cholesterol on synthetic DD K binding to egg lecithin liposomes was investigated by intrinsic fluorescence of tryptophan residue, measurements of kinetics of 5(6)-carboxyfluorescein (CF) leakage, dynamic light scattering and isothermal titration microcalorimetry. An 8 nm blue shift of tryptophan maximum emission fluorescence was observed when DD K was in the presence of lecithin liposomes compared to the value observed for liposomes containing 43 mol% cholesterol. The rate and the extent of CF release were also significantly reduced by the presence of cholesterol. Dynamic light scattering showed that lecithin liposome size increase from 115 to 140 nm when titrated with DD K but addition of cholesterol reduces the liposome size increments. Isothermal titration microcalorimetry studies showed that DD K binding both to liposomes containing cholesterol as to liposomes devoid of it is more entropically than enthalpically favored. Nevertheless, the peptide concentration necessary to furnish an adjustable titration curve is much higher for liposomes containing cholesterol at 43 mol% (2 mmol L(-1)) than in its absence (93 micromol L(-1)). Apparent binding constant values were 2160 and 10,000 L mol(-1), respectively. The whole data indicate that DD K binding to phosphatidylcholine liposomes is significantly affected by cholesterol, which contributes to explain the low hemolytic activity of the peptide.     

Cytotoxic effect of unilamellar detergent dialysis liposomes on Trypanosoma brucei and Trypanosoma congolense bloodstream forms in vitro

Liposomes from egg yolk lecithin and egg yolk lecithin/ganglioside are cytotoxic for Trypanosoma brucei and Trypanosoma congolense bloodstream forms in vitro. The trypanocidal effect is influenced by the liposome age and concentration. This effect is diminished in the presence of whole blood in vitro and could not be observed in vivo. Freeze-fractured parasite membrane showed an intramembranous particle aggregation after incubation with liposomes. Liposomes from egg yolk lecithin kill trypanosomes more rapidly than do liposomes from egg yolk lecithin/cholesterol.     

Reconstitution into liposomes of the glycoprotein of vesicular stomatitis virus by detergent dialysis.

The glycoprotein of vesicular stomatitis (VS) virus was selectively liberated from the virion membrane by the dialyzable nonionic detergent, beta-D-octylglucoside. The isolated viral glycoprotein could be rendered virtually free of phospholipid and detergent, under which conditions it formed tail-to-tail glycoprotein micelles in the form of rosettes. When mixtures of viral glycoprotein and egg lecithin were dialyzed free of octylglucoside, glycoprotein vesicles formed spontaneously with spikes protruding in the same external orientation as the VS virion membrane. The glycoprotein vesicles exhibited increased and uniform buoyant density, indicating relative homogeneity in the proportion of glycoprotein and phosphatidylcholine in each glycoprotein liposome. Evidence for similar insertion and orientation of VS viral glycoprotein in both phosphatidylcholine vesicles and virion membrane was substantiated by the finding that proteolytic digestion with thermolysin gave rise to hydrophobic glycoprotein tail fragments in vesicle or virion membranes that migrated identically in polyacrylamide gels.     

Kinetics of water penetration into unsonicated liposomes effects ofn-alkanols and cholesterol

Summary The rate of swelling of egg lecithin liposomes under osmotic shock has been studied employing a stopped-flow spectrophotometer. Incorporation of cholesterol and simple alcohols into the liposomal structure elicits a biphasic response in swelling rate: at low concentrations these additives increase but at high concentrations they decrease water permeabilty. For simplen-alkanols, the effects can be correlated with structure. Specifically, the concentration of alcohol required to elicit maximal permeability as well as the maximal permeability decreases with increasing length of the alcohol. These effects are accounted for on the basis of modification of the orientation and packing of lecithin molecules in the bilayer membrane of the liposome.     

