Effects of vitamin E on the response of the fetal middle cerebral artery to the pressure test.

OBJECTIVE: To examine the effects of the maternal administration of vitamin E on the vasoreactivity of the middle cerebral artery (MCA) in preterm fetuses. STUDY DESIGN: The vasoconstrictive response of the proximal segment of the MCA to brief and partial external occlusions of the umbilical vein (pressure test) was studied in 22 fetuses between 21 and 35 weeks of gestation, before, and 3 to 7 days after, the maternal administration of oral vitamin E (50 mg daily). RESULTS: The vasoconstrictive activity of the MCA was eliminated in 15 fetuses (68.2%), unchanged in six (27.3%), and decreased in one (4.5%). In the latter seven cases, vasoconstriction of the proximal MCA in response to the pressure test was eliminated by increasing the vitamin E dosage to 100 mg/day. CONCLUSION: Vitamin E administered to the mothers had a pronounced effect on the proximal MCA reactivity in preterm fetuses.

Influence of polymyxins on the structural dynamics of Escherichia coli lipid membranes.

Polymyxins are a family of nonribosomic cationic peptide antibiotics highly effective against Gram-negative bacteria. Two members of this family, Polymyxins B and E (PxB, PxE), form molecular vesicle-vesicle contacts and promote a selective exchange of phospholipids at very low concentrations in the membrane, a biophysical phenomenon that can be the basis of their antibiotic mode of action. To get more insight into the interaction of these antibiotics with the lipid membrane, their effect on the structural dynamics of bilayers prepared with lipids extracted from the membrane of Escherichia coli was determined using fluorescently labeled phopholipids. Steady-state anisotropy measurements with probes that localize at different positions in the membrane give information on the effects of polymyxins on the mobility of the phospholipids. Results with PxB, PxE, colymycin M and polymyxin B nonapeptide (PxB-NP), a deacylated derivative with no antibiotic properties, are compared. At low peptide concentrations (<2 mol%) PxB and PxE bind to the membranes superficially, affecting very slightly the ordering of the lipids at the outermost part of the bilayer. Above this concentration, PxB and PxE insert more deeply in the bilayer, increasing lipid order both in the gel and liquid-crystal states and modifying phase transitions. Fluorescence experiments with pyrene labeled phospholipids indicate that the increase in lipid packing is accompanied by an enrichment of phospholipids in the bilayers. In contrast, colymycin M and PxB-NP did not modify lipid packing or phase transition, nor did they induce microdomain formation. The possible significance of these results in the antibiotic mode of action of PxB and PxE is discussed. The combination of spectroscopic techniques described here can be useful as part of a general method of screening for new antibiotics that act on the membrane by the same mechanism as polymyxins.

Membrane fusion induced by a lipopeptidic epitope from VP3 capside protein of hepatitis A virus.

Palmitoyl-VP3(110--121) (PVP3) is a synthetic lipopeptide derivative of a continuous epitope from the VP3 capsid protein of hepatitis A virus, and it is highly immunogenic in vivo. We have investigated the interaction of PVP3 with lipid model membranes of varying surface charge. Binding of PVP3 to anionic vesicles of PC/SM/PE/PS; (PC) 1-palmitoyl-2-oleoyl-phosphatidylcholine, (SM) sphingomyelin, (PE) 1,2-dipalmitoyl-phosphatidylethanolamine and (PS) L-alpha-phosphatidyl-L-serine, a composition that mimics the lipid component of natural membranes, was determined by tryptophan fluorescence and quenching experiments. In addition, and given the anionic net charge of the lipopeptide, binding to zwitterionic (PC/SM/PE) and cationic PC/SM/PE/DOTAP (DOTAP) 1,2-dioleoyl-3-trimethylammonium-propane mixtures was also determined. PVP3 binds to all three types of vesicles, but it adopts different forms depending on the electrical charge of the interface. This conclusion is supported by the insertion of PVP3 into lipid monolayers of the same charges spread at the air-water interface. The bound lipopeptide has membrane-destabilizing effects in all three vesicle compositions, as demonstrated by leakage of vesicle contents, whereas lipid mixing only occurs in cationic liposomes. Our results provide useful information for the design of a liposomal system that promotes a direct delivery of the membrane-incorporated immunogen to the immunocompetent cells, potentially increasing the immune response from the host.

