PART111. INTERACTIONOF PROTEINS WITH LIPIDS
LIPID-LIPID AND LIPID-PROTEIN INTERACTION IN MODEL SYSTEMS AND MEMBRANES* L. L. M. van Deenen, J. de Gier, R. A. Demel, B. de Kruyff, M. C . Blok, E. C . M. van der Neut-Kok, C . W. M. Haest, P. H. J. Th. Ververgaert, and A. J. Verkleij Department of Biochemistry Univeuity of Utrecht Utrecht, The Netherlands
In 1925 Gorter and Grendel' proposed that in the erythrocyte membrane the lipid molecules are arranged in a bilayer. They spread the lipids extracted from a given quantity of red blood cells in a Langmuir trough at the air-water interface and observed that the area occupied by the lipids was twice the surface of the erythrocytes. It is not our intention to discuss the merits and pitfalls of this ingenuous approach. After 50 years of intensive research the concept of the presence of a lipid bilayer in membranes appears more generally accepted when compared with the situation some ten years ago. Indeed several membrane properties are understandable on the basis of a lipid bilayer structure with proteins both located at the surfaces and penetrating or spanning the hydrophobic lipid core. The present paper summarizes some studies from our laboratory on lipid mono- and bilayers (liposomes) in relation to biomembranes.
Action of Polyene Antibiotics on Membranes Polyene antibiotics can interact with both artificial lipid interfaces and biological membranes, and cause changes in permeability characteristics. It is well established that the presence of sterols in the membrane is a requirement for the action of these antibiotics,' which compounds have a macrolide ring structure with a varying number of double bonds and hyroxyl groups (FIGURE 1).
In a collaborative study with Dr. S. C . Kinsky it was demonstrated that the sterol requirement of polyene antibiotics is manifested in the simple system of lipid monolayers.'. ' Injection of these antibiotics underneath a lipid monolayer induces at low molar ratios of antibiotic/lipid a significant increase of surface pressure, but only when sterol is present in the lipid film above a given sterol/phospholipid ratio. Recent studies were aimed at understanding the nature of the molecular association between the antibiotics and sterols and at interpreting the permeability alterations occurring in the membrane. In this respect it is appropriate to give first some attention to studies on the interaction between phospholipids and various sterols in monolayers, liposomes, and natural membranes. It was found that only sterols having a 3P-OH group, a planar ring system, and a hydrophobic side chain at C-17 gave a reduction of the *This paper is dedicated to the memory of Dr. Y. London, who was killed at the Golan Heights in October 1973. 124
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FlLlPlN
+%--+OM
bM%OTERKIN 0
o w m
m m o a -
FIGURE 1. Structures proposed for a number of polyene antibiotics. molecular area of a monolayer of synthetic unsaturated lecithins." The same requirments for the sterol molecule were found in studies on liposomes which demonstrated the importance of these structural parameters to reduce the passive permeability of the lipid bilayers towards nonekctrolytes, such as erythritol, glycerol and glucose, and Rb'." That the observations on the artificial systems are relevant to biomembranes is supported by experiments that involved the replacement of the cholesterol in the erythrocyte membrane by various sterol analogs.' The structural requirement of sterol interaction with other lipid components of biological membranes was studied also after incorporation of various sterols in the cell membrane of Acholeplasma laidlawii.O*oThe effect of the sterol upon the permeability of this membrane to erythritol was found to follow to the same rules as have been established for liposomes and monolayers. The relations observed between model systems and natural membranes for lipid-sterol interaction stimulated similar studies on polyene antibiotic-sterol interaction. In monoIayers,10 liposomes,lo-l* and A. laidlawii membrane^^"^ it could be demonstrated that the sterol incorporated in the membrane required a planar ring system, a hydrophobic side chain, and a 3p-OH group for polyene action. It is intriguing that there is a clear relationship between the structural requirments involved in (phospho) -lipid-sterol interaction and polyene antibiotic-sterol interaction. The latter association apparently is stronger than the former one. This is demonstrated also by differential scanning calorimetry and spectroscopic studies,10. la which revealed that the presence of polyene antibiotic has withdrawn sterol from its interaction with other membrane lipids so as to form polyene antibiotic-sterol complexes of defined stoichiometry. This complex formation alters the permeability of both artificial and natural membranes in a similar manner. The various polyenes, with the exception "7
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of filipin, cause selective permeability changes, suggesting the formation of pores with a diameter of about 8 A, permitting nonelectrolytes of the size of or smaller than glucose and monovalent cations to pass. Models for these complexes have been proposed by various inve~tigators."~~' A space-filling model developed for the amphotericin B-cholesterol complex is visualized as a circular arrangement of eight amphotericin B and eight cholesterol molecules.'o A schematic representation (FIGURE2) shows that in such complexes, each forming
FIGURE 2. Schematic representation of conducting pores formed by amphotericin B and cholesterol in a lipid bilayer.''
a half pore, the inside is hydrophilic because of the presence of the hydroxyl groups of the polyene antibiotic. The cholesterol molecules fit in the ring system of the polyene antibiotic and are in close contact with the double bond system without having direct contact with the hydrophilic region(s) of the antibiotic. The hydroxyl group of cholesterol and the charged groups of amphotericin B are believed to be located at the membrane surfaces. Similar structures have been deduced for complexes formed by nystatin and etruscomycin." Furthermore, it was argued that the primaricin-cholesterol complex cannot form a conducting pore because the length of the two half-pores is limited when compared with the thickness of a lipid bilayer. Filipin is known to cause more drastic alterations in both artificial and natural membranes. The permeability changes (e.g., release of the enzyme glucose-6-phosphate dehydrogenase) suggest that significant structural alterations occur in the membrane by the formation of the filipin-cholesterol complex. Freeze-etch electron microscopy demonstrated that filipin induces the formation of aggregates 150-250 A in diameter in both biological membranes and liposomes which contain 3). At the etched face of the membrane "projections" cholesterol". Is (FIGURE of about 50 A in height are visible. N o change in fracture faces was observed after treatment of cholesterol-rich membranes with amphotericin B. A hypothetical model for filipin-cholesterol was proposed by De Kruyff 4. In contrast to the other polyene and Demell' and is reproduced in FIGURE antibiotics under discussion, filipin does not have a charged carboxyl and mycosamine group. As demonstrated with space-filling models filipin is most
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FIGURE3. Action of filipin" on the membrane of Acholeplasma laidlawii [inner fracture face and etch face (a), outer fracture face (b)], human erythrocytes [inner fracture face (c), etch face (d)] and liposomes of lecithin and cholesterol ( e ) .
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FIGURE 4. Schematic representation of a proposed complex of filipin and cholesterol in a lipid bilayer."
suitably packed in a parallel array. One side of such a planar aggregate is more hydrophobic (double bond system) and the other side hydrophilic (hydroxyl groups). A bilayer of filipin molecules exposing on both sides the double bond system to hydrophobic interaction with cholesterol molecules may give aggregates in the interior of the lipid core of the membrane, thereby leading to membrane deformation and local disruptures.