TritonX-100对含桐酸的卵磷脂脂质体的作用

本文研究了非离子型表面活性剂TritonX-100对含桐酸的卵磷脂脂质体的作用,结果表明,在TritonX-100对含桐酸的脂质体的作用中,存在一个TritonX-100的临界浓度,低于这个临界浓度时,TritonX-100的加入对脂质体的尺寸影响很小;当TritonX-100的浓度超过临界浓度时,脂质体迅速聚集成大团粒.     

Effect of pH on the control release of microencapsulated dye in lecithin liposomes. II

The objective of our work has been the microencapsulation of dyes with lecithin from soybean, with the formation of liposomes, as a substitute for synthetic auxiliaries so as to improve the quality of the effluent. Current scenarios promote the disintegration and leakage of the liposomes, such as, changes in temperature, pH, and the use of surfactants. Since dyeing process is a mix of all these parameters, we pretended to study each one separately. Changes in pH at constant temperature induce a release of dye similar with changes in temperature. In acid conditions, we found a very fast initial dye release which doesn't occur in basic conditions. Using carboxyfluorescein, as a pH fluorescence probe, we concluded that the liposome membrane doesn't protect the liposome interior from changes on the external pH.     

The outer membrane component of the multidrug efflux pump from Pseudomonas aeruginosa may be a gated channel.

OprM, the outer membrane component of the MexAB-OprM multidrug efflux pump of Pseudomonas aeruginosa, has been assumed to facilitate the export of antibiotics across the outer membrane of this organism. Here we purified to homogeneity the OprM protein, reconstituted it into liposome membranes, and tested its channel activity by using the liposome swelling assay. It was demonstrated that OprM is a channel-forming protein and exhibits the channel property that amino acids diffuse more efficiently than saccharides. However, antibiotics showed no significant diffusion through the OprM channel in the liposome membrane, suggesting that OprM functions as a gated channel. We reasoned that the protease treatment may cause the disturbance of the gate structure of OprM. Hence, we treated OprM reconstituted in the membranes with alpha-chymotrypsin and examined its solute permeability. The results demonstrated that the protease treatment caused the opening of an OprM channel through which antibiotics were able to diffuse. To elucidate which cleavage is intimately related to the opening, we constructed mutant OprM proteins where the amino acid at the cleavage site was replaced with another amino acid. By examining the channel activity of these mutant proteins, it was shown that the proteolysis at tyrosine 185 and tyrosine 196 of OprM caused the channel opening. Furthermore, these residues were shown to face into the periplasmic space and interact with other component(s). We considered the possible opening mechanism of the OprM channel based on the structure of TolC, a homologue of OprM.     

椒莪脂质体的制备及其质量标准的建立

目的:将椒莪油制成脂质体,优选制备工艺,建立质量标准。方法:采用薄膜超声法制备椒莪脂质体,通过正交实验优选处方和制备工艺,HPLC、GC建立其质量标准。结果:最佳处方为卵磷脂:胆固醇7:1,卵磷脂:油3.5:1;HPLC法测定脂质体中莪术油的含量,建立标准曲线,回归方程为Y=14958X+16795,r=0.9996;椒目仁油的测定方法同前文报道。得到的脂质体形态均一,包封率在75%左右。结论:建立的制备工艺简单,便于操作;检测方法的精密度、回收率均符合要求。     

Effect of pH on the Control Release of Microencapsulated Dye in Lecithin Liposomes. II

Abstract

The objective of our work has been the microencapsulation of dyes with lecithin from soybean, with the formation of liposomes, as a substitute for synthetic auxiliaries so as to improve the quality of the effluent. Current scenarios promote the disintegration and leakage of the liposomes, such as, changes in temperature, pH, and the use of surfactants. Since dyeing process is a mix of all these parameters, we pretended to study each one separately. Changes in pH at constant temperature induce a release of dye similar with changes in temperature. In acid conditions, we found a very fast initial dye release which doesn't occur in basic conditions. Using carboxyfluorescein, as a pH fluorescence probe, we concluded that the liposome membrane doesn't protect the liposome interior from changes on the external pH.     