Membrane fusion by an RGD-containing sequence from the core protein VP3 of hepatitis A virus and the RGA-analogue: implications for viral infection.

The interaction of an RGD-containing epitope from the hepatitis A virus VP3 capsid protein and its RGA-analogue with lipid membranes was studied by biophysical methods. Two types of model membrane were used: vesicles and monolayers spread at the air/water interface, with a composition that closely resembles the lipid moiety of hepatocyte membranes: PC/SM/PE/PC (40:33:12:15; PC: 1-palmitoyl-2-oleoylglycero-sn-3-phosphocholine; SM: sphingomyelin from chicken egg yolk; PE, 1,2-dipalmitoyl-phosphatidylethanolamine; PS: L-alpha-phosphatidyl-L-serine from bovine brain). In addition, zwitterionic PC/SM/PE (47:39:14) and cationic PC/SM/PE/DOTAP (40:33:12:15; DOTAP: 1,2-dioleoyl-3-trimethylammonium-propane) membranes were also prepared in order to dissect the electrostatic and hydrophobic components in the interaction. Changes in tryptophan fluorescence, acrylamide quenching, and resonance energy transfer experiments in the presence of vesicles, as well as the kinetics of insertion in monolayers, indicate that both peptides bind to the three types of membrane at neutral and acidic pH; however, binding is irreversible only at low pH. Membrane-destabilizing and fusogenic activities are triggered by acidification at pH 4-6, characteristic of the endosome. Fluorescence experiments show that VP3-RGD and VP3-RGA induce mixing of lipids and leakage or mixing of aqueous contents in anionic and cationic vesicles at pH 4-6, indicating leaky fusion. Interaction with zwitterionic vesicles (PC/SM/PE) results in leakage without lipid mixing, indicating pore formation. Replacement of aspartic acid in the RGD motif by alanine maintains the membrane-destabilizing properties of the peptide at low pH, but not its antigenicity. Since the RGD tripeptide is related to receptor-mediated cell adhesion and antigenicity, results suggest that receptor binding is not a molecular requirement for fusion. The possible involvement of peptide-induced membrane destabilization in the mechanism of hepatitis A virus infection of hepatocytes by the endosomal route is discussed.

Effect of the lipid interface on the catalytic activity and spectroscopic properties of a fungal lipase.

Lipase from the fungi Thermomyces (formerly Humicola) lanuginosa (TlL) is widely used in industry. This interfacial enzyme is inactive under aqueous conditions, but catalytic activation is induced on binding to a lipid-water interface. In order for protein engineering to design more efficient mutants of TlL for specific applications, it is important to characterize its interfacial catalysis. A complete analysis of steady-state kinetics for the hydrolysis of a soluble substrate by TlL has been developed using an interface different from the substrate. Small vesicles of 1-palmitoyl-2-oleoylglycero-sn-3-phosphoglycerol (POPG) or other anionic phospholipids are a neutral diluent interface for the partitioning of substrate and enzyme. TlL binds to these interfaces in an active or open form, thus implying a displacement of the helical lid away from the active site. A study of the influence of substrate and diluent concentration dependence of the rate of hydrolysis provides a basis for the determination of the primary interfacial catalytic parameters. The interfacial activation is not supported by zwitterionic vesicles or by large anionic vesicles of 100 nm diameter, although TlL binds to these interfaces. Using a combination of fluorescence-based techniques applied to several mutants of TlL with different tryptophan residues we have shown that TlL binds to phospholipid vesicles in different forms rendering different catalytic activities, and that the open lid conformation is achieved and stabilized by a combination of electrostatic and hydrophobic interactions between the enzyme's lipid-binding face and the interface.

Fluorescence study on the interaction of a multiple antigenic peptide from hepatitis A virus with lipid vesicles.