Eflects of Membrane Lipid Composition of Valinomycin-induced Cation Permeability
Excellent correlations have been found between passive permeability of liposomes and natural membranes of different lipid composition. The consequences of induction of chemical variations in lipid structure on membrane behavior could be predicted in many cases on the basis of the properties of the lipids in monomolecular films. As a follow-up of these studies on simple diffusion of nonelectrolytes the valinomycin-mediated cation transport was studied in liposomes and natural membranes which contained: ( 1 ) phospholipids with differently charged polar headgroups, (2) hydrophobic chains of a different degree of unsaturation, and (3) a variation in sterol content. A striking difference in the properties of polar headgroup exists between two closely related (bacterial) phospholipids: the negatively-charged phosphatidylglycerol and positively-charged lysylphosphatidylglycerol. The permeability of Rb' and K' was found to be higher for liposomes of phosphatidylglycerol than for those of lysylphosphatidylgly~erol.'~ Valinomycin was able to increase the transport of these cations in the liposomes of phosphatidylglycerol, but not across the barrier of the positively charged liposomes from lysylphosphatidylglyerol. In order to extrapolate these results from model studies to biological membranes the lipid composition of intact cells of Staphylococcus aureux was varied by means of the environmental pH. Indeed it was found that with an increasing lysylphosphatidylglycerol-to-phosphatidylglycerolratio
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the valinomycin-mediated exchange of "'Rb across the cell membrane was decreased. '' A second lipid parameter which was found to control the rate of valinomycin-mediated transport in black films'" and liposomesZ1 is the nature of the fatty acid constituents of the phospholipids. For instance, it was found that increasing unsaturation of the paraffin chain promotes the valinomycininduced cation leak. After variation of the fatty acid composition of the membrane lipids in A . Iaidlawii cells the valinomycin-induced K' and Rb' permeability was measured with three different techniques. In accordance with the experiments on liposomes it was found that the increase in the degree of unsaturation of the lipids causes a higher effectivity of valinomycin." Insertion of cholesterol in liposomes and in Acholeplastna laidlawii membranes causes a decrease in permeability. The observation that the lipid composition, e.g., the degree of unsaturation of fatty acids, influences the rate of valinomycin-mediated transport can be interpreted in different ways. The possibility exists that this ionophore displays a higher affinity for lipid bilayers with higher unsaturation of the phoxpholipids. Alternatively, the carrier may have a greater mobility within the unsaturated lipid bilayer. The kinetics of valinomycin-induced K' leak were studied on liposomes loaded with potassium thiocyanate.23 The lipophilic anion permits a considerable leakage in the absence of proton conductors, thus making unnecessary the addition of uncoupIers.*' The initial rate of K' leakage was found to be proportional to the valinomycin concentration. A rapid partition equilibrium of valinomycin occurs between the liposomal bilayer and the aqueous phase. A high affinity of valinomycin for the lipid bilayer was apparent and the partition constant has a negative temperature coefficient. The kinetic data support a model involving the formation of a ternary complex of valino5 ) . Using liposomes of phosphomycin with K' and a thiocyanate ion (FIGURE lipids with a different content of polyunsaturated fatty acids it was demonstrated that the rate of valinomycin-mediated transport increased with increasing unsaturation of the phospholipids; the affinity of the ionophore may even decrease somewhat with increasing unsaturation of the lipid b i l a ~ e r . ' In ~ short, it is most likely that the translocation of the complex is the rate-limiting step and that the turnover rate of more complicated transport systems also may
AL,K+
=VAL,@ -CN
CNS-
FIGURE 5. Model for the valinomycin-mediated K' leak from liposomes containing KCNS."
K+
OUTSIDE
MEMBRANE
INSIDE
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be controlled by the local lipid composition of the membrane. Insertion of cholesterol in liposomes" and A . laidlawii" was found to bring about a reduction of the valinomycin-mediated transport; current kinetic studies may give further insight in the molecular mechanism involved.
Eflects of Lipid-Phase Transition on Membrane Permeability and Lipid-Protein Interaction
In recent years much attention has been given to thermotropic transitions in biological membranes. The transition of lipids from the gel (solid) to the liquid-crystalline phase and vice versa can affect many properties of membranes. In A . laidlawii the cell barrier remains intact upon cooling below the transition temperature, although under these conditions the cells are more fragile when exposed to mechanical or osmotic forces. Passive permeation of nonelectrolytes through A . laidlawii membranes at temperatures in the range of lipid phase transition appears to occur predominantly in the regions of the m-mbrane that are still in liquid crystalline state.' It was reported that in black lipid membranes valinomycin was not capable of increasing the conductivity of the films at temperatures below the transition temperature of the lipids." In order to validate an extension of this conclusion to natural membranes, experiments with A . laidlawii were carried out at the extended region of the gel to liquid-crystalline phase transition." It can be seen that the valinomycin-induced efflux of K is (nearly) zero below the temperature of the gel-liquid crystalline phase transition (FIGURE 6 ) . In the temperature range above the phase transition the valinomycin-inducible K' leakage gradually increases with increasing temperature. The immobilization of the carrier function of valinomycin below the lipid phase transition could not be demonstrated in E. coli because a spontaneous loss of, for example, intracellular K' occurs when these cells are rapidly cooled below the transition temperature.*" Liposomes prepared from given synthetic phospholipids, such as (dimyristoyl)lecithin, also revealed a release of intracellular K' without valinomycin present when the lipid arrangement is transformed from the liquid-crystalline to the gel state." Current experiments showed that this phenomenon may depend u601
O V A L INDUCED K'RELEASE
FIGURE6. Leakage of K' from cells of A . faidhwii B in relation to the lipid phase transition." Elaidic and palmitic acid added to the growth medium.
10
0 TEMPERATURE *C
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to a significant extent on the nature of the lipids making up the bilayer, and the permeation alteration at the phase transition exhibits a certain selectivity even within the rang- of monovalent cations. Such alterations in ion permeability may find an explanation in the distortions of the lipid conformation during the phase transition. In this context it is of interest to refer to alterations in the architecture of biological and artificial membranes at lipid phase transition as visualized by freeze-etching electron micro~copy.~' This technique allows a direct observation of the transition from the liquid-crystalline to a gel state of synthetic phosphatidylcholines present in liposomes. Whereas band patterns in the former phase are smooth, quenching from below the transition temperature revealed 7). Liposomes characteristic band patterns with defined periodicities (FIGURE made of two phosphatidylcholine, which have a difference of no more than two CH. groups, exhibited a band pattern with a new periodicityz8(FIGURE 8). These mixtures display one thermotropic transition, and a homogeneous distribution of the phospholipid species below the temperature of the phase transition is likely. Mixtures of phospholipids with a difference in chain length of more than two CH2 groups or containing one saturated and one unsaturated phospholipid give two thermotropic peaks during differential scanning calorimetry; accordingly at temperatures between, these two phase transition regions of band patterns (phospholipid species in gel phase) and smooth area (phospholipid species in liquid8).'* At temperatures above both crystalline state) can be observed (FIGURE peaks the fracture faces are smooth; and when the mixture is quenched from a temperature below both peaks, the band patterns of both phosphatidylcholine species are visible. Thus the freeze-etching technique makes it possible to visualize the phenomenon of phase separation, which occurs in the temperature range between the phase transition of the two individual phospholipids. A discussion of the effects of different polar headgroups,"' mixtures of oppositely charged phospholipids,"' and the presence of cations on the images produced by freeze-etching" is beyond the scope of the present contribution. Many of these parameters are important when comparing results obtained on liporomes and biological membranes. Freeze-etching of natural membranes has demonstrated that a phase transition of the lipids is accompanied by an aggregation of the intercalated particles in A. laidlawii,". 53 E. coli,". 33 and Tetrahymena pyriformi~.~" The onset and the extent of aggregation have been found to be related to the beginning and degree of phase transition (liquid-crystalline to gel), respectively (FIGURE 9). It has been suggested that the aggregation of the protein particles occurs after partial solidification of the lipids, giving regions of lipid in the gel state and a concentration of the protein particles in the lipid regions remaining in the liquid-crystalline state. However, in some organisms ( S . aureus and Bacillus subtilis) particle aggregation was not observed in the fracture face although differential scanning calorimetry"' and breaks in Arrhenius plots of membrane-bound enzymes indicate that a phase The nonappeartransition of lipids occurred in the temperature region ~tudied.~' ance of particle aggregation could be explained by the presence of branchedchain fatty acids in the membrane lipids of these bacteria. Indeed, A . laidlawii cells that were grown in the presence of branched-chain fatty acids, and readily incorporated these constituents into membrane lipids, failed to give the particle reorganization induced by the lipid phase transition which was detected by differential scanning calorimetry." A different orientation of the branched-chain fatty acid constituents, giving also in the gel phase a rather loose packing,
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FIGURE 7. Band patterns observed by freeze-etch electron microscopy of synthetic phosphatidylcholines quenched from below the gel-liquid crystalline transition temperature.'' ( a ) (dilauroy1)-lecithin; (b) (dimyristoy1)-lecithin; ( c ) (l-palmitoyl-2oleoyl) -lecithin.
