Modulation of the Activity of Secretory Phospholipase A2 by Antimicrobial Peptides

The antimicrobial peptides magainin 2, indolicidin, and temporinsB and L were found to modulate the hydrolytic activity of secretoryphospholipase A₂ (sPLA₂) from bee venom and in human lacrimalfluid. More specifically, hydrolysis of phosphatidylcholine(PC) liposomes by bee venom sPLA₂ at 10 µM Ca²⁺ was attenuatedby these peptides while augmented product formation was observedin the presence of 5 mM Ca²⁺. The activity of sPLA₂ towardsanionic liposomes was significantly enhanced by the antimicrobialpeptides at low [Ca²⁺] and was further enhanced in the presenceof 5 mM Ca²⁺. Similarly, with 5 mM Ca²⁺ the hydrolysis of anionicliposomes was enhanced significantly by human lacrimal fluidsPLA₂, while that of PC liposomes was attenuated. These resultsindicate that concerted action of antimicrobial peptides andsPLA₂ could improve the efficiency of the innate response toinfections. Interestingly, inclusion of a cationic gemini surfactantin the vesicles showed an essentially similar pattern on sPLA₂activity, suggesting that the modulation of the enzyme activityby the antimicrobial peptides may involve also charge propertiesof the substrate surface.

INTRODUCTION

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Introduction

During the past 2 decades, living organisms of all types havebeen found to produce a large repertoire of gene-encoded antimicrobialpeptides that play an important role in innate immunity to microbialinvasion. Antimicrobial peptides can be synthesized at low metaboliccost and easily stored in large amounts and are readily availableshortly after an infection to rapidly neutralize a broad rangeof microbes. Magainins were discovered in the skin of the Africanclawed frog Xenopus laevis and show a broad spectrum of antimicrobial(40, 77, 78) and anticancer activities (4, 13) at nonhemolyticconcentrations. In a manner similar to that of magainins, indolicidinis active against both gram-negative and -positive bacteria(60) and fungi (1), as well as protozoa (2). Indolicidin iscytotoxic also to rat and human T lymphocytes (57), lyses redblood cells (1), and has activity against human immunodeficiencyvirus type 1 (55). Indolicidin is found in cytoplasmic granulesof bovine neutrophils (60) and has a unique amino acid compositionwith five Trp and three Pro residues in its 13-amino acid sequence.With a size similar to that of indolicidin, temporins are linear10- to 13-residue-long peptides isolated from the skin of theeuropean red frog, Rana temporaria (62). Temporin B has beenfound to be active against gram-positive bacteria and is nothemolytic (62). In contrast, temporin L is active against bothgram-positive and-negative bacteria, is hemolytic, and is toxicto cancer cells (54).

Antimicrobial peptides are considered to kill bacteria by permeabilizingand/or disrupting their membranes (39, 71, 83). The molecularbases for the activity and selectivity of these peptides havebeen intensively studied using model membranes (39, 70). Beingcationic, antimicrobial peptides interact preferentially withacidic lipids that are particularly abundant in bacteria (16,17, 51), thus providing a basis for differences in cell specificity.The interaction of antimicrobial peptides with bilayers altersthe organization of the membrane and makes them permeable toions, for instance (32, 69), causing membrane depolarization(42). Various models accounting for the peptide-induced membranepermeation process have been proposed (7, 61). It has been reportedthat not only the nature of the peptide but also the characteristicsof the cell membrane as well as the metabolic state of the targetcells determine the mechanism of action of antimicrobial peptides(37). However, the action of cationic antimicrobial peptidesis not limited to direct killing of microorganisms. Accordingly,they exhibit an impressive variety of additional activities,having an impact particularly on the quality and effectivenessof innate responses and inflammation (25). Stimulation of hostdefense mechanisms by antimicrobial peptides has been demonstrated(3, 74), and receptor-mediated signaling by some peptides havealso been reported (26, 75). Some of the antimicrobial peptidescould act synergistically with other host antimicrobial moleculesto kill microbes (11), and positive cooperativity has been reportedbetween peptides and lysozyme, as well as between differentantimicrobial peptides (25, 43). It is also becoming clear thatthere exists a certain degree of coupling between the innateand adaptive immune systems and that antimicrobial peptidesinfluence both the quality and effectiveness of immune and inflammatoryresponses.