Liposomes-mycobacteria incubation systems as a partial model of host-parasite interaction at cell membrane level

The multilamellar lecithin-cholesterol liposomes entrapping carboxyfluorescence as a specific marker were prepared to be incubated with mycobacteria. The extent of resulting liposomal lysis was measured by marker release. Mycobacteria were highly active in this respect regardless of the species. Staphylococci were much less active and E. coli was completely inactive. Molecular species of lecithin and their ratio to cholesterol were related with the liposome sensitivity to mycobacteria. Ultrastructural and biochemical study showed that liposomes can be in close contact with mycobacteria and lecithins are degraded so that released fatty acids are incorporated into bacterial lipids, especially in virulent species. Liposomes-mycobacteria interaction was discussed as a partial model of the phagocyte-parasite interaction at the membrane level.     

Immobilized artificial membrane chromatography: supports composed of membrane lipids

Cell membranes provide an environment for several types of molecular processes and we are attempting to mimic the cell membranes' environment on a chromatography solid support. Chromatography solid supports utilizing lecithin as the bonded phase were synthesized and the HPLC behavior of hydrophilic peptides evaluated. A diC14 lecithin containing a terminal carboxy group on the C2 fatty acid chain was amidated with the surface amines of Nucleosil-300 (7NH2) silica particles. Based on elemental analysis, lecithin was coupled to Nucleosil-300 (7NH2) at a surface density near that of lecithin found in biological membranes and this novel chromatographic support material is denoted as Nucleosil-lecithin, the prototype immobilized artificial membrane. Infrared difference spectra of Nucleosil-lecithin minus Nucleosil-300 (7NH2) clearly showed amide I (1653.1 cm-1) and amide II (1550.9 cm-1) bands, giving direct spectroscopic evidence for the amide linkage. Spectral deconvolution resolved two peaks for the amide I band, and three peaks for the amide II band. This demonstrates lecithin interchain amide hydrogen bonding and/or hydrogen bonds between the lecithin amide link and unreacted silica surface amines. Nucleosil-lecithin as a solid phase mimics membranes and can be used to study the interactions of biomolecules with membranes. Our primary objective is to develop HPLC methods for studying the interaction between cell membranes and peptide sequences found near the interfaces of cell membranes. A frequency distribution of amino acids bracketing approximately 400 transmembrane peptide sequences showed Cys to be the least frequently occurring amino acid at this putative interfacial membrane region. Hydrophilic peptide analogs bearing Cys were used as model compounds to test Nucleosil-lecithin solid supports. Small peptides, six to eight amino acids in length, containing Cys bind approximately 2X tighter to Nucleosil-lecithin compared to identical peptides without the Cys residue. Thus, Cys at the interface of cells may stabilize protein-lipid interactions.     

Lecithin:cholesterol acyltransferase. Functional regions and a structural model of the enzyme

The amino acid sequence of human lecithin:cholesterol acyltransferase has been determined by degradation and alignment of peptides obtained from tryptic and staphylococcal digestions and the cleavage with cyanogen bromide and consisted of 416 amino acid residues. All of the tryptic peptides of lecithin:cholesterol acyltransferase were isolated and sequenced. Peptides resulting from digestion by staphylococcal protease, cyanogen bromide cleavage, or the combination of the two methods were employed to find overlapping segments. The N terminus of human lecithin:cholesterol acyltransferase was determined to be phenylalanine by sequencing the whole protein up to 40 residues while the C terminus was identified as glutamic acid through carboxypeptidase Y cleavage. Cys50 and Cys74 and Cys313 and Cys356 were identified as the two disulfide bridges while the free sulfhydryl groups were located at positions 31 and 184. The N-glycosylated sites of the protein were assigned to asparagines at positions 20, 84, 272, and 384. The active site of lecithin:cholesterol acyltransferase was identified as serine on position 181 according to its homology with other serine-type esterases which have a common structure of glycine-variable amino acid-active serine-variable amino acid-glycine (Gly-X-Ser-X-Gly) with the variable amino acids disrupting the homology. No long internal repeats or homologies with apolipoproteins were found. The secondary structure is consistent with the results of predictive algorithms. A simple model of the enzyme is proposed on the basis of available chemical data and predictive methods.     