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FIGURE8. Freeze-etch electron micrographs of equimolar mixtures of synthetic ( A ) (dilauroy1)-lecithin and (dimyristoy1)-lecithin quenched phosphatidyl~holines.~~ from below the thermotropic peak. ( B ) (dipalmitoy1)-lecithin and (l-palmitoyl-2oleoy1)-lecithin quenched from between the two thermotropic peaks as measured by differential scanning calorimetry.
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FIGURE9. Structures of inner faces. E . coli cells demonstrated by freeze-fracturing of membranes quenched from above (a), within (b), and below (c) the lipid phase transition."
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may explain why the protein particles are not squeezed out and remain homogeneously localized in the hydrocarbon region of the membrane. In short, lipid phase transition can cause lipid-protein segregation when the lipids attain the gel state, but not when membrane lipids contain branchedchain fatty acids. Oppositely, proteins can affect the transition temperature of lipid? and create an ordering effect on the lipid core, particularly when ionic forces are predominantly involved in the interaction between lipids and proteins. The Nature of Associations between Proteins and Lipids In biological membranes a number of proteins can be located at either surface of the lipid bilayer, and this lipid-protein interaction may involve principally electrostatic attractions. Proteins that deeply penetrate or even span the lipid bilayer may associate mainly by hydrophobic forces with lipids. It is further realized that protein-lipid associations occur that to a varying degree depend on a subtle interplay between both electrostatic and hydrophobic interaction. For the understanding of membrane architecture and its function it is relevant to investigate the interaction of defined proteins and lipids under well-controlled conditions. The system of monomolecular layers of lipids allows study of the interaction with proteins injected underneath the lipid film by recording changes in surface pressure (at constant surface area) or changes in surface area (at constant pressure). These approaches can be combined with measurements of surface radioactivity by means of labeled protein. Furthermore, it is possible to study the effects of cations on the interaction of protein with lipid as well as the competition between two proteins for a particular lipid. Finally, it is possible to use proteolytic enzymes and phospholipases in combination with lipid-protein monolayers to obtain information about the nature of interaction and to determine which functional groups of both partners are involved. Some examples demonstrating these applications are briefly discussed. Interaction between A, basic protein from CNS myelin membrane and monolayers of various lipid classes demonstrated a pronounced increase of surface pressure (at constant area) with monolayers made of negativelycharged lipids. Of particular interest is the strong reaction between the basic protein and a film of cerebroside sulphate,a' a lipid that is abundant in this membrane. The results suggested the importance of coulombic interactions for the high affinity between basic protein and negatively-charged lipids, and in this respect it was considered of interest to study the possible effect of charge inhibition by Caz+. When A, protein is bound to the lipid monolayer, no binding of T a 2 +could be detected, but injection of basic protein underneath a lipid-'sCa'+ monolayer causes a decrease of surface radioactivity such that T a 2 +is completely expelled from the interface (FIGURE 10). The phenomenon is accompanied by an increase in surface pressure, which is considered to be due to penetration of the protein into the lipid phase. This finding supports the view that electrostatic attractions are effective for the association between lipid and this protein. Measurement of the surface radioactivity using 'SII-labeled AI basic protein showed that the amount of basic protein bound to the lipid film correlates well with the increase in surface pressure. That hydrophobic interactions in
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FIGURE10. The effect of A, basic protein on the interaction of Caz+ with a monolayer of cerebroside sulphate. The desorption of T a Z +was measured by monitoring the surface radioactivity; the penetration of protein was measured by the change in surface p~essure.~' 60 TIME (MINI
addition to electrostatic interaction play a part in the formation of this lipidprotein system was suggested by an influence of fatty acid length. Perhaps more conclusive in this respect is the observation that at constant film pressure a most significant increase in surface area (of 200%400%) was found after injection of A1 basic protein underneath a monolayer of cerebroside sulphate. This result indicates also that the lipid monolayer is penetrated and that the protein may extend into the apolar region of the film. Experiments were undertaken to elucidate which parts of the protein are particularly involved in the association with the negatively-charged lipids. The lipid-protein layer was treated with proteolytic enzymes such as trypsin, chymotrypsin, subtilopeptidase, or pronase, which were injected underneath the complex.as It was found that these enzymes were much less effective in the degradation of the protein after its interaction with lipid monolayers. It could be concluded that certain regions of the protein are protected by the association with lipid. To determine which peptide linkages of the Al basic protein were preserved from proteolytic attack, the lipid-protein complex was collected from the interface after the action of trypsin. Peptide maps demonstrated that six peptide bonds located at the N-terminal region between position 23 and 113 of the protein were protected in the protein-lipid complex; a tentative model in which these regions of the protein are considered to penetrate into the lipid phase was postulated (FIGURE 11). A second myelin protein, the Folch-Lees proteolipid, shows affinity for a variety of lipids and most remarkably for cholesterol as well.3nThis interaction was demonstrated to be dependent on the sterol structure. That these two major myelin proteins have different preferences for associating with lipids was demonstrated by experiments in which both proteins compete for interac12). A subsequent injection of A1 basic tion with a lipid monolayer (FIGURE protein and Folch-Lees proteolipid underneath a cholesterol monolayer shows a preferential binding of the proteolipid to cholesterol and a desorption of Al basic protein from the interface. In similar experiments it was demonstrated that the basic protein had the highest affinity for cerebroside sulphate when compared with Folch-Lees proteolipid. The preferences observed suggest that specific associations of these proteins with particular lipid classes may also occur in the myelin membrane. Extrapolation of these results to the native membrane supports the concept of both lipid and protein asymmetry in this membrane.'" An asymmetric distribution of lipids in the erythrocyte membrane has been established on the basis of studies combining the action of phospholipases and freeze-etch electron m i c r o s c ~ p y .The ~ ~ outer monolayer of the human
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FIGURE1 1. Schematic representation of binding sites and conformation of A, basic protein after association with myelin lipids."
'01
4 MSlC PO-
30 TIME IN MlHVTEI
eo
80
PROTEIN
10
10
00 TlME IN MINUTES
w
FIGURE12. Competition between A, basic protein and Folch-Lees proteolipid in their association with monolayers of lipids. (Left) subsequent injection of A, basic protein and Folch-Lees protein underneath a cholesterol monolayer. (Right) reversed sequence of protein injection."
