Interaction of class A amphipathic helical peptides with phospholipid unilamellar vesicles

The exchangeable apolipoproteins are important in determining the structure/function properties of lipoproteins. These proteins typically contain varying amounts of amphipathic helices. Five model peptides, 18A, Ac-18A-NH2, Ac-18R-NH2, 37pA, and 37aA, have been designed to investigate variations of the amphipathic alpha-helix structural motif on their lipid-binding properties. These include the 18-residue peptides, 18A and Ac-18A-NH2, examples of class A helices, and Ac-18R-NH2, which has the positions of acidic and basic residues interchanged relative to 18A. Three larger peptides were also studied: 36A, a dimer of 18A, 37pA and 37aA, dimers of 18A coupled by Pro (18A-Pro-18A) and Ala (18A-Ala-18A), respectively. We report here the results of a thermodynamic characterization of the binding properties of these peptides to small unilamellar vesicles of POPC. Partition coefficients, Kp, were determined by fluorescence spectroscopy and binding enthalpies, deltaH, by titration calorimetry. These parameters were used to obtain the free energies, deltaG0, and entropies, deltaS0, of binding. The results of this study indicate Kp values on the order of 10(5), with interactions being enthalpically but not entropically favored in all cases. The presence of positively charged residues at the interface (18A and Ac-18A-NH2) enhances binding but has little effect on the extent of bilayer penetration. The presence of tandem repeats decreases lipid affinities for these small, highly curved bilayers. Our results are consistent with the idea that interaction appears to be confined largely to the surface, with some degree of penetration of the hydrophobic face of the helix into the interior of the bilayer.     

Interaction of wheat alpha-thionin with large unilamellar vesicles

The interaction of the wheat antibacterial peptide alpha-thionin with large unilamellar vesicles has been investigated by means of fluorescence spectroscopy. Binding of the peptide to the vesicles is followed by the release of vesicle contents, vesicle aggregation, and lipid mixing. Vesicle fusion, i.e., mixing of the aqueous contents, was not observed. Peptide binding is governed by electrostatic interactions and shows no cooperativity. The amphipatic nature of wheat alpha-thionin seems to destabilize the membrane bilayer and trigger the aggregation of the vesicles and lipid mixing. The presence of distearoylphosphatidylethanolamine-poly(ethylene glycol 2000) (PEG-PE) within the membrane provides a steric barrier that inhibits vesicle aggregation and lipid mixing but does not prevent leakage. Vesicle leakage through discrete membrane channels is unlikely, because the release of encapsulated large fluorescent dextrans is very similar to that of 8-aminonaphthalene-1,3,6,trisulfonic acid (ANTS). A minimum number of 700 peptide molecules must bind to each vesicle to produce complete leakage, which suggests a mechanism in which the overall destabilization of the membrane is due to the formation of transient pores rather than discrete channels.     

Binding of dipyridamole to phospholipid vesicles: a fluorescence study

Binding and localization of the vasodilator and antitumor drug coactivator dipyridamole (DIP) and one of its derivatives, RA25, to phospholipid vesicles of DMPC (dimyristoylphosphatidylcholine) and DPPC (dipalmitoylphosphatidylcholine) was studied using fluorescence spectroscopy as well as quenching of fluorescence. The analysis of fluorescence data indicates that neutral dipyridamole binds to the phospholipids in their liquid crystalline phase with an association constant of 950 M(-1) and 1150 M(-1) to DMPC and DPPC, respectively. Protonation of DIP leads to a 3-fold reduction of the association constant. For the gel phospholipid phase, the binding is smaller (a factor of 2), independently of pH, suggesting that the more flexible lipid packing in the liquid crystalline phase facilitates the binding of the drug. The association constant of RA25 neutral form is considerably lower than for DIP, being around 295 M(-1). Fluorescence quenching with nitroxides TEMPO and stearic acid doxyl derivatives suggests the localization of DIP to be closer to the 5th carbon of alkyl chain. The quenching effect of 5-DSA below the lipid phase transition suggests that a strong static quenching may be operative. The quenching effect of 16-DSA is almost as great as that for 5-DSA below the phase transition, being even higher above the phase transition. This effect is probably due to the trans-gauche isomerization of the stearic acid nitroxide, making the encounter of its paramagnetic fragment with the DIP chromophore possible. Our data are consistent with DIP location close to the bilayer surface in the border of hydrophobic-polar heads interface which is similar to the data in micellar systems. In the case of RA25, the drug is in the outer part of the head group interface being much exposed to the aqueous phase and being significantly less accessible to the membrane nitroxide quenchers.     