The relationship of protein and lipid synthesis during the biogenesis of mitochondrial membranes

Summary Rat liver mitochondria were fractionated into inner and outer membrane components at various times after the intravenous injection of¹⁴C-leucine or¹⁴C-glycerol. The time curves of protein and lecithin labeling were similar in the intact mitochondria, the outer membrane fraction, and the inner membrane fraction. In rat liver slices also, the kinetics of³H-phenylalanine incorporation into mitochondrial KCl-insoluble proteins was identical to that of¹⁴C-glycerol incorporation into mitochondrial lecithin. These results suggest a simultaneous assembly of protein and lecithin during membrane biogenesisThe proteins and lecithin of the outer membrane were maximally labeledin vivo within 5 min after injection of the radioactive precursors, whereas the insoluble proteins and lecithin of the inner membrane reached a maximum specific acitivity 10 min after injection.Phospholipid incorporation into mitochondria of rat liver slices was not affected when protein synthesis was blocked by cycloheximide, puromycin, or actinomycin D. The injection of cycloheximide 3 to 30 min prior to¹⁴C-choline did not affect thein vivo incorporation of lecithin into the mitochondrial inner or outer membranes; however treatment with the drug for 60 min prior to¹⁴C-choline resulted in a decrease in lecithin labeling. These results suggest that phospholipid incorporation into membranes may be regulated by the amount of newly synthesized protein available.When mitochondria and microsomes containing labeled phospholipids were incubated with the opposite unlabeled fractionin vitro, a rapid exchange of phospholipid between the microsomes and the outer membrane occurred. A slight exchange with the inner membrane was observed.     

Restoration of Ca2+-transport in sarcoplasmic reticulum ATPase solubilized with octaethyleneglycol mono n-dodecyl ether

One mg protein/ml of sarcoplasmic reticulum (SR) membranes isolated from rabbit skeletal muscle were solubilized with 50 mg/ml of octaethyleneglycol mono n-dodecyl ether (C12E8) in a solution containing 5 mM CaCl2, 0.1 M KCl, and 20% glycerol at pH 7.5. When 30 mg/ml of soybean lecithin was added to this mixture and then incubated with Bio-beads SM-2 at 20 degrees C for 1.5 h to remove the detergent from the mixture, proteoliposomes were formed. This process restored Ca2+-uptake activity to approximately 50% of that of control sR. However, Ca2+-transport was not observed when SR membranes were formed without the addition of soybean lecithin. The reconstituted vesicles also catalyze Ca2+-release, which is coupled to the backward reaction which forms ATP from ADP and P1 in the presence of a Ca2+-gradient across the membrane. When the reconstituted vesicles were subjected to equilibrium centrifugation in a 5 to 25% glycerol density gradient, all of the Ca2+-transport activity was closely associated with the fraction containing soybean liposome.     

Permeability of amino acids into liposomes.

1. A simple and rapid assay for the measurement of permeability of amino acids into liposome membrane was carried out by using the liposomes trapping D-amino acid oxidase (D-amino acid: O2 oxidoreductase (deaminating), EC 1.4.3.3) inside the membrane. 2. Permeability of amino acids into liposomes depended on the lipid composition of the membrane. Permeability of amino acids into phosphatidylcholine-cholesterol liposomes depended critically on temperature. 3. Permeability also depended on the structure of amino acids. The order of permeability was norvaline greater than isoleucine greater than leucine greater than phenylalanine greater than tryptophan greater than methionine greater than tyrosine, valine greater than threonine greater than serine greater than alanine greater than glycine.