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erythrocyte is considered to consist mainly of phosphatidylcholine and sphingomyelin and there is an additional 20% of phosphatidylethanolamine. The inner layer is composed of phosphatidylethanolamine,phosphatidylserine, and a small fraction of choline-containing phospholipids. These studies make one aware of the fact that certain phospholipases can act on the phospholipids present in the erythrocyte membrane, whereas others fail to do so. Various interpretations can be made and it was thought to be of interest to make a comparative study of a series of phospholipases on monolayers of phospholipids adjusted at various surface pressures. To date only those phospholipases that are capable of acting on highly compressed phospholipid monolayers (above 30 dynes/cm) were found to be able to attack their substrates in the intact erythrocyte membrane." This relationship permits estimation of the virtual surface pressure of the outer lipid layer of the erythrocyte. This parameter, which gives information about the tightness of the packing of the lipid molecules, is important for the evaluation of the classic experiment of Gorter and Grendel. In addition it seems plausible to conclude that at the erythrocyte surface not all polar headgroups of phospholipids are "occupied" by strong electrostatic interactions with proteins and that not all phospholipid ester linkages susceptible to phospholipase action are shielded off. A different situation exists for the binding of phospholipid to a pure protein from beef liver, which specifically catalyzes the transfer of phosphatidylcholine between both natural and artificial membranes." This protein was demonstrated to function as a carrier in the transfer of phosphatidylcholine between two separate monomolecular films." The carrier protein contains one mole of phosphatidylcholine per mole of protein. Various phospholipases were unable to hydrolyze this phosphatidylcholine, indicating that at least the polar moiety of the phospholipid molecule is deeply bound into the carrier protein. Only after perturbation with organic solvents or detergents could hydrolysis of phosphatidylcholine occur." The examples given above may indicate that studies in monomolecular layers in combination with proteolytic and lipolytic enzymes may supply useful information about the molecular segments of proteins and lipids that are involved in their mutual interaction.
References 1. GORTER, E. & F. GRENDEL. 1925. On bimolecular layers of lipids on the chromocytes of the blood. J. Exp. Med. 41: 439-443. 2. KINSKY,S. C. 1970. Antibiotic interaction with model membranes. Ann. Rev. Pharmacol. 10: 119-142. 3. DFMEL,R. A., L. L. M. VAN DEENEN& S. C. KINSKY.1965. Penetration of lipid monolayers by polyene antibiotics. J. BioI. Chem. 240 2749-2753. L. L. M. VAN DEENEN& S. C. KINSKY. 4. DEMEL,R. A., F. J. L. CROMBRAG, 1968. Interaction of polyene antibiotics with single and mixed lipid monomolecular layers. Biochim. Biophys. Acta 150: 1-14. 5. DEMEL,R. A., K. R. BRUCKDORFER & L. L. M. VAN DEENEN.1972. Structural
requirements of sterols for the interaction with lecithin at the air-water interface. Biochim. Biophys. Acta 255: 3 11-320. 6. DEMEL,R. A., K. R. BRUCKDORFER& L. L. M. VAN DEENEN.1972. The effect of sterol structure on the permeability of liposomes to glucose, glycerol and Rb'. Biochim. Biophys. Acta 2 5 5 321-330.
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BRUCKDORFER, K. R., R. A. DEMEL,J. DE GIER& L. L. M. VAN DEENEN. 1969. The effect of partial replacements of membrane cholesterol by other sterols on the osmotic fragility and glycerol permeability of erythrocytes. Biochim. Biophys. Acta 181: 334-345. DEKRUYFF,B., R. A. DEMEL& L. L. M. VAN DEENEN.1972. The effect of cholesterol and epicholesterol incorporation on the permeability and on the phase transition of intact Acholeplasma laidlawii cell membranes and derived liposomes. Biochim. Biophys. Acta 255: 33 1-347. DE KRUYFF, B., W,1. DE GREEF,R. V. M. VAN EYK,R. A. DEMEL& L. L. M. VAN DEENEN. 1973. The effect of different fatty acid and sterol compositions on the erythritol flux through the cell membrane of Acholeplasma laidlawii. Biochim. Biophys. Acta 298: 479-499. NORMAN, A. W., R. A. DEMEL,B. DE KRUYFF& L. L. M. VAN DEENEN.1972. Studies on the biological properties of polyene antibiotics; evidence for the direction interaction of filipin with cholesterol J. Biol. Chem. 247: 1918-1929. NORMAN, A. W., R. A. DEMEL,B. DE KRUYFF,W. S. M. GEURTS VAN KESSEL & L. L. M. VAN DEENEN.1972. Studies on the biological properties of polyene antibiotics: Comparison of other polyenes with filipin in their ability to interact specifically with sterol. Biochim. Biophys. Acta 290: 1-14. DE KRUYFF, B., W. J. GERRITSEN, A. OERLEMANS, R. A. DEMEL& L. L. M. VAN DEENEN. 1974. Polyene antibiotic-sterol interactions in membranes of Acholeplasma laidlawii cells and lecithin liposomes. I. Specificity of the membrane permeability changes induced by the polyene antibiotics. Biochim. Biophys. Acta 339: 30-43. DE KRUYFF, B., W. H. GERRITSEN, A. OERLEMANS, P. W. M. VAN DIJK,R. A. DEMEL& L. L. M. VAN DEENEN.1974. Polyene antibiotic-sterol interactions in membranes of Acholeplasma laidlawii cells and lecithin liposomes. 11. Temperature dependence of the polyene antibiotic-sterol complex formation. Biochim. Biophys. Acta 339: 44-56. FINKELSTEIN, A. & R. HOLZ.1973. Aeueous pores created in this lipid membrane by the polyene antibiotics nystatin and amphotericin B. In Membranes. G. Eisenman, Ed. Vol. 2: 377-407. Marcel Dekker, lnc. New York, N.Y. ANDREOLI, T. E. 1973. On the anatomy of amphotericin B-cholesterol pores in lipid bilayer membranes. Kidney Int. 4: 337-345. DE KRUYFF, B. & R. A. DEMEL.1974. Polyene antibiotic-sterol interactions in membranes of AchoZeplmma Zaidlawii cells and lecithin liposomes. 111. Molecular structure of the polyene antibiotic-cholesterol complexes. Biochim. Biophys. Acta 339: 57-70. VERKLEIJ, A. J., B. DE KRWFF, W. F. GERRITSEN, R. A. DEMEL,L. L. M. VAN DEENEN & P. H. J. TH. VERVERGAERT. 1973. Freeze-etch electron microscopy of erythrocytes, Acholeplasma laidlawii cells and liposomal membranes after the action of filipin and amphotericin B. Biochim. Biophys. Acta 291: 577-581. TILLACK, T. W. & S. C. KINSKY. 1973. A freeze-etch study of the effects of filipin on liposomes and human erythrocyte membranes. Biochim. Biophys. Acta 323: 43-54. HAEST,C. W. M., T. DE GIER,J. A. F. OP DEN KAMP, P. BARTELS & L. L. M. VAN DEENEN.1972. Change in permeability of S ~ Q ~ ~ Y ~ O C Oaureus CCUS and derived liposomes with varying lipid composition. Biochim. Biophys. Acta 255: 720-733. EISENMAN, G., G. SZABO,S. CIANI,S. Mc LANCHLIN & S. KRASNE.1973. Ion binding and ion transport produced by neutral lipid-soluble molecules. I n Progress in Surface and Membrane Science. Vol. 6 139-241. Academic Press. New York, N.Y. DE GIER,J., c. W. M. HAEST,J. G . MANDERSLOOT & L. L. M. VAN DEENEN. 1970. Valinomycin-induced permeation of “Rb+ through the bilayers of liposomes with varying composition. Biochim. Biophys. Acta 211: 373-375.
