Phospholipid vesicles (liposomes) as models for biological membranes: Their properties and interactions with cholesterol and proteins

P H O S P H O L I P I D VESICLES ( L I P O S O M E S ) AS M O D E L S FOR BIOLOGICAL M E M B R A N E S : THEIR PROPERTIES A N D I N T E R A C T I O N S W I T H CHOLESTEROL A N D PROTEINS DEMETRIOSPAPAHADJOPOULOS

Department of Experimental Pathology and HAROLD K. KIMELBERG

Department of Neurosurgery, Roswell Park Memorial Institute, 666 Elm Street, Buffalo, New York 14203

CONTENTS 1. Introduction 2. Methodological Aspects A. Preparation of Large Multilamellar Vesicles B. Preparation of Unilamellar Vesicles C. Permeability Measurements 3. Physicochemical Properties A. Phase Transitions: Molecular Packing and Clustering B, Fluid State: Molecular Mobility and Orientation C. Gross Morphology and Orientation of Polar Groups D. Effects of Sonication 4. Permeability Properties A. Cations B. Anions C. Role of Fixed Charges D. Water and Non-electrolytes E. Effect of Chain Length, Unsaturation and Phase Transitions F. Effects of Bi- and Multi-valent Metals 5, Role of Cholesterol A. Physical Studies B. Permeability Studies C. Biological Implications: Aging and Atherosclerosis 6. Interactions with Proteins A. Binding Studies B. Effects of Proteins on Permeability of Vesicles C. Structure o(Protein-Vesiele Complexes D. Interactions of Enzymes with Vesicles E. Encapsulation of Proteins and Enzymes as Models for Cellular Organelles 141

142 145 145 147 148 149 149 156 159 162 164 164 168 169 171 172 177 180 180 184 188 190 190 193 201 205 212

142

D. PAPAHADJOPOULOS AND H. K. KIMELBERG

F. Biological Significance of Protein-Vesicle Interactions Postscript Abbreviations Acknowledgments References

1.

215 217 218 218 218

INTRODUCTION

Model membranes consisting of a lipid bilayer were introduced in the early 1960stI,2~ and immediately became the subject of intensive study. It is an interesting coincidence that, during the same period, serious objections were raised concerning the existence of a lipid bilayer as a structural component of biological membranes. Several models were introduced, which markedly revolutionized the simple, and hitherto unchallenged, bilayer sandwich representation of membrane architecture33' 4~ Basically, the new ideas consisted of two basic premises: one, that membranes could be a two-dimensional grouping of small lipoprotein subunits, and two, that protein could be present wholly or partly within the low dielectric environment of the lipid layer, rather than exclusively occupying the interface between lipids and water. Recent reviews have elaborated on the arguments introduced by the proponents of each hypothesis35-9~ The present authors' appreciation of the problem, based on the available data, is that a lipid bilayer is probably a structural element present in most membranes, although its continuity could be interrupted to varying degrees by non-bilayer regions,tl°~ The chemical and functional diversity of most membranes makes it quite conceivable that many of its components are topologically segregated. Certain functions of a particular membrane could, therefore, be localized in specialized domains, and each such area could be undergoing structural fluctuations associated with its function. It would thus appear that the most relevant questions concerning membrane structure would be: how much does a given membranes' area deviate from the overall time-average structure (bilayer?) and how a particular function of the membrane relates to the dynamic interrelationship between bilayer and non-bilayer structure. The use of phospholipid bilayer membranes provides the opportunity