Phospholipases A₂ (PLA₂s) are ubiquitous enzymes, which catalyzethe hydrolysis of the sn-2 ester bond of phospholipids to releasefree fatty acids and lysophospholipids (for a review, see reference15). PLA₂s play a key role in various biological processes,including homeostasis of cellular membranes, lipid digestion,host defense, signal transduction, and production of lipid mediatorssuch as eicosanoids and lysophospholipid derivatives, whichexhibit diverse and potent biological actions (15, 44). Interestingly,it has been shown that secretory PLA₂ (sPLA₂) has antibacterialactivity and represents an important host defense molecule (9,50, 53). Mammalian group IIA sPLA₂s have been found at highlevels in inflammatory conditions and have the ability to killgram-positive bacteria (9, 24). High concentrations of theseenzymes showed some bactericidal effect also against gram-negativebacteria. Likewise, they are able to act as acute-phase proteins,in concert with other antibacterial proteins (9). Belongingto group IIA sPLA₂s, the lacrimal fluid sPLA₂ has been identifiedas the principal mediator of antistaphylococcal activity (53).Similarly to group II PLA₂, mammalian group V PLA₂ has beenfound to be bactericidal against gram-positive bacteria (24),suggesting potential as therapeutic agents against bacterialinfections. The mechanisms of regulation of PLA₂ activity havebeen subjected to intense research, and the control of phospholipidderivatives produced by its action has long been consideredin the treatment of related diseases (18, 76). The discoveryof specific inhibitory peptides has given some insight intothe regulation of the activity of intracellular PLA₂s (68).

Mellitin, a 26-amino-acid peptide present in bee venom, hasbeen previously found to enhance the activity of sPLA₂s (11).Yet, mellitin is also profoundly hemolytic (7, 11), which wouldeventually limit its possible application as an antimicrobialagent. In order to study if the activation of sPLA₂ is a moregeneral property of antimicrobial peptides and if the hemolyticactivity is independent from this effect, we compared the influenceof magainin 2, temporins B and L, and indolicidin on the activityof sPLA₂s of bee venom and human lacrimal fluid. The two formerpeptides are nonhemolytic in contrast to indolicidin and temporinL (1, 54, 62, 77, 78). Our results reveal significant impactof these peptides on the hydrolytic activity of sPLA₂.

MATERIALS AND METHODS

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Materials and Methods

Materials. Bee venom PLA₂ was purchased from Sigma Chemical Co. (St. Louis,Mo.). 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG)was from Avanti Polar Lipids (Alabaster, Ala.), and the fluorescentphospholipid analogs 1-palmitoyl-2-[6-(pyren-1-yl)]hexanoyl-sn-glycero-3-phosphocholine(PPHPC) and 1-palmitoyl-2-[6-(pyren-1-yl)]hexanoyl-sn-glycero-3-phosphomonomethylester(PPHPM) from K&V Bioware (Espoo, Finland). Concentrationsof lipids were determined gravimetrically with a high-precisionelectrobalance (Cahn, Cerritos, Calif.), and those of the fluorescentlipids were determined spectrophotometrically by using 42,000cm^-1 at 342 nm as the molar extinction coefficient for pyrene.The gemini surfactant (2S,3R)-2,3-dimethoxy-1,4-bis(N-hexadecyl-N,N-dimethylammonnium)butane dibromide (SR-1) was synthesized as described previously(12), and its purity was verified by nuclear magnetic resonance.This compound was kindly provided by S. Borocci and G. Mancini(University of Rome, Rome, Italy). The purity of other lipidswas checked by thin-layer chromatography on silicic acid-coatedplates (Merck, Darmstadt, Germany) developed with chloroform/methanol/water(65:25:4 [vol/vol/vol]). Examination of the plates after iodinestaining and, when appropriate, upon UV illumination revealedno impurities. Magainin 2 was from Sigma, indolicidin from Bachem(Bubendorf, Switzerland), temporin L from Synpep (Dublin, Calif.),and temporin B from Tana Laboratories (Houston, Tex.). The puritiesof the above synthetic peptides (>99, >95, >94, and>90% for magainin 2, indolicidin, and temporins L and B,respectively) were assessed by high-performance liquid chromatography,and their sequences were verified by both automated Edman degradationand mass spectrometry. Peptide concentrations were determinedgravimetrically and by quantitative ion-exchange column chromatographyand ninhydrin derivatization.

Collection of lacrimal fluid. Tears were collected from healthy donors briefly exposed tothe vapors of freshly minced onions. The collected fluid wasstored at -20°C until used.