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NEUT-KOK,E. C. M., J. DE GIER, E. J. MIDDELBEEK & L. L. M. VAN DEENEN. Valinomycin-induced potassium and rubidium permeability of intact cells of Acholeplasma laidlawii B. Biochim. Biophys. Acta 332: 97-103. 23. BLOK,M. C., J. DE GIER & L. L. M. VAN DEENEN. 1974. Kinetics of the valinomycin-induced potassium ion leak from liposomes with potassium thiocyanate enclosed. Biochim. Biophys. Acta 367: 210-224. 24. BLOK,M. C., J. DE GIER & L. L. M. VAN DEENEN.1974. Some factors affecting the valinomycin-induced leak from liposomes. Biochim. Biophys. Acta 367: 202-209. 25. KRASNE,S., G. EISENMAN& G . SZABO.1971. Freezing and melting of lipid bilayers and the mode of action of nonactin, valinomycin and gramicidin. Science 174: 412-418. 26. HAEST,C. W. M., J. DE GIER, G. A. VAN ES, A. J. VERKLEIJ,L. L. M. VAN DEENEN.1972. Fragility of the permeability barrier of Escherichia coli. Biochim. Biophys. Acta 288: 43-53. L. L. M. VAN DEENEN& P. F. 27. VERKLEIJ, A. J., P. H. J. TH. VERVERGAERT, ELBERS.1972. Phase transition of phospholipid bilayers and membranes of Acholeplasma laidlawii B visualized by freeze fracturing electron microscopy. Biochim. Biophys. Acta 288: 326-332. P. H. J. TH., A. J. VERKLEIJ,P. F. ELBERS & L. L. M. VAN 28. VERVERCAERT, DEENEN. 1973. Analysis of the crystalization process in lecithin liposomes: A freeze-etch study. Biochim. Biophys. Acta 311: 320-329. B. DE KRUYFF& L. L. M. VAN 29. VERKLEIJ,A. J., P. H. J. TH. VERVERGAERT, DEENEN.1974. The distribution of cholesterol in bilayers of phosphatidylcholines as visualized by freeze fracturing. Biochim. Biophys. Acta 373: 495-501. 30. TOCANNE, J. F., P. H. J. TH. VERVERGAERT, A. J. VERKLEIJ & L. L. M. VAN DEENEN.1974. A monolayer and freeze-etching study of charged phospholipids. I. Effects of ions and pH on the ionic properties of phosphatidylglycerol and lysylphosphatidylglycerol. Chem. Phys. Lipids 12: 201-219. A. J. VERKLEIJ& L. L. M. VAN 31. TOCANNE,J. F., P. H. J. TH.VERVERGAERT, DEENEN.1974. A monolayer and freeze-etching study of charged phospholipids. 11. Ionic properties of mixtures of phosphatidylglycerol and lysylphosphatidylglycerol. Chem. Phys. Lipids 12: 220-23 1. J. F. TOCANNE 32. VERKLEIJ,A. J., B. DE KRUYFF,P. H. J. m VERVERGARET, & L. L. M. VAN DEENEN.1974. The influence of pH, Gazf and protein on the thermotropic behaviour of the negatively charged phospholipid, phosphatidylglycerol. Biochim. Biophys. Acta 3 3 9 432-437. 33. JAMES,R. & D. BRANTON.1973. Lipid and temperature-dependent structural changes in Acholeplasma laidlawii cell membranes. Biochim. Biophys. Acta 323: 378-390. 34. HAEST, C. W. M., A. J. VERKLEIJ,J. DE GIER, R. SCHEEK,P. H. J. TH. VERVERGAERT & L. L. M. VAN DEENEN.1974. The effect of lipid phase transition on the architecture of bacterial membranes. Biochim. Biophys. Acta 356: 17-26. 35. KLEEMANN, W. & H. M. MCCONNELL. 1974. Lateral phase separation in Escherichia coli membranes. Biochim. Biophys. Acta 345: 220-230. 36. SPETHS,V. & F. WUNDERLICH. 1973. Membranes of Tetrahymena 11. Direct visualization of reversible transitions in biomembrane structure induced by temperature. Biochim. Biophys. Acta 291: 621-628. 3 7. DEMEL,R. A., Y.LONDON, W. S . M. GEURTSVAN KESSEL.F. G. A. VOSSENBERG & L. L. M. VAN DEENEN.1973. The specific interaction of myelin basic protein with lipids at the air-water interface. Biochim. Biophys. Acta 311: 507-519. 38. LONDON,Y., R. A. DEMEL,W. S. M. GEURTSVAN KESSEL,F. G. A. VOSSENBERG & L. L. M. VAN DEENEN. 1973. The protection of A, myelin basic protein against the action of proteolytic enzymes after interaction of the protein with lipids at the air-water interface. Biochim. Biophys. Acta 311: 520-530. 22.
VAN DER
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39. LONDON,Y.,R. A. DEMEL,W. S. M. GEURTSVAN KESSEL,P. ZAHLER & L. L. M. VAN DEENEN.1974. The interaction of the “Folch Lees” protein with lipids at the air-water interface. Biochim. Biophys. Acta 332: 69-84. 40. VERKLEIJ,A. J., R. F. A. ZWAAL,B. ROELOFSEN, P. COMFURIUS, D. KASTELIJN & L. L. M. VAN DEENEN.1973. The asymmetric distribution of phospholipids in the human red cell membrane. A combined study using phospholipases and freeze-etch electron microscopy. Biochim. Biophys. Acta 323: 178-193. W. S. M. GEURTSVAN KESSEL 41. DEMEL,R. A., R. F. A. ZWAAL,B. ROELFSEN, & L. L. M.VAN DEENEN.Unpublished results. 42. KAMP,H. H., K. W. A. WIRTZ& L. L. M. VAN DEENEN.1973. Some properties of phosphatidylcholine exchange protein purified from beef liver. Biochim. Biophys. Acta 318: 313-325. 43. DEMEL,R. A., K. W. A. WIRTZ,H. H. KAMP,W. S. M. GEURTSVAN KESSEL & L. L. M. VAN DEENEN.1973. Phosphatidylchoiine exchange protein from beef liver. Nature New Biol. 2 4 6 102-105. 44. KAMP,H. H., E. D. SPRENGERS, J. WESTERMAN, K. W. A. WIRTZ& L. L. M. VAN DEENEN. Unpublished results.
LIPID-LIPID AND LIPID-PROTEIN INTERACTION IN MODEL SYSTEMS AND MEMBRANES* L. L. M. van Deenen, J. de Gier, R. A. Demel, B. de Kruyff, M. C . Blok, E. C . M. van der Neut-Kok, C . W. M. Haest, P. H. J. Th. Ververgaert, and A. J. Verkleij Department of Biochemistry Univeuity of Utrecht Utrecht, The Netherlands
In 1925 Gorter and Grendel' proposed that in the erythrocyte membrane the lipid molecules are arranged in a bilayer. They spread the lipids extracted from a given quantity of red blood cells in a Langmuir trough at the air-water interface and observed that the area occupied by the lipids was twice the surface of the erythrocytes. It is not our intention to discuss the merits and pitfalls of this ingenuous approach. After 50 years of intensive research the concept of the presence of a lipid bilayer in membranes appears more generally accepted when compared with the situation some ten years ago. Indeed several membrane properties are understandable on the basis of a lipid bilayer structure with proteins both located at the surfaces and penetrating or spanning the hydrophobic lipid core. The present paper summarizes some studies from our laboratory on lipid mono- and bilayers (liposomes) in relation to biomembranes.
Action of Polyene Antibiotics on Membranes Polyene antibiotics can interact with both artificial lipid interfaces and biological membranes, and cause changes in permeability characteristics. It is well established that the presence of sterols in the membrane is a requirement for the action of these antibiotics,' which compounds have a macrolide ring structure with a varying number of double bonds and hyroxyl groups (FIGURE 1).