The interaction of the multiple antigenic peptide MAP4VP3 with lipid membranes has been studied by spectroscopic techniques. MAP4VP3 is a multimeric peptide that corresponds to four units of the sequence 110-121 of the capsid protein VP3 of hepatitis A virus. In order to evaluate the electrostatic and hydrophobic components on the lipid-peptide interaction, small unilamelar vesicles of different compositions, including zwitterionic dipalmitoylphosphatidylcholine (DPPC), anionic dipalmitoylphosphatidylcholine/phatidylinositol (DPPC:PI 9:1), and cationic dipalmitoylphosphatidylcholine/stearylamine (DPPC:SA 9.5:0.5), were used as membrane models. Intrinsic tryptophan fluorescence changes and energy transfer experiments show that MAP4VP3 binds to all three types of vesicles with the same stoichiometry, indicating that the electrostatic component of the interaction is not important for binding of this anionic peptide. Steady-state polarization experiments with vesicles labeled with 1,6-diphenyl-1,3,5-hexatriene or with 1-anilino-8-naphtalene sulphonic acid indicate that MAP4VP3 induces a change in the packing of the bilayers, with a decrease in the fluidity of the lipids and an increase in the temperature of phase transition in all the vesicles. The percentage of lipid exposed to the bulk aqueous phase is around 60% in intact vesicles, and it does not change upon binding of MAP4VP3 to DPPC vesicles, indicating that the peptide does not alter the permeability of the membrane. An increase in the amount of lipid exposed to the aqueous phase in cationic vesicles indicates either lipid flip-flop or disruption of the vesicles. Binding to DPPC vesicles occurs without leakage of entrapped carboxyfluorescein, even at high mol fractions of peptide. However, a time-dependent leakage is seen with cationic DPPC/SA and anionic DPPC/PI vesicles, indicating that the peptide induces membrane destabilization and not lipid flip-flop. Resonance energy transfer experiments show that MAP4VP3 leakage from cationic vesicles is due to membrane fusion, whereas leakage from anionic vesicles is not accompanied by lipid mixing. Results show that MAP4VP3 interacts strongly with the lipid components of the membrane, and although binding is not of electrostatic nature, the bound form of the peptide has different activity depending on the membrane net charge; thus, it is membrane disruptive in cationic and anionic vesicles, whereas no destabilizing effect is seen in DPPC vesicles.

Cationic peptide antimicrobials induce selective transcription of micF and osmY in Escherichia coli.

Cationic antimicrobial peptides, such as polymyxin and cecropin, activated transcription of osmY and micF in growing Escherichia coli independently of each other. The micF response required the presence of a functional rob gene. It is intriguing that in this and other assays an identical response profile was also seen with hyperosmotic salt or sucrose gradient, two of the most commonly used traditional food preservatives. The osmY and micF transcription was not induced by hypoosmotic gradient, ionophoric peptides, uncouplers, or with other classes of membrane perturbing agents. The antibacterial peptides did not promote transcription of genes that respond to macromolecular or oxidative damage, fatty acid biosynthesis, heat shock, or depletion of proton or ion gradients. These and other results show that the antibacterial cationic peptides induce stasis in the early growth phase, and the transcriptional efficacy of antibacterial peptides correlates with their minimum inhibitory concentration, and also with their ability to mediate direct exchange of phospholipids between vesicles. The significance of these results is developed as the hypothesis that the cationic peptide antimicrobials stress growth of Gram-negative organisms by making contacts between the two phospholipid interfaces in the periplasmic space and prevent the hyperosmotic wrinkling of the cytoplasmic membrane. Broader significance of these results, and of the hypothesis that the peptide mediated contacts between the periplasmic phospholipid interfaces are the primary triggers, is discussed in relation to antibacterial resistance.

Interfacial control of lid opening in Thermomyces lanuginosa lipase.