Assay for PLA₂. PLA₂ activity was determined by a kinetic assay described previously(45, 46, 65-67). Appropriate amounts of the lipid stock solutionswere mixed in chloroform to obtain the desired compositionsand then dried under a stream of nitrogen followed by high vacuumfor a minimum of 2 h. The lipid residues were subsequently dissolvedin ethanol to yield a lipid concentration of 50 µM, andsmall unilamellar liposomes were formed by rapidly injectingthis lipid ethanolic solution into the buffer (5). The lipidconcentration was 1.25 µM for both PPHPC and PPHPM/POPG(5:5 [molar ratio]) liposomes in a total volume of 2 ml. Allfluorescence measurements were performed in magnetically stirredquartz cuvettes (with 1-cm path length) at 37°C. After 15min of equilibration the reactions were initiated by adding50 ng of bee venom sPLA₂ or 0.5 µl of human lacrimal fluid.The progress of phospholipid hydrolysis was followed by measuringthe pyrene monomer intensity at 400 nm as a function of time.Fluorescence intensities were measured with a Perkin-Elmer LS50B spectrometer with an excitation wavelength of 344 nm andboth emission and excitation band-passes set at 4 nm. The assaywas calibrated by adding known picomolar aliquots of (pyren-1-yl)hexanoateinto the reaction mixture in the absence of enzyme while detectingpyrene monomer emission intensity. The activity of the enzymewas calculated from the initial velocity of the reaction kineticcurves and converted to the amount of product released per timeunit. The assays were carried out both with and without added5 mM Ca²⁺. Under the former conditions approximately 10 µMresidual Ca²⁺ was present, as determined by titration with EDTA.

RESULTS

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Results

Effects of antimicrobial peptides on the activity of bee venom sPLA_2. Increasing the concentrations of the antimicrobial peptidesmagainin 2, indolicidin, and temporins B and L progressivelyattenuated the hydrolysis of zwitterionic PPHPC liposomes bythis enzyme at a low [Ca²⁺] (Fig. 1A). Indolicidin was mosteffective, and the hydrolytic activity of the enzyme decreasedby approximately 86% at 0.4 µM peptide. Inhibition ofthe activity of sPLA₂ by the antimicrobial peptides decreasedin the sequence indolicidin > temporin L ${approx}$ magainin 2 >temporin B. Interestingly, in the presence of 5 mM Ca²⁺, theabove peptides had the opposite effect and the hydrolysis ofPPHPC liposomes was enhanced (Fig. 1B), the activation beingaugmented with increasing concentrations of the peptides. Theefficiency of equimolar concentrations of these antimicrobialpeptides to activate bee venom sPLA₂s increased in the sequencemagainin < temporin B < indolicidin < temporin L.

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FIG. 1. Effects of magainin 2 ( ${blacksquare}$ ), indolicidin (•), and temporins L ( ${blacktriangleup}$ ) and B ( ${blacktriangledown}$ ) on the hydrolysis of PPHPC liposomes by bee venom sPLA₂ without added Ca²⁺ (A) and in the presence of 5 mM Ca²⁺ (B). Final lipid concentration is 1.25 µM in 2 ml of 50 mM Tris-HCl, 150 mM NaCl, pH 7.4. The reactions were started by adding 50 ng of PLA₂ into the cuvette, while temperature was maintained at 37°C with a circulating water bath. Each data point represents the mean of triplicate measurements. The standard deviation is less than 1 nmol of fatty acid/min and for the sake of clarity is not shown.

The outer leaflet of mammalian plasma membranes is exclusivelycomposed of zwitterionic phospholipids, whereas bacterial membranescontain large amounts of negatively charged phospholipids (16,17, 51). Accordingly, we studied the effects of these peptideson the hydrolysis of anionic liposomes by bee venom sPLA₂. Inbehavior similar to that of other sPLA₂s (66), this enzyme hasbeen shown to hydrolyze the anionic liposomes more rapidly thanneutral vesicles, with Ca²⁺ activating the enzyme (Fig. 1 and2; see references 8 and 72). At 10 µM Ca²⁺, all four peptidesactivated the enzyme, this effect being augmented in the sequenceindolicidin < temporin B < magainin 2 ${approx}$ temporin L (Fig.2A). These antimicrobial peptides activated bee venom sPLA₂also in the presence of 5 mM Ca²⁺ with essentially no differencesbetween the four peptides (Fig. 2B). Compared to that of zwitterionicvesicles, the activation by the antimicrobial peptides of sPLA₂action on anionic liposomes with 5 mM Ca²⁺ was more pronounced(Fig. 1B and 2B), being maximally ${approx}$ 400 and 136%, respectively.