In a collaborative study with Dr. S. C . Kinsky it was demonstrated that the sterol requirement of polyene antibiotics is manifested in the simple system of lipid monolayers.'. ' Injection of these antibiotics underneath a lipid monolayer induces at low molar ratios of antibiotic/lipid a significant increase of surface pressure, but only when sterol is present in the lipid film above a given sterol/phospholipid ratio. Recent studies were aimed at understanding the nature of the molecular association between the antibiotics and sterols and at interpreting the permeability alterations occurring in the membrane. In this respect it is appropriate to give first some attention to studies on the interaction between phospholipids and various sterols in monolayers, liposomes, and natural membranes. It was found that only sterols having a 3P-OH group, a planar ring system, and a hydrophobic side chain at C-17 gave a reduction of the *This paper is dedicated to the memory of Dr. Y. London, who was killed at the Golan Heights in October 1973. 124
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FlLlPlN
+%--+OM
bM%OTERKIN 0
o w m
m m o a -
FIGURE 1. Structures proposed for a number of polyene antibiotics. molecular area of a monolayer of synthetic unsaturated lecithins." The same requirments for the sterol molecule were found in studies on liposomes which demonstrated the importance of these structural parameters to reduce the passive permeability of the lipid bilayers towards nonekctrolytes, such as erythritol, glycerol and glucose, and Rb'." That the observations on the artificial systems are relevant to biomembranes is supported by experiments that involved the replacement of the cholesterol in the erythrocyte membrane by various sterol analogs.' The structural requirement of sterol interaction with other lipid components of biological membranes was studied also after incorporation of various sterols in the cell membrane of Acholeplasma laidlawii.O*oThe effect of the sterol upon the permeability of this membrane to erythritol was found to follow to the same rules as have been established for liposomes and monolayers. The relations observed between model systems and natural membranes for lipid-sterol interaction stimulated similar studies on polyene antibiotic-sterol interaction. In monoIayers,10 liposomes,lo-l* and A. laidlawii membrane^^"^ it could be demonstrated that the sterol incorporated in the membrane required a planar ring system, a hydrophobic side chain, and a 3p-OH group for polyene action. It is intriguing that there is a clear relationship between the structural requirments involved in (phospho) -lipid-sterol interaction and polyene antibiotic-sterol interaction. The latter association apparently is stronger than the former one. This is demonstrated also by differential scanning calorimetry and spectroscopic studies,10. la which revealed that the presence of polyene antibiotic has withdrawn sterol from its interaction with other membrane lipids so as to form polyene antibiotic-sterol complexes of defined stoichiometry. This complex formation alters the permeability of both artificial and natural membranes in a similar manner. The various polyenes, with the exception "7
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of filipin, cause selective permeability changes, suggesting the formation of pores with a diameter of about 8 A, permitting nonelectrolytes of the size of or smaller than glucose and monovalent cations to pass. Models for these complexes have been proposed by various inve~tigators."~~' A space-filling model developed for the amphotericin B-cholesterol complex is visualized as a circular arrangement of eight amphotericin B and eight cholesterol molecules.'o A schematic representation (FIGURE2) shows that in such complexes, each forming
FIGURE 2. Schematic representation of conducting pores formed by amphotericin B and cholesterol in a lipid bilayer.''
a half pore, the inside is hydrophilic because of the presence of the hydroxyl groups of the polyene antibiotic. The cholesterol molecules fit in the ring system of the polyene antibiotic and are in close contact with the double bond system without having direct contact with the hydrophilic region(s) of the antibiotic. The hydroxyl group of cholesterol and the charged groups of amphotericin B are believed to be located at the membrane surfaces. Similar structures have been deduced for complexes formed by nystatin and etruscomycin." Furthermore, it was argued that the primaricin-cholesterol complex cannot form a conducting pore because the length of the two half-pores is limited when compared with the thickness of a lipid bilayer. Filipin is known to cause more drastic alterations in both artificial and natural membranes. The permeability changes (e.g., release of the enzyme glucose-6-phosphate dehydrogenase) suggest that significant structural alterations occur in the membrane by the formation of the filipin-cholesterol complex. Freeze-etch electron microscopy demonstrated that filipin induces the formation of aggregates 150-250 A in diameter in both biological membranes and liposomes which contain 3). At the etched face of the membrane "projections" cholesterol". Is (FIGURE of about 50 A in height are visible. N o change in fracture faces was observed after treatment of cholesterol-rich membranes with amphotericin B. A hypothetical model for filipin-cholesterol was proposed by De Kruyff 4. In contrast to the other polyene and Demell' and is reproduced in FIGURE antibiotics under discussion, filipin does not have a charged carboxyl and mycosamine group. As demonstrated with space-filling models filipin is most
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FIGURE3. Action of filipin" on the membrane of Acholeplasma laidlawii [inner fracture face and etch face (a), outer fracture face (b)], human erythrocytes [inner fracture face (c), etch face (d)] and liposomes of lecithin and cholesterol ( e ) .
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FIGURE 4. Schematic representation of a proposed complex of filipin and cholesterol in a lipid bilayer."
suitably packed in a parallel array. One side of such a planar aggregate is more hydrophobic (double bond system) and the other side hydrophilic (hydroxyl groups). A bilayer of filipin molecules exposing on both sides the double bond system to hydrophobic interaction with cholesterol molecules may give aggregates in the interior of the lipid core of the membrane, thereby leading to membrane deformation and local disruptures.
Eflects of Membrane Lipid Composition of Valinomycin-induced Cation Permeability
Excellent correlations have been found between passive permeability of liposomes and natural membranes of different lipid composition. The consequences of induction of chemical variations in lipid structure on membrane behavior could be predicted in many cases on the basis of the properties of the lipids in monomolecular films. As a follow-up of these studies on simple diffusion of nonelectrolytes the valinomycin-mediated cation transport was studied in liposomes and natural membranes which contained: ( 1 ) phospholipids with differently charged polar headgroups, (2) hydrophobic chains of a different degree of unsaturation, and (3) a variation in sterol content. A striking difference in the properties of polar headgroup exists between two closely related (bacterial) phospholipids: the negatively-charged phosphatidylglycerol and positively-charged lysylphosphatidylglycerol. The permeability of Rb' and K' was found to be higher for liposomes of phosphatidylglycerol than for those of lysylphosphatidylgly~erol.'~ Valinomycin was able to increase the transport of these cations in the liposomes of phosphatidylglycerol, but not across the barrier of the positively charged liposomes from lysylphosphatidylglyerol. In order to extrapolate these results from model studies to biological membranes the lipid composition of intact cells of Staphylococcus aureux was varied by means of the environmental pH. Indeed it was found that with an increasing lysylphosphatidylglycerol-to-phosphatidylglycerolratio
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the valinomycin-mediated exchange of "'Rb across the cell membrane was decreased. '' A second lipid parameter which was found to control the rate of valinomycin-mediated transport in black films'" and liposomesZ1 is the nature of the fatty acid constituents of the phospholipids. For instance, it was found that increasing unsaturation of the paraffin chain promotes the valinomycininduced cation leak. After variation of the fatty acid composition of the membrane lipids in A . Iaidlawii cells the valinomycin-induced K' and Rb' permeability was measured with three different techniques. In accordance with the experiments on liposomes it was found that the increase in the degree of unsaturation of the lipids causes a higher effectivity of valinomycin." Insertion of cholesterol in liposomes and in Acholeplastna laidlawii membranes causes a decrease in permeability. The observation that the lipid composition, e.g., the degree of unsaturation of fatty acids, influences the rate of valinomycin-mediated transport can be interpreted in different ways. The possibility exists that this ionophore displays a higher affinity for lipid bilayers with higher unsaturation of the phoxpholipids. Alternatively, the carrier may have a greater mobility within the unsaturated lipid bilayer. The kinetics of valinomycin-induced K' leak were studied on liposomes loaded with potassium thiocyanate.23 The lipophilic anion permits a considerable leakage in the absence of proton conductors, thus making unnecessary the addition of uncoupIers.*' The initial rate of K' leakage was found to be proportional to the valinomycin concentration. A rapid partition equilibrium of valinomycin occurs between the liposomal bilayer and the aqueous phase. A high affinity of valinomycin for the lipid bilayer was apparent and the partition constant has a negative temperature coefficient. The kinetic data support a model involving the formation of a ternary complex of valino5 ) . Using liposomes of phosphomycin with K' and a thiocyanate ion (FIGURE lipids with a different content of polyunsaturated fatty acids it was demonstrated that the rate of valinomycin-mediated transport increased with increasing unsaturation of the phospholipids; the affinity of the ionophore may even decrease somewhat with increasing unsaturation of the lipid b i l a ~ e r . ' In ~ short, it is most likely that the translocation of the complex is the rate-limiting step and that the turnover rate of more complicated transport systems also may
AL,K+
=VAL,@ -CN
CNS-
FIGURE 5. Model for the valinomycin-mediated K' leak from liposomes containing KCNS."