Small unilamelar vesicles of anionic phospholipids (SUV), such as 1-palmitoyl-2-oleoylglycero-sn-3-phosphoglycerol (POPG), provide an interface where Thermomyces lanuginosa triglyceride lipase (TlL) binds and adopts a catalytically active conformation for the hydrolysis of substrate partitioned in the interface, such as tributyrin or p-nitrophenylbutyrate, with an increase in catalytic rate of more than 100-fold for the same concentration of substrate [Berg et al. (1998) Biochemistry 37, 6615-6627.]. This interfacial activation is not seen with large unilamelar vesicles (LUV) of the same composition, or with vesicles of zwitterionic phospholipids such as 1-palmitoyl-2-oleoylglycero-sn-3-phosphocholine (POPC), independently of the vesicle size. Tryptophan fluorescence experiments show that lipase binds to all those types of vesicles with similar affinity, but it adopts different forms that can be correlated with the enzyme catalytic activity. The spectral change on binding to anionic SUV corresponds to the catalytically active, or "open" form of the enzyme, and it is not modified in the presence of substrate partitioned in the vesicles, as demonstrated with inactive mutants. This indicates that the displacement of the lid characteristic of lipase interfacial activation is induced by the anionic phospholipid interface without blocking the accessibility of the active site to the substrate. Experiments with a mutant containing only Trp89 in the lid show that most of the spectral changes on binding to POPG-SUVs take place in the lid region that covers the active site; an increase in Trp anisotropy indicates that the lid becomes less flexible in the active form, and quenching experiments show that it is significantly buried from the aqueous phase. On the other hand, results with a mutant where Trp89 is changed to Leu show that the environment of the structural tryptophans in positions 117, 221, and 260 is somehow altered on binding, although their mobility and solvent accessibility remains the same as in the inactive form in solution. The form of TlL bound to POPC-SUV or -LUV vesicles as well as to LUV vesicles of POPG has the same spectral signatures and corresponds to an inactive or "closed" form of the enzyme. In these interfaces, the lid is highly flexible, and Trp89 remains accessible to solvent. Resonance energy transfer experiments show that the orientation of TlL in the interface is different in the active and inactive forms. A model of interaction consistent with these data and the available X-ray structures is proposed. This is a unique system where the composition and physical properties of the lipid interface control the enzyme activity.

Cecropins induce the hyperosmotic stress response in Escherichia coli.

Cecropin A and B, below or near their minimum inhibitory concentrations in viable Escherichia coli, interfered with the rapid NaCl-induced hyperosmotic shrinkage of the cytoplasmic volume (plasmolysis), and also activated the promoter of the hyperosmotic stress gene osmY. The same promoter was also expressed by hyperosmolar NaCl or sucrose, two of the most commonly used antimicrobial food preservatives. Stress responses were monitored during the logarithmic growth phase of E. coli strains that contain specific promoters fused to a luxCDABE operon on a plasmid. The luminescence assay, developed to monitor the transcriptional response to stresses, is based on the premise that organisms often respond and adapt to sublethal environmental adversities by increased expression of stress proteins to restore homeostasis. The luminescence response from these fusion strains to a specific stress occurs as the transcription at the promoter site is activated. Cecropins induced luminescence response only from the osmY-luxCDABE fusion, but not the corresponding stress promoter activation associated with macromolecular or oxidative damage, or leakage of the cytoplasmic content including the proton gradient. The inhibitory effect of cecropins on plasmolysis is interpreted to suggest that the primary locus of action of these antimicrobial peptides in the periplasmic space is on the coupling between the inner and outer membrane.

Calcium-triggered selective intermembrane exchange of phospholipids by the lung surfactant protein SP-A.

It is shown that human lung surfactant protein (SP-A) mediates selective exchange of phospholipid probes with unlabeled phospholipid in excess vesicles in the presence of calcium and NaCl. The exchange occurs without leakage of vesicle contents, or transbilayer movement (flip-flop) of the phospholipid probes, or fusion of vesicles. Individual steps preceding the exchange are dissected by a combination of protocols, and the results are operationally interpreted in terms of a model where a calcium-dependent change in SP-A triggers aggregation of vesicles followed by probe exchange between the vesicles in contact through SP-A. The contacts remain stable in the presence of calcium; i.e., the vesicles in contact do not change their partners on the time scale of several minutes. The binding of SP-A to vesicles and the aggregation of vesicles are rapid, and the aggregation is rapidly reversed by EGTA; i.e., both the forward and reverse aggregation reactions are complete in about 1 min. The exchange rate of the various probes between aggregated vesicles below 1 mM calcium in the presence of NaCl shows selectivity, i.e., a modest dependence on the net anionic charge on vesicles and for the headgroup of the probe. Exchange with lower selectivity is seen at >2 mM Ca in the absence of NaCl. SP-A binding to vesicles does not show an absolute specificity for the phospholipid structure, but the time course of the subsequent changes does. The results suggest that SP-A contacts between phospholipid interfaces could mediate the exchange of phospholipid species (trafficking and sorting) between lung surfactant pools in the hypophase and all accessible phospholipid interfaces of the alveolar space.

Osmotic stress in viable Escherichia coli as the basis for the antibiotic response by polymyxin B.