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FIG. 2. Effects of magainin 2 ( ${blacksquare}$ ), indolicidin (•), and temporins L ( ${blacktriangleup}$ ) and B ( ${blacktriangledown}$ ) on the hydrolysis of PPHPM/POPG (50:50, molar ratio) liposomes by bee venom sPLA₂ without added Ca²⁺ (A) and in the presence of 5 mM Ca²⁺ (B). Otherwise, conditions were as described in the legend for Fig. 1.

In order to exclude the possibility that trace amounts of Ca²⁺may be present in the peptides used, which could enhance PLA₂activity, we measured enzyme activity in the presence of increasingconcentrations of EDTA. Both with and without peptides, EDTAabolished sPLA₂ activity at 10 µM (Fig. 3), thus suggestingthe antimicrobial peptides to be essentially free of Ca²⁺.

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FIG. 3. Inhibition of EDTA on the hydrolysis of PPHPM/POPG (50:50 [molar ratio]) liposomes by bee venom sPLA₂ without antimicrobial peptides ( ${blacksquare}$ ) and in the presence of 0.2 µM magainin 2 (•), indolicidin ( ${blacktriangleup}$ ), and temporins L ( ${blacktriangledown}$ ) and B ( ${diamondsuit}$ ). Otherwise, conditions were as described in the legend for Fig. 1.

Effects of antimicrobial peptides on the activity of sPLA₂ from human tears. Human sPLA₂ is present in many tissues and secretions, includingrheumatoid synovial fluid (59), platelets (33), Paneth cells(49), neutrophils (56), and lacrimal glands (48). High concentrationsof group II sPLA₂ are present in human lacrimal fluid, an importanthost defense mechanism in eyes that kills gram-positive bacteria(53). This sPLA₂ requires Ca²⁺ as an essential active site cofactorand, in keeping with the catalytic activity of this enzyme beingimportant to its antimicrobial activity, also the latter isCa²⁺ dependent (53). As reported previously (6) human lacrimalfluid sPLA₂ has a marked preference for anionic phospholipids,hydrolyzing anionic vesicles more rapidly than neutral vesicles(Fig. 3). The hydrolysis of neutral liposomes by lacrimal fluidsPLA₂ was inhibited by all four antimicrobial peptides, withtemporin L being most effective (Fig. 4). Similar to the activityof bee venom sPLA₂, the activity of lacrimal fluid enzyme towardsanionic liposomes was significantly enhanced by the antimicrobialpeptides (Fig. 4). The enzyme activity was significantly increasedby magainin 2 and temporins B and L, and there were essentiallyno differences between these three peptides, while indolicidinwas least effective.

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FIG. 4. Effects of 0.2 µM magainin 2, indolicidin, and temporins L and B on the hydrolysis of 1.25 µM PPHPC (crosshatched) and PPHPM/POPG (50:50, molar ratio, hatched) liposomes by human lacrimal fluid sPLA₂ in the presence of 5 mM Ca²⁺. The reactions were started by adding 0.5 µl of human lacrimal fluid into the cuvette. *, P < 0.05. Each data point represents mean plus or minus standard error of the mean (n = 5).

Effects of cationic gemini surfactant on the activity of sPLA₂. The above results readily showed that the positively chargedantimicrobial peptides have pronounced effects on the activityof sPLA₂. Interestingly, an essentially similar pattern wasevident for the impact of other membrane-partitioning agents(45-47, 65) on sPLA₂ activity. In order to elucidate if thepositive charges of these cationic perturbants play a role intheir effects on the activity of sPLA₂, we included a smallamount (5 mol%) of the cationic gemini surfactant SR-1 in thevesicles. Strikingly, this cationic surfactant showed qualitativelysimilar effects on sPLA₂ activity (Fig. 5), suggesting thatthe positive charges of these perturbants play an importantrole in their modulation of sPLA₂ activity.

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FIG. 5. Hydrolysis of 1.25 µM PPHPC and PPHPM/POPG (50:50, molar ratio) liposomes by bee venom (A) and human lacrimal fluid (B) sPLA_2. The vesicle compositions were PPHPC with 10 µM Ca²⁺ (a), PPHPC with 5 mM Ca²⁺ (b and e), PPHPM/POPG with 10 µM Ca²⁺ (c), and PPHPM/POPG with 5 mM Ca²⁺ (d and f), in the presence (5 mol%, crosshatched) and absence (hatched) of the cationic gemini surfactant SR-1. *, P < 0.05. Each data point represents mean plus or minus standard error of the mean (n = 5).