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143

to investigate how membrane proteins, enzymes, metals, etc., interact with lipids, and how the interaction affects the structure and function of each component. Because of their simplicity and their known constitution, such model membranes can be powerful tools for probing into the molecular mechanism of specific functions. Furthermore, they can provide knowledge on the function of specific chemical constituents present within the heterogeneous membrane network. Finally, model membranes can, and have, already been used for the study of pharmacological action of drugs, hormones, vitamins, etc. Liposomes are simply the liquid-crystalline particles obtained when amphipathic lipids, such as phospholipids, are dispersed in excess water. Their existence, and some of their properties, were known long before their description as a possible model for membrane permeability. Studies by X-ray diffraction tl 1,12~ and electron microscopy <13-tin had shown a lamellar structure, with lipid bilayers intercalated by a water space of variable thickness. The effect of various phospholipid dispersions on certain enzymatic reactions was also well recognized. However, the potentiality of the system as a model membrane was not appreciated until Bangham and his co-workers t17~ produced evidence indicating that each bilayer lamella of the multilamellar particles is a completely enclosed sac. With such an onion-skin-type structure, it became possible to study the permeability properties of the lipid bilayers by determination of the efflux of small molecules trapped inside the aqueous compartments of the particles. The multilamcllar vesicles produced by simple mechanical agitation are heterogeneous in size, shape and number of lamellac per vesicle. This heterogeneity makes it very difficult to estimate the total surface area available for diffusion studies. Although this problem is not very important for studying the effects of a particular compound on the permeability properties of the membranes, it is a serious shortcoming in cases where diffusion coefficients are desired, and also where the kinetics of the multi-compartment system becomes an obstacle. However, the uncertainties concerning surface area and geometry have been largely elimated by the developmertt of unilamellar vesicles, tl 8-2o~ These speroidal, single-sheU sacs of fairly uniform size' (approx. 250 A diameter) can be produced from the larger multilamellar particles by exposure to ultrasonic radiation. They arc currently being studied

144

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D. PAPAHADJO~IOJILOSAND H. K. KIMELBERG

extensivelyin terms of their structure, permeability and other physicochemical properties. Electron micrographs of phospholipid vesicles are shown in Fig. 1. Another widely used model membrane system is the black lipid films (bilayers), described originally by Mueller et al. (1~ a few years prior to the description of liposomes. The two systems offer mutually complementing advantages, and should best be used in conjunction. Furthermore, insight into some of their properties could be gained by parallel studies involving monomolecular films. Bilayers are best suited for studying electrical properties. On the other hand, liposomes, with much larger available surface area, are best suited for diffusional flux studies. Other advantages of the liposomes stem from the absence of extraneous hydrocarbon or other materials needed for the formation of the black films. Thus, the membrane composition can be precisely and easily defined. Finally, the liposomes (vesicles) are very stable and amenable to numerous chemical and physical manipulations, such as centrifugation, passage through columns, electrophoresis, spectrophotometry, fluorescence, nuclear magnetic and electron spin resonance and microcalorimetry. The literature covering the field of phospholipid model membranes is so extensive that no attempt will be made to cover the subject in its totality. Instead, after a discussion of some general properties, we will concentrate on two important aspects, which have not yet been adequately reviewed, namely, the role of cholesterol as a membrane component, and the effects of proteins on the structure and function of lipid membranes. The reader is referred to numerous recent reviews covering the general aspects of model membranes, including bilayers (vesicles) by Bangham,(2x) Rothfield and Finkelstein,(22) Reeves, (23) Thompson and Henn, (24) Bangham C9) and Papahadjopoulos. C'°'2s) Physical properties of phospholipid membranes such as nuclear magnetic resonance (NMR), fluorescence and calorimetry were reviewed by Steim,t26) Ladbrooke and Chapman, t27) Waggoner and Stryer,(2s) Chapman and Dodd, (29) Oldfield and Chapman, (a°~ Horwitz(a') and studies with electron spin resonance (ESR) probes by Jost et a/.,(32) Smith, (aa) Mehlhorn and Keith.~34) Several specific topics have also been reviewed recently. These include the interaction of phospholipid membranes with antibiotic ionophores,taS,a6) the

::ki?.' ;../::.