Cationic antimicrobial peptides, such as polymyxin B (PxB), below growth inhibitory concentration induce expression of osmY gene in viable E. coli without leakage of solutes and protons. osmY expression is also a locus of hyperosmotic stress response induced by common food preservatives, such as hypertonic NaCl or sucrose. High selectivity of PxB against Gram-negative organisms and the basis for the hyperosmotic stress response at sublethal PxB concentrations is attributed to PxB-induced mixing of anionic phospholipid between the outer layer of the cytoplasmic membrane with phospholipids in the inner layer of the outer membrane. This explanation is supported by PxB-mediated rapid and direct exchange of anionic phospholipid between vesicles. This mechanism is consistent with the observation that genetically stable resistance against PxB could not be induced by mutagenesis.

Interfacial activation of triglyceride lipase from Thermomyces (Humicola) lanuginosa: kinetic parameters and a basis for control of the lid.

A strategy is developed to analyze steady-state kinetics for the hydrolysis of a soluble substrate partitioned into the interface by an enzyme at the interface. The feasibility of this approach to obtain interfacial primary kinetic and equilibrium parameters is demonstrated for a triglyceride lipase. Analysis for phospholipase A2 catalyzed hydrolysis of rapidly exchanging micellar (Berg et al. (1997) Biochemistry 36, 14512-14530) and nonexchangeable vesicular (Berg et al., (1991) Biochemistry 30, 7283-7291) phospholipids is extended to include the case of a substrate that does not form the interface. The triglyceride lipase (tlTGL) from Thermomyces (formerly Humicola) lanuginosa hydrolyzes p-nitrophenylbutyrate or tributyrin partitioned in the interface of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) vesicles at a rate that is more than 100-fold higher than that for the monodispersed substrate or for the substrate partitioned into zwitterionic vesicles. Catalysis and activation is not seen with the S146A mutant without the catalytic serine-146; however, it binds to the POPG interface with the same affinity as the WT. Thus POPG acts as a diluent surface to which the lipase binds in an active, or "open", form for the catalytic turnover; however, the diluent molecules have poor affinity for the active site. Analysis of the substrate and the diluent concentration dependence of the rate of hydrolysis provides a basis for the determination of the primary interfacial catalytic parameters. As a competitive substrate, tributyrin provided a check for the apparent affinity parameters. Nonidealities from the fractional difference in the molecular areas in interfaces are expressed as the area correction factor and can be interpreted as a first-order approximation for the interfacial activity coefficient. The basis for the interfacial activation of tlTGL on anionic interface is attributed to cationic R81, R84, and K98 in the "hinge" around the 86-93 "lid" segment of tlTGL.

pH-induced destabilization of lipid bilayers by a peptide from the VP3 protein of the capsid of hepatitis A virus.

The membrane destabilizing and fusogenic properties of the synthetic peptide VP3(110-121), corresponding to an immunogenic sequence of the hepatitis A virus (HAV) VP3 capsid protein, were studied. By tryptophan fluorescence and acryalmide quenching it was demonstrated that the peptide binds liposomes of POPC-SM-DPPE (47 + 39 + 14) and POPC-SM-DPPE-DOTAP (40 + 33 + 12 + 15) and penetrates the membrane, at both neutral and acidic pH (POPC = 1-palmitoyl-2-oleoylglycero-sn-3-phosphocholine; SM = sphingomyelin; DPPE = 1,2-dipalmitoylphosphatidylethanolamine; DOTAP = 1,2-dioleoyl-3-trimethylammoniumpropane). VP3(110-121) did not have membrane-destabilizing properties at neutral pH. Acid-induced destabilization of the vesicles was demonstrated by fluorescence techniques and dynamic light scattering. VP3(110-121) induced aggregation of POPC-SM-DPPE-DOTAP (40 + 33 + 12 + 15) vesicles, lipid mixing and leakage of vesicle contents, all consistent with fusion of vesicles. In POPC-SM-DPPE (47 + 39 + 14) vesicles, at acidic pH, VP3(110-121) induced membrane destabilization with leakage of contents but without aggregation of vesicles or lipid mixing. The peptide only showed fusogenic properties when bound to the vesicles at neutral pH before acidification to pH below 6.0, and no effect was seen if the peptide was added to vesicles already set at acidic pH. These results may have physiological significance in the mechanism of infection of host hepatic cells by HAV.

Use of an imperfect neutral diluent and outer vesicle layer scooting mode hydrolysis to analyze the interfacial kinetics, inhibition, and substrate preferences of bee venom phospholipase A2.