K+
OUTSIDE
MEMBRANE
INSIDE
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be controlled by the local lipid composition of the membrane. Insertion of cholesterol in liposomes" and A . laidlawii" was found to bring about a reduction of the valinomycin-mediated transport; current kinetic studies may give further insight in the molecular mechanism involved.
Eflects of Lipid-Phase Transition on Membrane Permeability and Lipid-Protein Interaction
In recent years much attention has been given to thermotropic transitions in biological membranes. The transition of lipids from the gel (solid) to the liquid-crystalline phase and vice versa can affect many properties of membranes. In A . laidlawii the cell barrier remains intact upon cooling below the transition temperature, although under these conditions the cells are more fragile when exposed to mechanical or osmotic forces. Passive permeation of nonelectrolytes through A . laidlawii membranes at temperatures in the range of lipid phase transition appears to occur predominantly in the regions of the m-mbrane that are still in liquid crystalline state.' It was reported that in black lipid membranes valinomycin was not capable of increasing the conductivity of the films at temperatures below the transition temperature of the lipids." In order to validate an extension of this conclusion to natural membranes, experiments with A . laidlawii were carried out at the extended region of the gel to liquid-crystalline phase transition." It can be seen that the valinomycin-induced efflux of K is (nearly) zero below the temperature of the gel-liquid crystalline phase transition (FIGURE 6 ) . In the temperature range above the phase transition the valinomycin-inducible K' leakage gradually increases with increasing temperature. The immobilization of the carrier function of valinomycin below the lipid phase transition could not be demonstrated in E. coli because a spontaneous loss of, for example, intracellular K' occurs when these cells are rapidly cooled below the transition temperature.*" Liposomes prepared from given synthetic phospholipids, such as (dimyristoyl)lecithin, also revealed a release of intracellular K' without valinomycin present when the lipid arrangement is transformed from the liquid-crystalline to the gel state." Current experiments showed that this phenomenon may depend u601
O V A L INDUCED K'RELEASE
FIGURE6. Leakage of K' from cells of A . faidhwii B in relation to the lipid phase transition." Elaidic and palmitic acid added to the growth medium.
10
0 TEMPERATURE *C
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to a significant extent on the nature of the lipids making up the bilayer, and the permeation alteration at the phase transition exhibits a certain selectivity even within the rang- of monovalent cations. Such alterations in ion permeability may find an explanation in the distortions of the lipid conformation during the phase transition. In this context it is of interest to refer to alterations in the architecture of biological and artificial membranes at lipid phase transition as visualized by freeze-etching electron micro~copy.~' This technique allows a direct observation of the transition from the liquid-crystalline to a gel state of synthetic phosphatidylcholines present in liposomes. Whereas band patterns in the former phase are smooth, quenching from below the transition temperature revealed 7). Liposomes characteristic band patterns with defined periodicities (FIGURE made of two phosphatidylcholine, which have a difference of no more than two CH. groups, exhibited a band pattern with a new periodicityz8(FIGURE 8). These mixtures display one thermotropic transition, and a homogeneous distribution of the phospholipid species below the temperature of the phase transition is likely. Mixtures of phospholipids with a difference in chain length of more than two CH2 groups or containing one saturated and one unsaturated phospholipid give two thermotropic peaks during differential scanning calorimetry; accordingly at temperatures between, these two phase transition regions of band patterns (phospholipid species in gel phase) and smooth area (phospholipid species in liquid8).'* At temperatures above both crystalline state) can be observed (FIGURE peaks the fracture faces are smooth; and when the mixture is quenched from a temperature below both peaks, the band patterns of both phosphatidylcholine species are visible. Thus the freeze-etching technique makes it possible to visualize the phenomenon of phase separation, which occurs in the temperature range between the phase transition of the two individual phospholipids. A discussion of the effects of different polar headgroups,"' mixtures of oppositely charged phospholipids,"' and the presence of cations on the images produced by freeze-etching" is beyond the scope of the present contribution. Many of these parameters are important when comparing results obtained on liporomes and biological membranes. Freeze-etching of natural membranes has demonstrated that a phase transition of the lipids is accompanied by an aggregation of the intercalated particles in A. laidlawii,". 53 E. coli,". 33 and Tetrahymena pyriformi~.~" The onset and the extent of aggregation have been found to be related to the beginning and degree of phase transition (liquid-crystalline to gel), respectively (FIGURE 9). It has been suggested that the aggregation of the protein particles occurs after partial solidification of the lipids, giving regions of lipid in the gel state and a concentration of the protein particles in the lipid regions remaining in the liquid-crystalline state. However, in some organisms ( S . aureus and Bacillus subtilis) particle aggregation was not observed in the fracture face although differential scanning calorimetry"' and breaks in Arrhenius plots of membrane-bound enzymes indicate that a phase The nonappeartransition of lipids occurred in the temperature region ~tudied.~' ance of particle aggregation could be explained by the presence of branchedchain fatty acids in the membrane lipids of these bacteria. Indeed, A . laidlawii cells that were grown in the presence of branched-chain fatty acids, and readily incorporated these constituents into membrane lipids, failed to give the particle reorganization induced by the lipid phase transition which was detected by differential scanning calorimetry." A different orientation of the branched-chain fatty acid constituents, giving also in the gel phase a rather loose packing,
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FIGURE 7. Band patterns observed by freeze-etch electron microscopy of synthetic phosphatidylcholines quenched from below the gel-liquid crystalline transition temperature.'' ( a ) (dilauroy1)-lecithin; (b) (dimyristoy1)-lecithin; ( c ) (l-palmitoyl-2oleoyl) -lecithin.
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FIGURE8. Freeze-etch electron micrographs of equimolar mixtures of synthetic ( A ) (dilauroy1)-lecithin and (dimyristoy1)-lecithin quenched phosphatidyl~holines.~~ from below the thermotropic peak. ( B ) (dipalmitoy1)-lecithin and (l-palmitoyl-2oleoy1)-lecithin quenched from between the two thermotropic peaks as measured by differential scanning calorimetry.
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FIGURE9. Structures of inner faces. E . coli cells demonstrated by freeze-fracturing of membranes quenched from above (a), within (b), and below (c) the lipid phase transition."