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FIG. 1. Electron micrographs of phospholipid vesicles. A. Egg phosphatidylcholine dispersed by hand-shaking in 145 mM ammonium acetate, and subsequently stained with isoosmolar salt solution containing ammonium molybdate, in order to prevent osmotic lysis. B. Egg phosphatidylcholine dispersed by hand-shaking in 2 % (w/v) a m m o n i u m molybdate. C. Brain phosphatidylinositol dispersed in 145 mM ammonium acetate by sonication in a bath-type sonicator for 50 minutes and subsequently stained with. isoosmolar salt solution containing ammonium molybdate. D. Beef heart cardiolipin vesicles obtained by sonication in bath-type sonicator for 15 minutes in 145 mM N a C l ' a n d subsequently fixed by osmium tetroxide (0.3% final concentration of Os04 for 5-10 minutes at 0°C). Marker: 1000 A. (0.1p). (Pictures A. B. C. from Papahadjopoulos and Miller. (18) Picture D from Black et al. (137))

SS4/2--fp 144

PHOSPHOLIPIDVESICLES(LIPO3OMES)

145

effects of steroids and other pharmacological compounds,C37) the use of liposomes as models for antigen-antibody reactions¢38) and the use of model membranes for the study of the mechanism of action of local anesthetics.~9) Several other reviews dealt mostly with the properties of bilayer black films.~4°--42) 2.

METHODOLOGICAL

ASPECTS

A. Preparation of Large Multilamellar Vesicles The preparation of liposomes is relatively simple. When dried preparations of naturally occurring phospholipids are allowed to come in contact with water or aqueous salt solution, they swell to form a variety of liquid crystalline structures. The phenomenon is analogous to the lyotropic mesomorphism observed with the soaps of long-chain acids and other amphiphatic molecules. The threedimensional configuration of the resulting hydrated crystals depends on the amount of water and temperature. A detailed description of the subject is given by Dervichian, t43) Bangham,t21) Luzzatit44) and Chapman and Wallach.t4s) In most cases where phospholipids are equilibrated with an excess amount of water (more than 50 ~o by weight), they assume a unique structure of lamellar configuration, the smectic mesophase. This phase can be visualized as stacked phospholipid bilayers, with water taking up the space between bilayers. The ability of phospholipids to form liquid-crystalline particles of concentric folded lamellae is related to the transition temperature (T~, melting of the hydrocarbon chains). Thus, natural phospholipids, which are usually above this transition point at ambient temperatures, will form these structures spontaneously. The transition temperature depends largely on the chain length, unsaturatiorl and branching of the acyl esters. Synthetic phospholipids with long-chain saturated acyl esters will form similar structures, if the temperature is raised close to the transition point, t46) The o~iginal preparation of multilamellar vesicles t2) was made by simple mechanical shaking of dry egg phosphatidyl choline (PC) with an aqueous salt solution in a round-bottom flask. This method produces a heterogeneous mixture of closed multilamellar vesicles of

146 D. PAPAHADJOPOULOS AND H. K, KIMELBERG varying size. Most phospholipids studied so far have been found to produce such vesicles, including purified preparations or total lipid extracts from tissues. Difficulties have been encountered with phosphatidylethanolamine (PE), which tends to give suspensions that aggregate easily with very low capture of ions, when dispersed in physiological salt solutions (0.1 M NaC1, pH 7.0). t471 However, stable swollen vesicles can be obtained ifPE is dispersed in low ionic strength (1.0-10 m_MNaCI) and pH above 8.0.t18~The presence of bivalent metals can interfere with the preparations of vesicles from acidic phospholipids. In this case, large aggregates are obtained which cannot be dispersed, even with sonication. Bivalent or higher valency metals could be present, either as counterions to the extracted acidic phospholipids or as contaminants from the preparatory procedures or the salts used for the suspension. Washing the lipid preparations with HC1 or EDTA, t18~ or the inclusion of small amounts of EDTA in the suspending solution eliminates this problem. ~48~ Other amphiphatic lipid compounds such as glyceroglycoli, pids, sphingoglycolipids, etc., will probably form similar liquid-crystalline structures, ~49'5°~ although their behavior has not been studied extensively as yet. Cholesterol, which by itself does not form liquid crystals, can be incorporated into the phospholipid lamellae up to a 1 : 1 molar ratio. Considerable amounts of other long-chain amphiphatic molecules such as dicetyl phosphate, or stearyl amine, can also be incorporated. Mixing PE with PC also facilitates the suspension of the former phospholipid into swollen vesicles.C47~ It is obvious that the behavior of each lipid compound in water will be determined by the balance of the energies of interactions of water with the hydrocarbon part and the polar group. A useful classification of the different lipid compounds, in terms of their interaction with water, was given by Small3 s 1