Temperature-controlled release property of phospholipid vesicles bearing a thermo-sensitive polymer

The supraspinatus, infraspinatus, teres minor and subscapu?aris muscles form a musculotendinous rotator cuff that provides dynamic stability to the shoulder joint. Symptoms of rotator cuff injury include limitation of motion, weakness and pain that often radiates down the upper arm and is present at night. Examination may reveal deltoid and rotator cuff atrophy, tenderness, limited passive range of motion and weakness on abduction and external rotation. Radiographs may show degenerative changes of the acromion or acromioclavicular joint, cysts, sclerosis and spurs of the greater tuberosity, and calcific deposits within the supraspinatus tendon. In most patients with subacromial impingement, conservative management, including physical therapy, nonsteroidal anti-inflammatory drugs and subacromial injections, is successful. Failure of conservative therapy after six to 12 weeks merits further evaluation with magnetic resonance imaging or arthrography, and consideration of surgery.     

Fusion and protein-mediated phospholipid exchange studied with single bilayer phosphatidylcholine vesicles of different density

A novel method has been developed for the study of phospholipid exchange and fusion of phospholipid vesicles. Two homogeneous populations of single bilayer phosphatidylcholine vesicles of similar size but markedly different density have been prepared. "Dense" vesicles were made from brominated dioleoyl phosphatidylcholine. "Light" vesicles were prepared from dioleoyl phosphatidylcholine. The two populations were easily separated by density gradient centrifugation. Phosphatidylcholine exchange protein from beef liver was used to promote lecithin exchange between the vesicle populations. Only the lecithin of the external monolayers of the vesicles was available for exchange by exchange protein, implying that flip-flop of vesicle phosphatidylcholine did not take place at a detectable frequency. No spontaneous intervesicle phosphatidylcholine exchange was observed. However, the dense and light vesicles did spontaneously fuse, over several hours, to produce particles of hybrid density.     

Cholesteryl ester transfer from phospholipid vesicles to human density lipoproteins

The exchange of cholesteryl esters between different lipoproteins was reported to bae mediated by a protein present in human plasma. In this study wer have examined the movement of cholesteryl ester from unilamellar phospholipid vesicles to high density lipoprotein (HDL). Experimental conditions were establisehd so that vesicles containing egg yolk lecithin and cholesteryl oleatea (molar ratio of 86:1) could be incubated with human HDL so that neaither disruption of particles nor transfer of lipid occurred. Addition of human lipoprotein-deficient plasma to the system promoted the transfer of cholesteryl oleate, but not phospholipid, from vesicles to HDL. Cholesteryl oleate transfer was dependent upon amount of HDL or lipoproteain-deficient plasma added and occurred when either HDL2 or HDL3 were present. Addition of unesterified cholesterol to the vesicles did not influence lcholesteryl ester transfer to HDL. When phospholipid vesicles containing both cholesteryl oleate and triolein (molar ratio 86:1:1) were incubated with HDL and lipoprotein-deficient plasma, only cholesteryl oleate was transferred from the vesicles to HDL. Lipoprotein-deficient plasma derived from rabbits promoted the selective transfer of cholesteryl oleate from these visicles, but rat plasma did not cause any movement of cholesteryl oleate or triolein from vesicles to HDL. HDL containing labeled cholesteryl esters was prepared and incubated with vesicles containing unlabeled cholesteryl esters or phospholipid alone. Addition of lipoprotein-deficient plasma did not promote transfer of cholesteryl esters from HDL to vesicles, whereas transfer from HDL to low density lipoprotein was readily observed. The results indicated that a protein present in rabbit and human plasma is effective in the selective, unidirectional transport of cholesteryl esters from a phospholipid bilayer to a plasma lipoprotein.     