DISCUSSION

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Discussion

sPLA₂s constitute a large family of structurally related enzymes,which are found in numerous organisms, including mammalian tissues,venoms, and plants, and have been classified into 12 groups(I to XII) primarily based on sequence homology (14). GroupIII sPLA₂ was first identified in bee venom (34) and is oneof the most thoroughly studied of this group. This enzyme hasbeen cloned (34), and its tertiary structure revealed an overallscaffold similar to that of group I and group II sPLA₂ (58).Group IIA sPLA₂s are secreted by a variety of cells involvedin the inflammatory response. The presence of these enzymesin human macrophages, the Paneth cells of the intestine, andthe lacrimal glands is significant because all these cells havean established antibacterial function (9). Human lacrimal fluidsPLA₂ binds avidly to phosphatidylglycerol-rich membranes ofbacteria, and quantities as low as 1 ng per ml are sufficientto kill gram-positive bacteria (53). The activity of this sPLA₂against gram-negative bacteria is considerably enhanced in thepresence of agents that disrupt their lipopolysaccharide coat(73). It is apparent that, in order to hydrolyze the bacterialcell membrane, the enzyme must first penetrate the anionic peptidoglycancell wall. This is related to the cell wall structures characterizingthis bacterial species and is affected by factors such as growthstate and cell wall-degrading enzymes and substrates. Accordingly,the sensitivity of gram-positive bacteria to sPLA₂ varies greatlybetween species (19, 53). Agents disrupting the cell wall and/orfacilitating the access of PLA₂ to the membrane could be anticipatedto promote the bactericidal effect of sPLA₂.

Antimicrobial peptides show a broad range of activity againstgram-positive and-negative bacteria, fungi, mycobacteria, andsome enveloped viruses (79). With a net positive charge, antimicrobialpeptides are characterized by their preference for anionic interfacesthat confers relative specificity on the bacterial membranesas opposed to the zwitterionic membranes of host cells (35,39, 41, 52). The same charge characteristics are inherent tohuman group IIA sPLA₂s, which have a large number of cationicresidues on their substrate binding surface (63). Our data demonstratea pronounced influence of the antimicrobial peptides on theactivities of sPLA₂s, suggesting possible synergistic actionof these two antibacterial components, irrespective of the hemolyticactivity of some of the peptides. The effects of the four peptideson sPLA₂ activity depend on lipid composition in a manner showinga bacterial membrane to be susceptible to enhanced hydrolysis,while the host cell membrane would remain intact. Our resultsthus highlight the potential of concerted action between sPLA₂and antimicrobial peptides as part of the innate response toinfections, particularly when more antibiotic-resistant bacterialstrains are emerging.

A variety of membrane perturbants have been shown to affectthe adsorption and action of PLA₂ (e.g., references 11, 29,and 45). In keeping with this, our present results demonstratethe profound impact of the four antimicrobial peptides studiedon the sPLA₂ reaction. In distinction from enzymes acting onmonomeric soluble substrates, PLA₂s have access to their substrateonly within the membrane phase and are sensitive to the curvatureand as well as physicochemical characteristics of the phospholipidmembrane (10, 20, 21, 27, 36, 64). Also, "structural defects"in membranes have been proposed to affect PLA₂ activity (31),with subtle changes in the structure of phospholipids havinga profound influence on the affinity of the interface bindingsite of the enzyme to the substrate surface (30). Accordingly,defects in membranes caused by antimicrobial peptides couldenhance the penetration of sPLA₂ and the hydrolysis of vesicleswould proceed at a much higher rate with augmented catalyticturnover of the bound enzyme. For kinetic analysis two-dimensional"scooting" of sPLA₂ in the substrate surface has been suggested,with very slow exchange of PLA₂ or phospholipid between thephospholipid vesicles (21, 22, 30). The antimicrobial peptidescould modulate sPLA₂s activity by substrate replenishment throughpeptide-mediated vesicle-vesicle contacts, similarly to mellitin(11). The effects of the four antimicrobial peptides on thestructure of anionic membranes in particular are pronounced(38, 80, 81). As suggested for magainin 2 and indolicidin (80),the peptide would first bind to the outer leaflet of the bilayerand subsequently cosegregate in a cooperative manner with thebound acidic phospholipids into microdomains. Reorientationof peptides and formation of "channel"- or "pore"-like structurescould occur, and the bilayer structure could become locallydestabilized. Reorientation of temporin B (81) and two statesof temporin L in bilayers (82) was also suggested. These peptidesare likely to induce complex dynamic structures in the membraneswith a variety of substrate configurations. Different effectsof these peptides on membrane properties may thus influencesPLA₂ activity to different degrees (Fig. 1 to 3).