B. Preparation of Unilamellar Vesicles Exposure of the coarse phospholipid suspensions to ultrasonic radiation tends to break up the large multilamellar particles into small, unilamellar vesicles318-a°' s2) Prolonged sonication results in a fairly homogenous population of spheroidal closed sacs of approximately

PHOSPHOLIPID VESICLES (LIPOSOlVIES)

147

250/k d i a m e t e r , each one c o n t a i n i n g an a q u e o u s space enclosed by a lipid bilayer. T h e limiting size is p r o b a b l y defined b y the c o n s t r a i n t of the small r a d i u s of c u r v a t u r e on the p a c k i n g o f the p h o s p h o l i p i d molecules. The t i m e o f s o n i c a t i o n r e q u i r e d for the c o m p l e t e c o n v e r s i o n into h o m o g e n e o u s small vesicles d e p e n d s on the type of lipid, (¢s) ionic strength, ~s3) a n d p e r h a p s t e m p e r a t u r e . S e p a r a t i o n of the uni'lamellar vesicles from the large l i p o s o m e s can be a c h i e v e d by gel-filtration on Sepharose 4B. (19' s4, 4a) Recent N M R studies h a v e i n d i c a t e d t h a t the p a c k i n g of the p h o s p h o l i p i d m o l e c u l e s is n o t d r a s t i c a l l y a l t e r e d by the process of ~onication, Csz'55~ a l t h o u g h s o m e evidence i n d i c a t e s an i n c r e a s e d

D A

Sontcation

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Gel-filtration .

,

FIG. 2. Schematic representation of procedure for production of unilamellar vesicles by sonication. A. Multilamellar vesicle obtained by shaking dry phospholipid in aqueous solution. Black dots indicate partial entrapment of a solute within the aqueous interlamellar spaces. B. Formation of unilamellar vesicles after exposure to ultrasonic waves. Solute molecules still inside and outside the vesicles. C. Vesicles after passage through a gel-filtration column. Inset indicates the bilayer structure. The non-trapped solute molecules have been removed from the bulk phase during the passage through the column. Vesicles are now ready for determination of diffusion rates. D. Diagram of sonicating procedure in a bath-type sonicator. The phospholipid sample is evaporated to dryness in glass tube (I) under vacuum. The initial coarse, suspension is made after the addition of the salt solution, by shaking under nitrogen. The tube is then positioned in the sonicating bath (2). The temperature of the bath is controlled by circulating cold water (4} through a peristaltic pdmp (3). (From Papahadjopoulos." o))

148

D. PAPAHADJOPOULOSAND H. K. KIMELBERG

degree of disorder. (56) However, hydrolytic and oxidative degradation can be produced during the procedure, tsT, so) with pronounced effects on the physical properties and permeability behavior ¢sg~of the vesicles. The auto-oxidation of the double bounds can be minimized by an atmosphere of nitrogen or argon ; chilling of the sonicating probe and the suspension would also minimize the thermal degradation.(6°) However, the use of bath-type sonicator, where the lipid dispersion comes in contact only with glass under a controlled temperature and nitrogen atmosphere, avoids these difficulties, and has been shown to give nearly quantitative production of unilamellar vesicles348) A schematic diagram of the procedure is given in Fig. 2. Two other methods for the preparations of uni- or pauci-lamellar large vesicles have been reported. Mueller and Rudin c6a)have produced large spheres (many microns in diameter) surrounded by a thin (60100/~) membrane, by a procedure involving emulsification of total brain or heart lipids in hexane with a small vohime of protein solution. Although it was reported that these cellules show electrical properties similar to those of planar bilayer membranes, they have not yet been studied in detail. Reeves and Dowben c62) reported a procedure similar to that of Bangham et al. c2) In this case, however, the phospholipids were-thinly spread on a large area of a glass vessel and allowed to equilibrate with distilled water or a solution of non-electrolytes. Shaking or vibration was avoided during the equilibration. The procedure resulted in formation of large vesicles (0.5 to 10 microns in diameter) surrounded by one or more bilayers. These membranes were shown to be stable on centrifugation and osmotically sensitive.