Interfacial catalytic constants for bee venom phospholipase A2 (bvPLA2) have been obtained for its action on vesicles of the anionic phospholipid 1,2-dimyristoylphosphatidylmethanol (DMPM) in the highly processive scooting mode. Spectroscopic measurements which directly measure transbilayer movement of membrane components show that this exchange does not occur in anionic vesicles that have undergone complete bvPLA2-catalyzed hydrolysis of all phospholipids in the outer vesicle monolayer. 3-Hexadecyl-sn-glycero-1-phosphocholine (D-LPC) is an adequate neutral diluent for bvPLA2, which is defined as an amphiphile that forms an aggregate to which enzyme binds but neutral diluent molecules bind weakly in the enzyme's active site. D-LPC has weak affinity for the active site of bvPLA2, and theory and protocols are developed that allow its use to determine equilibrium dissociation constants for competing active site ligands. Some of the properties of bvPLA2 are shared by other 14 kDa PLA2s. (1) Ca2+ is required for binding of ligands to the active site but not for the binding of enzyme to the interface. (2) bvPLA2 does not significantly discriminate between phospholipids with different polar head groups or acyl chains. (3) bvPLA2 does not bind to phosphatidylcholine vesicles, and binding occurs if anionic amphiphiles are present in the vesicle. Novel features of bvPLA2 include the following: (1) Neutral diluents for other 14 kDa phospholipases A2 are not neutral diluents for bvPLA2. (2) Saturation of the active site with a variety of different ligands does not completely prevent histidine alkylation by 2-bromo-4'-nitroacetophenone, and Ca2+ binding does not change the rate of histidine alkylation. Finally, the carbohydrate portion of bvPLA2 does not alter the interfacial catalytic properties of the enzyme. Kinetic analysis of bvPLA2 in the scooting mode together with previous studies with other 14 kDa PLA2s provides a paradigm for the quantitative analysis of interfacial catalysis.

Synergism between mellitin and phospholipase A2 from bee venom: apparent activation by intervesicle exchange of phospholipids.

Mellitin, a cationic amphiphilic peptide, has an apparent activating effect on interfacial catalysis by phospholipase A2 (PLA2) of bee venom on zwitterionic vesicles of 1-palmitoyl-2-oleoylglycero-sn-3-phosphocholine (POPC) and on anionic vesicles of 1,2-dimyristoylglycero-sn-3-phosphomethanol (DMPM), as well as on covesicles of POPC/DMPM (3:7). On the other hand, mellitin-induced increase in the rate of pig pancreatic PLA2 is seen only on anionic vesicles. Interfacial kinetic protocols and spectroscopic methods show that the activation is due to enhanced substrate replenishment resulting from intervesicle exchange of zwitterionic or anionic phospholipids through vesicle-vesicle contacts established by mellitin. It is shown that as the hydrolysis on POPC vesicles progresses, due to a high propensity of bee PLA2 for binding to the product containing zwitterionic vesicles, most of the enzyme in the reaction mixture is trapped on few vesicles that are initially hydrolyzed, and thus reaction ceases. Under these conditions, mellitin promotes substrate replenishment by direct exchange of the products of hydrolysis from the enzyme-containing vesicles with the substrate present in excess vesicles which have not been hydrolyzed. Pig PLA2 has poor affinity for POPC vesicles, and the affinity is only modestly higher in the presence of low mole fractions of the products of hydrolysis; therefore, the enzyme is not trapped on those vesicles. Biophysical studies confirm that the phospholipid exchange occurs through stable intervesicle contacts formed by low mole fractions of mellitin, without transbilayer movement of phospholipids or fusion of vesicles. At high mole fraction (> 1.5%) mellitin induces leakage in POPC vesicles and does not form additional contacts. In POPC/DMPM vesicles, the contacts are formed even at high mole fractions of mellitin. Changes in intrinsic tryptophan fluorescence of mellitin indicate that bound mellitin exists in at least two different functional forms depending on the lipid composition and on the lipid:peptide ratio. A model is proposed to accommodate amphiphilic mellitin as a transmembrane channel or an intervesicle contact.

Salt-triggered intermembrane exchange of phospholipids and hemifusion by myelin basic protein.