Fusion of phospholipid vesicles induced by the ribosome inactivating protein saporin

The single chain ribosome-inactivating protein Saporin-S6 (SO-6) induces the fusion of acid phospholipid vesicles. The extent of fusion was measured by resonance energy transfer assay between the N-(7-nitro-2-1,3-benzoxadiazol-4-yl)-dimyristoylphosphatidyl lithanolamine (NBD-PE)(donor) and N-(lissamine rhodamine B sulphonyl)-diacylphoshaidylethanolamine (Rh-PE) (acceptor) incorporated in the vesicle. The saturated lipid/protein molar ratio is approx. 100:1. The time course of fusion of vesicles induced by the protein showed that the process was completed within 10 minutes, and the size of the particles in the medium was enlarged which conforms the occurrence of the fusion occurring. The fusion is temperature dependent and the liquid-crystalline state lipid is more apt to fuse than the gel phase lipid. The effect of SO-6 is also dependent on ionic strength and pH, high salt concentration and basic pH may abolish fusion, which suggests that both electrostatic and hydrophobic components may be involved in the process.     

Interaction and orientation of an alpha-aminoisobutyric acid- and tryptophan-containing short helical peptide pore-former in phospholipid vesicles, as revealed by fluorescence spectroscopy

The interaction and orientation of a membrane protein ion channel model, an alpha-aminoisobutyric acid analogue of gramicidin B (GBA), in egg yolk phosphatidylcholine vesicles was studied by means of fluorescence spectroscopic techniques. GBA helices form stable ion-conducting pores in membranes [Jelokhani-Niaraki et al. (1995) J. Chem. Soc. Perkin Trans. 2, 801-808]. In an alpha-helical model for the peptide, all Trp residues (intrinsic fluorophores) are distributed near the C-terminus. Fluorescence quenching experiments revealed the exposure of the helical peptides' C-termini to aqueous environments. Dansyl-labeled vesicles were used to investigate the GBA dynamism of the interaction with membranes. It was shown that considerable amounts of peptide reside on and in the vicinity of the outer surface of lipid bilayers. The transmembrane transfer to the inner layer is slow due to the high affinity of Trp residues for bilayer interfaces which anchor the peptide to the outer surface. A structural-functional interpretation of the GBA interaction with membranes is presented.     

Influence of lipid composition on pediocin PA-1 binding to phospholipid vesicles

Pediocin PA-1 bound to anionic lipid vesicles with saturated or unsaturated fatty acid chains in a lipid concentration-dependent fashion. Little change in binding parameters was observed for zwitterionic lipid vesicles. Decreasing the anionic lipid content of the vesicles gave a higher relative dissociation constant for the peptide-lipid interactions and further supports the electrostatic interaction model of binding.     

The characterization of liposomal glucose oxidase electrodes for the measurement of glucose

Many biosensors have been described for the measurement of glucose in order to monitor diabetic patients. Glucose oxidase has been used commonly in the construction of glucose sensors but the performance of this enzyme is limited by enzyme saturation kinetics, which restrict the measurement of clinically relevant glucose concentrations (0 to 25 mM). Diffusion limiting membranes have been described that result in the exposure of the enzyme to lower concentrations of glucose than are present in the bulk test solution. Recently a liposomal enzyme electrode was reported whereby glucose oxidase was encapsulated within liposomes so that the lipid bilayer was the diffusion limiting membrane. It was shown that the electrode response was defined by the lipid constituents of the liposome, and that a linear response to glucose could be achieved up to 40 mM. This paper describes research undertaken to improve the methods of production of a liposomal enzyme electrode. Improved immobilization of liposomes is demonstrated with the use of poly-L-lysine solution. The variation in electrode response with respect to the amount of glucose oxidase liposomally encapsulated is reported. The new method allows a greater number of sensors to be produced from a single batch of liposomes. Studies also show the biofouling effects of the lipid constituents of ruptured liposomes on the response of the electrode to glucose over time.     