C. Permeability Measurements Several methods have been described for the study of the permeability • properties of lipid vesicles. The original method introduced by Bangham et al. ¢2~ involves the use of isotope tracers and has been used extensively for the study of effiux rates for 22Na, 42K, 36C1, a6Rb, t4C-glucose. The tracer is added in the salt solution during the formation of the vesicles, when it can be trapped within the aqueous interior of the closed membranes. The vesicles with trapped tracer are then separated

PHOSPHOLIPIDVESICLES(LIPOSOMES)

149

from the untrapped species either by dialysis or by passage through a gel-filtration column (usually Sephadex G-50). The efflux of the trapped tracers is then followed by sequential dialysis of the vesicles against the same salt solution. The method could be adaptable for measuring the influx of isotopic tracers, although no such studies have been reported. This method is particularly suited for studying the efflux of slowly diffusing solutes. (*s) A modification of this method has been used by Kinsky and his colleagues, who measured the efflux of glucose by following spectrophotometrically the reduction of NAD with suitable enzyme systems utilizing glucose as substrate363) Other, non-isotopic markers, such as (CrO2 ions), have been used to study the non-specific effects of several pharmacological agents on the permeability of liposomes36.) A method particularly suited to fast-diffusing solutes, such as water, glycerol, urea, etc., has also been developed by Bangham36s) It depends on measurements of the change in optical density of liposome suspensions as the osmolarity of the aqueous environment changes in respect to the interior of the liposomes. Shrinking (in hypertonic solutions) and swelling (in hypotonic solutions) is accompanied by an increase and decrease in optical density, respectively. This method has been used extensively by DeGier, Van Deenen and colleagues in studies of glycerol and gly¢ol permeability of liposomes of different fatty acid chains, c66) A promising new method, with perhaps still limited applicability, involves the use of ion-specific electrodes for continuous recording of efflux or influx of solutes. It has been applied to studies of H + and K + fluxes. ~67'6s) As specific electrodes become more sensitive and practical, this method offers several advantages over tracer dialysis or photometry.

3,

PHYSICOCHEMICAL

PROPERTIES

A. Phase Transitions: Molecular Packing and Clustering

Phospholipids are amphipathic molecules of approximately cylindrical shape. The polar group is located at one end of the long axis of the

150

D. PAPAHADJOPOULOSAND H. K. KIMELBERG

cylinder. This molecular arrangement provides a clear-cut differentiation between the hydrophilic (phosphorylchlorine, ethanolamine, etc.) and hydrophobic (hydrocarbon chains) parts of the molecule. It also predetermines the tendency of phospholipid molecules to associate in the form of stacked bilayers. X-ray diffraction studies by Luzatti and his colleagues (44) have revealed that phospholipids can undergo a number of phase transitions (polymorphism) in relation to changes in temperature (thermotropic) and water content (lyotropic). Some of these phases are lamellar, although non-lamellar phases, such as hexagonal and cubic, have also been observed in the presence of only small amounts of water. Phase equilibria of egg lecithin in water are shown in Fig. 3. t69) In the hexagonal phase, the phospholipid molecules associate in the form of long cylinders containing water and lined by the polar head-groups of the phospholipids. Transitions from a lamellar to a non-lamellar (porous) phase might be very important for the function ofphospholipids in membranes by modulating the permeability barrier. However, it should be stressed that the predominant structural arrangement obtained, when phospholipids are dispersed in excess water, is lamellar, consisting of stacked bilayers. For a more detailed account of phospholipid polymorphism and other physicochemical properties the reader is referred to reviews by Bangham, ~21) Dervichian, ~43) Williams and Chapman. c7°~ For the purpose of this review, the most relevant structural arrangement is the liquid-crystalline phase, which is obtained spontaneously when phospholipids are brought in contact with excess water at a temperature close to or above the melting point for the acyl chains. This transition temperature (T~) is characteristic for each phospholipid species, and is defined mostly by the configuration of the acyl chain, but also by the degree of hydration and the chemistry of the headgroups370.71~ Thus, the presence of cis-double bonds, branching and decreasing chain length tend to reduce the T~. This phenomenon is in accord with the diminishing van der Waals interactions obtained under these conditions3 TM A list of transition temperatures obtained by differential scanning calorimetry with different phospholipids is shown in Table 1. Lines 1-5 indicate the effect of different acyl-chains on the T~for PC. Lines 4 and 7 show that the T~ for PE is 25 ° higher than that of PC with identical