Intervesicle phospholipid exchange through molecular contacts induced by the C1 molecular species of myelin basic protein (MBP) are characterized by using methods that amplify the effect of MBP-membrane interaction. The effect of salt concentration (KCl) on the vesicle-vesicle interaction of anionic sonicated covesicles of 30% 1-palmitoyl-2-oleoylglycero-sn-3-phosphocholine and 70% 1,2-dimyristoylglycero-sn-3-phosphomethanol (POPC/DMPM) by MBP is dissected by a combination of protocols into individual steps: aggregation of vesicles, apposition and contact formation, and hemifusion. Scattering and resonance energy transfer measurements reveal that, in the absence of KCl, MBP promotes rapid aggregation of the vesicles without lipid mixing. At >40 mM KCl, the extent of aggregation is larger and time-dependent. Fluorescence dequenching due to dilution of labeled phospholipids indicates that on a somewhat slower time scale, hemifusion of vesicles is triggered by salt, with mixing of the outer monolayer lipids but without flip-flop of phospholipids and without mixing or leakage of the aqueous contents. The exchange and hemifusion are seen with anionic vesicles; the effect of the structure of phospholipid, composition of vesicles, and the protein/lipid ratio is primarily on the kinetics of these and other competing processes. Thus, at 0.022 mol % of MBP and less than 100 mM KCl, it is possible to uncouple three sequential steps: (1) aggregation of vesicles by MBP; (2) apposition of bilayers and selective lipid exchange through vesicle-vesicle contacts established by MBP, i.e., anionic and zwitterionic phospholipids exchange, but cationic probes are excluded; and (3) hemifusion and lipid mixing of contacting monolayers of vesicles.

A fluorescence and CD study on the interaction of synthetic lipophilic hepatitis B virus preS(120-145) peptide analogues with phospholipid vesicles.

The interaction of the immunogenic peptide of human hepatitis B virus (HBV) preS(120-145), including B and T epitopes, with phospholipid vesicles has been studied by fluorescence techniques and CD. In addition, interaction of three lipopeptides derived from preS(120-145) containing stearoyl, cholanoyl, and tripalmitoyl-S-glyceryl-cysteine (Pam3C) SS moieties with dipalmitoylphosphatidylcholine (DPPC) has been investigated by polarization fluorescence spectroscopy. Fluorescence experiments showed an increase in fluorescence intensity and a blue shift of the maximum emission wavelength upon interaction of preS(120-145) with DPPC vesicles below the transition temperature (Tc), indicating that the tryptophan moiety enters a more hydrophobic environment. Moreover, fluorescence polarization experiments showed that the peptide decreased the membrane fluidity at the hydrophobic core, increasing the Tc of the lipid and decreasing the amplitude of the change of fluorescence polarization associated with the cooperative melting of 1,6-diphenyl-1,3,5-hexatriene labeled vesicles. The absence of leakage of vesicle-entrapped carboxyfluorescein indicates that the peptide did not promote vesicle lysis. Besides, the three lipopeptides derived from preS(120-145) showed a more pronounced rigidifying effect at the hydrophobic core of the bilayer, with a significative increase in the Tc. Stearoyl- and cholanoyl-preS(120-145) restricted the motion of lipids also at the polar surface, whereas Pam3CSS-preS(120-145) did not alter the polar head group order. Finally, CD studies in 2,2,2-trifluoroethanol or in presence of vesicles suggested that the bound peptide adopted amphiphilic alpha-helical and beta-sheet structures, with an important contribution of the beta-turn. It is concluded that preS(120-145) can interact with the lipid membrane through the formation of an amphipathic structure combination of beta-sheet and alpha-helix aligned parallel to the membrane surface, involving the N-terminal residues, and penetrating only a short distance into the hydrophobic core. The C-terminal part, with a combination of beta-turn and beta-sheet structure, remains at the outer part of the bilayer, being potentially accessible to immunocompetent cells. Furthermore, coupling of an hydrophobic moiety to the N-terminal part of the peptide favors anchoring to the membrane, probably facilitating interaction of the peptide with the immunoglobulin receptor. These results are in agreement with the induction of immune response by preS(120-145) and with the enhanced immunogenicity found in general for lipid-conjugated immunopeptides.

Specificity for the exchange of phospholipids through polymyxin B mediated intermembrane molecular contacts.