Direct observation of lipid hydroperoxides in phospholipid vesicles by electrospray mass spectrometry

Positive ion electrospray ionization mass spectrometry was used to obtain a lipid profile of vesicles prepared from egg yolk lethicin and enriched with arachidonylstearoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine. The vesicles were oxidized by treatment with tert-butylhydroperoxide and iron (II) sulfate, and the formation of hydroperoxides of the polyunsaturated lipid arachidonylstearoyl phosphatidylcholine was observed. The native lipid signal at 832 a.m.u. decreased and new signals appeared at 864, 896, and 928 a.m.u., corresponding to the addition of one (+32), two (+64), and three (+96) molecules of dioxygen. The dihydroperoxide was found to be the most favourable peroxide product, but it appeared that a degradation of the hydroperoxides was occurring concomitant with their formation, and only their net formation was observed. The rate of depletion of the polyunsaturated lipid and the rate of accumulation of the hydroperoxides was found to increase with the Fe2+ concentration between 10 microM and 2 mM, and was also dependent on the tert-butylhydroperoxide concentration. This is the first report of analysis of lipid hydroperoxides by electrospray mass spectrometry, showing that technique offers a sensitive, direct, and informative approach to the study of oxidative damage to biological membranes.     

Interaction of glucose and metformin with isolated red cell membrane

Isolated human erythrocyte membranes (red blood cell (RBC) ghosts) were incubated with glucose at 5, 10, 20 and 100 mmol/l concentrations, with insulin (0.01 to 200 mU/l) and metformin (CAS 657-24-9) 0.5 up to 50.0 mumol/l). Binding studies with 14C-glucose and subsequent gel electrophoresis revealed 60% of the radioactivity around ban 4.2-4.5 at 5 mmol/l, whereas a random distribution of radioactivity over all protein bands of the RBC membrane was found at 20 mmol/l concentration after incubation for 30 min or 48 h. Metformin does not bind covalently to RBC membranes, however, after photochemical linkage of 14C-metformin via the aminoreactive linker azidophenylglyoxal the highest radioactivity (21%) was counted in the range of band 4.2-4.5. In parallel with an increase of order parameters of 5-doxyl-stearic acid the thiol status of the membranes decreases as determined by monobromobimane fluorescence. 20 and 100 mmol/l concentrations of glucose decrease the reactivity of membrane thiols towards bromobimane significantly to 73 and 62% of the controls. Concomitantly, membrane fluidity at polar sites is diminished as measured by order parameters of spin label 5-doxyl stearic acid. In RBC membranes pretreated with 20 mmol/l glucose the decreased fluorescence is significantly raised again by insulin and metformin. This effect is even more pronounced, if insulin and metformin are incubated together. Reaction of membrane thiols with a maleimido spin label detects modification in the ratio of mobile and immobilized spin label populations in the electron paramagnetic resonance signal under the above conditions, indicative of conformational changes of membrane proteins.     

Interaction of nonachlazine and noradrenaline on a model phospholipid membrane

The binding of nonachlazine (NCL), imipramine (IMP), and noradrenaline (NA) with the model phospholipid membrane vesicles--liposomes--was studied. The binding was determined by the qunching effect of these substances on the fluorescence of 3-methoxybenzantrone (MBA) present in the membrane of the fluorescent probe. A method rendering possible calculation of the binding of the preparations under study with the membrane of the basis of the fluorescence changes was developed. The binding constant of the NCL, IMP, and NA interaction with the membrane was equal to (4.3 +/- 0.3)-10(3)M-1, (2.7 +/- 0.2)-10(3)M-1, and (0.7 +/- 0.15)-10(3)M-1, respectively. It was shown that NCL and IMP could compete with NA for the membrane binding centers. Such competitive interactions could be regarded as a probable mechanism of the block of the reverse NA transport through the synaptic and the vesicular membranes characteristic of NCL and IMP.     

19F-NMR studies of retinol transfer between cellular retinol binding proteins and phospholipid vesicles

The cellular retinol binding proteins, CRBP and CRBP II, are implicated in the cellular uptake of retinol and intracellular trafficking of retinol between sites of metabolic processing. 19F-NMR studies of retinol transfer between CRBP and CRBP II and phospholipid vesicles, using either fluorine-labeled ligand or protein, demonstrated that there was significantly more transfer of retinol from CRBP II to lipid vesicles than from CRBP. Differences in how readily protein-bound retinol is released to lipid bilayers may lead to differences in how these two proteins modulate intracellular retinol metabolism.