PHOSPHOLIPID VESICLES (LIPOSOMES)

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t phase (liquid)

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151

2 phoses (2 liquids)

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FIG. 3. The binary phase diagram of egg phosphatidylcholine and water as a function of temperature.V.l.represents viscous isotropic phase obtained between 0 ~ and 1 5 ~ water (face-centred cubic by X-ray diffraction). Neat represents an anisotropic liquid crystalline phase (hexagonally packed cylinders, containing water and lined by the polar head-groups of phospholipids, as indicated by X-ray diffraction). The cross-hatched area between 0 and 5 % water from 45 ° to 90°C represents an area in which lamellar liquid-crystalline phase may coexist with another liquid crystalline phase. Neat (lamellar) represents the liquid crystalline phase (Let) with stacked lamellae intercalated with water and shown in Fig. 5. The same phase exists in the presence of excess water to the right of the diagram. The line marked as T, is the ill-defined boundary of crystal-to-liquid-crystal phase transition. The diagonally hatched area below the T line represents solid lamellar phases, LI~ and L . The crystallographic characteristics ofeach phase were discussed and defin~ by Luzzati. (44) (From Small.(69)).

152

D. PAPAHADJOPOULOS AND H. K, KIMELBERG

acyl-chain configuration. In this case, it is possible that interactions between adjacent opposite charges increase the ~, presumably by increasing the cohesive forces lateral to the plane of the interface. On the contrary, the presence of a single negative charge (as in PG, for phosphatidylglycerol) tends to reduce the T~ compared to the values for similar PC (lines 3 and 8, also 4 and 10). This effect is presumably due to charge repulsion on the plane of the membrane. Screening or neutralization of the negative charges by Mg 2+ gives a large increase in Tc (lines 8 and 9). The condensing effect of Mg 2 ÷ had been observed earlier with monolayersJ TM Finally, as shown in Table 1, natural phospholipids such as egg PC, exhibit Tc well below ambient temperatures, presumably due to the presence of double bonds. TABLE l. THERMAL TRANSITIONSOF DIFFERENTPHOSPHOLIPIDS'I"

Lipid

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Dibehenoyl (C22) PC (a) Distearoyl (C18) PC (a) Dipalmitoyl (C16) PC (a) Dimyristoyl (C12) PC (a) Dioleyl (C18: 1) PC (a) Egg PC (a) Dimyristoyl (C12) PE (b) Dipalmitoyl (C16) PG, Na ÷ (c) Dipalmitoyl (C16) PG, Mg 2+ (c) Dimyristoyl (C12) PG, Na ÷ (c)

Temperature for gel-toliquid crystal (lamellar) T in (°C) 75 58 41 23 -22

AH (kcal/mole) 14.9 10.7 8.7 6.7 7.6

-15/-7 48 38 47 21

1 Obtained from differential scannin calorimetry in the presence of excess water. (a) Ladbrooke and Chapman, ~2~ (b) Keough and Chapman, ~496) quoted by Oldfield and Chapman, ~3°~(c) Papahadjopoulos, unpublished observations.