Structural specificity for the direct vesicle-vesicle exchange of phospholipids through stable molecular contacts formed by the antibiotic polymyxin B (PxB) is characterized by kinetic and spectroscopic methods. As shown elsewhere [Cajal, Y., Rogers, J., Berg, O.G., & Jain, M.K. (1996) Biochemistry 35, 299-308], intermembrane molecular contacts between anionic vesicles are formed by a small number of PxB molecules, which suggests that a stoichiometric complex may be responsible for the exchange of phospholipids. Larger clusters containing several vesicles are formed where each vesicle can make multiple contacts if sterically allowed. In this paper we show that the overall process can be dissected into three functional steps: binding of PxB to vesicles, formation of stable vesicle-vesicle contacts, and exchange of phospholipids. Polycationic PxB binds to anionic vesicles. Formation of molecular contacts and exchange of monoanionic phospholipids through PxB contacts does not depend on the chain length of the phospholipid. Only monoanionic phospholipids (with methanol, serine, glycol, butanol, or phosphatidylglycerol as the second phosphodiester substituent in the head group) exchange through these contacts, whereas dianionic phosphatidic acid does not. Selectivity for the exchange was also determined with covesicles of phosphatidylmethanol and other phospholipids. PxB does not bind to vesicles of zwitterionic phosphatidylcholine, and its exchange of covesicles is not mediated by PxB. Vesicles of dianionic phospholipids, like phosphatidic acid, bind PxB; however, this phospholipid does not exchange. The structural features of the contacts are characterized by the spectroscopic and chemical properties of PxB at the interface. PxB in intermembrane contacts is readily accessible from the aqueous phase to quenchers and reagents that modify amino groups. Results show that PxB at the interface can exist in two forms depending on the lipid/PxB ratio. Additional studies show that the stable PxB-mediated vesicle-vesicle contacts may be structurally and functionally distinct from "stalks", the putative transient intermediate for membrane fusion. The phenomenon of selective exchange of phospholipids through peptide-mediated contacts could serve as a prototype for intermembrane targeting and sorting of phospholipids during their biosynthesis trafficking in different compartments of a cell. The protocols and results described here also extend the syllogistic foundation in interfacial equilibria and catalysis.

Intermembrane molecular contacts by polymyxin B mediate exchange of phospholipids.

Direct intermembrane exchange of dimyristoylphosphatidylmethanol is mediated by polymyxin B (PxB), a cationic amphipathic cyclic decapeptide. The possibility that the phospholipid exchange is mediated by solubilization of phospholipids or by fusion of vesicles is ruled out. By kinetic and spectroscopic methods it is shown that the exchange occurs directly through vesicle-vesicle contacts formed by a few PxB molecules. The contact is stable on the time scale of several minutes such that neither PxB nor the vesicles in the pair forming a contact exchange with excess vesicles. Several contacts may be formed on a vesicle, which leads to the formation of a cluster of vesicles, and the lipid molecules on the outer monolayers of vesicles exchange throughout the cluster. Kinetics of substrate replenishment during processive interfacial catalysis suggests that the exchange of anionic lipids over the contact occurs at a rate considerably faster than 300 s-1. The exchange through the contact is specific for certain lipids, and phospholipids with a modified head group or phospholipase A2 bound to a vesicle are not transferred to the other vesicle in contact. Since this phenomenon has not been described before, possible implications of direct vesicle-vesicle exchange of phospholipids through peptide-mediated molecular contacts are discussed. Such a mechanism for intermembrane transfer of phospholipids could be responsible for intracellular trafficking and sorting of phospholipids; it could be a necessary first step for the sequence of events leading to budding, vesiculation, and secretion; and PxB-mediated transfer between the inner and outer membranes of Gram-negative bacteria could also account for its antibiotic action.

Direct vesicle-vesicle exchange of phospholipids mediated by polymyxin B.

Direct and rapid intermembrane exchange of phospholipids without fusion is shown to occur across stable vesicle-vesicle contacts formed by stoichiometric amounts of polymyxin B. The exchange is selective for monoanionic, but not dianionic, glycerophospholipids irrespective of their chain length or phase properties. Selective transfer mediated by protein contacts between membranes could serve as a model for stable fusion intermediates and provide a structural and organizational basis for direct phospholipid transfer as implicated in entry of enveloped viruses, secretion, endocytosis, and intracellular transport and targeting of lipids. It is also proposed that the loss of the specificity of phospholipid composition in the inner and outer membranes of gram negative bacteria mediated by polymyxin B could be the basis for its bactericidal action.