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may explain why the protein particles are not squeezed out and remain homogeneously localized in the hydrocarbon region of the membrane. In short, lipid phase transition can cause lipid-protein segregation when the lipids attain the gel state, but not when membrane lipids contain branchedchain fatty acids. Oppositely, proteins can affect the transition temperature of lipid? and create an ordering effect on the lipid core, particularly when ionic forces are predominantly involved in the interaction between lipids and proteins. The Nature of Associations between Proteins and Lipids In biological membranes a number of proteins can be located at either surface of the lipid bilayer, and this lipid-protein interaction may involve principally electrostatic attractions. Proteins that deeply penetrate or even span the lipid bilayer may associate mainly by hydrophobic forces with lipids. It is further realized that protein-lipid associations occur that to a varying degree depend on a subtle interplay between both electrostatic and hydrophobic interaction. For the understanding of membrane architecture and its function it is relevant to investigate the interaction of defined proteins and lipids under well-controlled conditions. The system of monomolecular layers of lipids allows study of the interaction with proteins injected underneath the lipid film by recording changes in surface pressure (at constant surface area) or changes in surface area (at constant pressure). These approaches can be combined with measurements of surface radioactivity by means of labeled protein. Furthermore, it is possible to study the effects of cations on the interaction of protein with lipid as well as the competition between two proteins for a particular lipid. Finally, it is possible to use proteolytic enzymes and phospholipases in combination with lipid-protein monolayers to obtain information about the nature of interaction and to determine which functional groups of both partners are involved. Some examples demonstrating these applications are briefly discussed. Interaction between A, basic protein from CNS myelin membrane and monolayers of various lipid classes demonstrated a pronounced increase of surface pressure (at constant area) with monolayers made of negativelycharged lipids. Of particular interest is the strong reaction between the basic protein and a film of cerebroside sulphate,a' a lipid that is abundant in this membrane. The results suggested the importance of coulombic interactions for the high affinity between basic protein and negatively-charged lipids, and in this respect it was considered of interest to study the possible effect of charge inhibition by Caz+. When A, protein is bound to the lipid monolayer, no binding of T a 2 +could be detected, but injection of basic protein underneath a lipid-'sCa'+ monolayer causes a decrease of surface radioactivity such that T a 2 +is completely expelled from the interface (FIGURE 10). The phenomenon is accompanied by an increase in surface pressure, which is considered to be due to penetration of the protein into the lipid phase. This finding supports the view that electrostatic attractions are effective for the association between lipid and this protein. Measurement of the surface radioactivity using 'SII-labeled AI basic protein showed that the amount of basic protein bound to the lipid film correlates well with the increase in surface pressure. That hydrophobic interactions in
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FIGURE10. The effect of A, basic protein on the interaction of Caz+ with a monolayer of cerebroside sulphate. The desorption of T a Z +was measured by monitoring the surface radioactivity; the penetration of protein was measured by the change in surface p~essure.~' 60 TIME (MINI
addition to electrostatic interaction play a part in the formation of this lipidprotein system was suggested by an influence of fatty acid length. Perhaps more conclusive in this respect is the observation that at constant film pressure a most significant increase in surface area (of 200%400%) was found after injection of A1 basic protein underneath a monolayer of cerebroside sulphate. This result indicates also that the lipid monolayer is penetrated and that the protein may extend into the apolar region of the film. Experiments were undertaken to elucidate which parts of the protein are particularly involved in the association with the negatively-charged lipids. The lipid-protein layer was treated with proteolytic enzymes such as trypsin, chymotrypsin, subtilopeptidase, or pronase, which were injected underneath the complex.as It was found that these enzymes were much less effective in the degradation of the protein after its interaction with lipid monolayers. It could be concluded that certain regions of the protein are protected by the association with lipid. To determine which peptide linkages of the Al basic protein were preserved from proteolytic attack, the lipid-protein complex was collected from the interface after the action of trypsin. Peptide maps demonstrated that six peptide bonds located at the N-terminal region between position 23 and 113 of the protein were protected in the protein-lipid complex; a tentative model in which these regions of the protein are considered to penetrate into the lipid phase was postulated (FIGURE 11). A second myelin protein, the Folch-Lees proteolipid, shows affinity for a variety of lipids and most remarkably for cholesterol as well.3nThis interaction was demonstrated to be dependent on the sterol structure. That these two major myelin proteins have different preferences for associating with lipids was demonstrated by experiments in which both proteins compete for interac12). A subsequent injection of A1 basic tion with a lipid monolayer (FIGURE protein and Folch-Lees proteolipid underneath a cholesterol monolayer shows a preferential binding of the proteolipid to cholesterol and a desorption of Al basic protein from the interface. In similar experiments it was demonstrated that the basic protein had the highest affinity for cerebroside sulphate when compared with Folch-Lees proteolipid. The preferences observed suggest that specific associations of these proteins with particular lipid classes may also occur in the myelin membrane. Extrapolation of these results to the native membrane supports the concept of both lipid and protein asymmetry in this membrane.'" An asymmetric distribution of lipids in the erythrocyte membrane has been established on the basis of studies combining the action of phospholipases and freeze-etch electron m i c r o s c ~ p y .The ~ ~ outer monolayer of the human
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FIGURE1 1. Schematic representation of binding sites and conformation of A, basic protein after association with myelin lipids."
'01
4 MSlC PO-
30 TIME IN MlHVTEI
eo
80
PROTEIN
10
10
00 TlME IN MINUTES
w
FIGURE12. Competition between A, basic protein and Folch-Lees proteolipid in their association with monolayers of lipids. (Left) subsequent injection of A, basic protein and Folch-Lees protein underneath a cholesterol monolayer. (Right) reversed sequence of protein injection."
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erythrocyte is considered to consist mainly of phosphatidylcholine and sphingomyelin and there is an additional 20% of phosphatidylethanolamine. The inner layer is composed of phosphatidylethanolamine,phosphatidylserine, and a small fraction of choline-containing phospholipids. These studies make one aware of the fact that certain phospholipases can act on the phospholipids present in the erythrocyte membrane, whereas others fail to do so. Various interpretations can be made and it was thought to be of interest to make a comparative study of a series of phospholipases on monolayers of phospholipids adjusted at various surface pressures. To date only those phospholipases that are capable of acting on highly compressed phospholipid monolayers (above 30 dynes/cm) were found to be able to attack their substrates in the intact erythrocyte membrane." This relationship permits estimation of the virtual surface pressure of the outer lipid layer of the erythrocyte. This parameter, which gives information about the tightness of the packing of the lipid molecules, is important for the evaluation of the classic experiment of Gorter and Grendel. In addition it seems plausible to conclude that at the erythrocyte surface not all polar headgroups of phospholipids are "occupied" by strong electrostatic interactions with proteins and that not all phospholipid ester linkages susceptible to phospholipase action are shielded off. A different situation exists for the binding of phospholipid to a pure protein from beef liver, which specifically catalyzes the transfer of phosphatidylcholine between both natural and artificial membranes." This protein was demonstrated to function as a carrier in the transfer of phosphatidylcholine between two separate monomolecular films." The carrier protein contains one mole of phosphatidylcholine per mole of protein. Various phospholipases were unable to hydrolyze this phosphatidylcholine, indicating that at least the polar moiety of the phospholipid molecule is deeply bound into the carrier protein. Only after perturbation with organic solvents or detergents could hydrolysis of phosphatidylcholine occur." The examples given above may indicate that studies in monomolecular layers in combination with proteolytic and lipolytic enzymes may supply useful information about the molecular segments of proteins and lipids that are involved in their mutual interaction.
References 1. GORTER, E. & F. GRENDEL. 1925. On bimolecular layers of lipids on the chromocytes of the blood. J. Exp. Med. 41: 439-443. 2. KINSKY,S. C. 1970. Antibiotic interaction with model membranes. Ann. Rev. Pharmacol. 10: 119-142. 3. DFMEL,R. A., L. L. M. VAN DEENEN& S. C. KINSKY.1965. Penetration of lipid monolayers by polyene antibiotics. J. BioI. Chem. 240 2749-2753. L. L. M. VAN DEENEN& S. C. KINSKY. 4. DEMEL,R. A., F. J. L. CROMBRAG, 1968. Interaction of polyene antibiotics with single and mixed lipid monomolecular layers. Biochim. Biophys. Acta 150: 1-14. 5. DEMEL,R. A., K. R. BRUCKDORFER & L. L. M. VAN DEENEN.1972. Structural
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