Due to the neutral charge and high curvature of PPHPC smallunilamellar liposomes, the four antimicrobial peptides studiedcan be expected to bind weakly to the zwitterionic membranesurface without inserting deeply into the hydrophobic core ofthe bilayers. At low [Ca²⁺], the coverage of the interface bythe peptides would decrease the area available for the bindingof sPLA₂, resulting in inhibition due to a smaller fractionof the enzyme being bound to the interface. Repulsion betweenCa²⁺ and membrane-bound, positively charged antimicrobial peptidescould also affect the binding of sPLA₂ to the membrane. However,in the presence of 5 mM Ca²⁺, the activity of bee venom sPLA₂towards PPHPC was enhanced by all four antimicrobial peptides.Ca²⁺ is essential for the activity of PLA₂s (8), and activationof bee venom PLA₂ by this ion could exceed the inhibition causedby the antimicrobial peptides with zwitterionic phosphatidylcholine(PC) as a substrate. Interestingly, hydrolysis of PC membraneby bee venom sPLA₂ in the presence of 5 mM Ca²⁺ was augmentedby the antimicrobial peptides, while under the same conditionsthe activity of lacrimal fluid sPLA₂ was attenuated. Comparedto that of the group III sPLA₂s, the interface binding surfaceof group II sPLA₂s contains a significantly larger number ofbasic amino acids and has a highly cationic character (23, 50).Accordingly, the effects of cationic antimicrobial peptideson the binding of these two enzymes to the substrate could differ,resulting in different effects on their impact on the catalyticactivities of bee venom and lacrimal fluid sPLA₂.

Interestingly, while magainin 2, indolicidin, and temporinsB and L differ in structural parameters, they had qualitativelyidentical effects on the hydrolysis of zwitterionic and anionicvesicles by sPLA₂ measured with and without added Ca²⁺. Intriguingly,comparison of zwitterionic and anionic vesicles as substratesfor sPLA₂ has revealed essentially similar pattern for the effectsof adriamycin (45), phorbol esters (46), and polyamines (65)on the activity of PLA₂. Modulation of sPLA₂ activity via similarchanges induced in the substrate by these peptides is thus suggested,implying a lack of a direct and specific effect on the enzyme.The enzyme action is thus likely to be affected via the physicalproperties of the substrate. Importantly, the cationic geminisurfactant SR-1 added to the vesicles also showed a similarpattern on sPLA₂ activity (Fig. 5), indicating that modulationof sPLA₂ reaction by the above cationic perturbants could involvealterations in the electric properties of the substrate surface.Subsequently, changes in the extent of the association of theenzyme to substrate could be involved, in keeping with the suggestionthat the initial binding of the enzyme to the interface is limitingand that conditions favoring its surface association enhancethe overall rate of hydrolysis (28). Possible variation in thebilayer curvature and presence of multilamellar vesicles wouldbe unlikely to influence the comparison of the measured effectsdue to progressively increasing concentrations of the addedantimicrobial peptides on the enzyme activity.

Due to the inherent complexity of the physicochemical characteristicsof the dynamic phospholipid/water interface harboring the siteof the catalytic action of PLA₂, it is difficult to distinguishat this stage between the various mechanisms outlined above.Furthermore, there is no reason to assume the above possibilitiesto be mutually exclusive. More thorough studies are thus warrantedto establish the molecular level mechanism(s) involved. Yet,taken the importance of the development of novel means to fightemerging antibiotic-resistant strains, such efforts are clearlyneeded.

ACKNOWLEDGMENTS

This study was supported by the Technology Development Fund(TEKES) and the Finnish Academy. Memphys is supported by theDanish National Research Council.

We thank S. Borocci and G. Mancini (University of Rome) forkindly providing the gemini surfactant SR-1. The skillful technicalassistance of Kaija Niva is appreciated.

FOOTNOTES

* Corresponding author. Mailing address: Helsinki Biophysics & Biomembrane Group, Institute of Biomedicine, P.O. Box 63 (Haartmaninkatu 8), FIN-00014 University of Helsinki, Finland. Phone: 358-9-19125400. Fax: 358-9-19125444. E-mail: Paavo.Kinnunen{at}Helsinki.Fi.

REFERENCES

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