The large endothermic transition revealed by DSC (Table 1) is usually attributed to the melting of the acyl chains. C7°) In the case of PC, this transition is preceded by a much smaller transition which is thought to indicate structural rearrangements of the polar head-group and water at the interface. {26,27,747 Studies with a very sensitive calorimeter and dilute suspensions of synthetic phospholipid vesicles{741 indicate that the main transition is truly isothermal (peak width 0.2 ° for 10 ~o to 90 conversion for dimyristoyl PC). The apparent heat capacities and other

PHOSPHOLIPID VESICLES(LIPOSOMES)

153

thermodynamic parameters of the lipids in the liquid-crystalline state (above T~) indicate that the aliphatic chains are much less mobile than the corresponding liquid normal paraffins. (74, 7s) Phospholipid membranes composed of mixed fatty acyl chains, or containing more than one head-group, exhibit very complex, but

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FIG. 4. Differential scanning calorimetry of different phospholipids and mixtures. A. Different aqueous suspensions (50° w/w) of phosphatidyl cholines (a) dioleyl, (b) distearoyl, (c) equimolar mixture of distearoyl/dioleyl, (d) 1-stearoyl-2-oleyl,(e) 1-stearoyl-2-elaidoyl.B. Different aqueous suspensions (excess water) of phosphatidylcholines (PC) and phosphatidylethanolamines (PE): (a) Dimyristoyl PC, 10070, (b) dimyristoyl PC, 9070 + dimyristoyl PE, 10/o,°/(c)dimyristoylPC, 70 70 + dimyristoylPE, 30 70,(d) equimolar mixture of above, (e) dimyristoyl PE, 10070. A: from Phillips et al. (~7) B: from Keough and Chapman,~496) quoted by Oldfield and Chapman.t3°) characteristic, transitions (Fig. 4). When the individual phospholipids in the mixture have widely different transition temperatures (Dioleyl PC and Distearoyl PC), two transitions are observed, although the

154

D . P A P A H A D J O P O U L O S A N D H . K. K I M E L B E R G

higher temperature transition is broadened and appears at somewhat lower temperatures, t76' 77) This indicates that, at the temperature region between the two transitions, the membrane is composed of clusters (domains), some of which are liquid and others crystalline. When, however, the individual chains have similar melting points ideal mixing is observed. If the different chains are esterfied in the same molecule (1-stearoyl-2-oleyl PC) a sharp transition at an intermediate temperature is observed. Broad intermediate peaks, indicating both mixing and clustering, were observed with membranes composed of mixtures of Dimyristoyl PE and PC. t3°~Phase separation and clustering of lipids in membranes is of considerable biological importance, since biological membranes show very broad transitions close to the ambient temperatures378, 79, 3o) The implications of phase separation in terms of permeability will be discussed in § 4E. Phase-transitions in phospholipid membranes have been studied by several other methods. Studies with X-ray diffraction indicate that a number of well-defined structural changes occur at the transition temperature. When oriented phospholipid membranes are cooled below To the broad diffraction in the region of 4.6 A is replaced by a sharp diffraction at approximately 4.2A. t44,71,s°'81) The crystallographic characteristics of the membranes below T indicate the formation of a crystalline structure within the hydrocarbon interior of each lamella in which the long chains are fully extended (trans-trans), packed in a hexagonal array and nearly perpendicular to the plane of the membranes.ta2, a3) In some cases the difference between the experimentally observed thickness of the lipid layer and the expected one, from fully extended perpendicular chains, has been explained in terms of an average tilt of 20-32 ° from the perpendicular, t71, al) As the membranes crystallize, the width of each bilayer increases by a few angstroms (from 39 A to 47 A or 48 A for Dipalmitoyl lipids), as would be expected by the closer lateral packing. The values of the overall width of each bilayer corresl~ond fairly well to double the length of the fatty acid chains, and a constant width for each head-group layer of approximately 10/~k. ta4'71,s0) Nuclear relaxation (7"1) measurements indicate considerable motion for the choline group at temperatures below the To when the hydrocarbon chains are frozen/sS' s6) However, considerable + broadening in the line widths of the --N(CH3) 3 proton NMR spectra

PHOSPHOLIPIDVESICLES(LIPOSOMES)

155

at temperatures below the T can be taken as an indication of decreased motional freedom or configurational change. A schematic representation of the above is shown in Fig. 5. O

58 A 2

48

O

47 A

/////?/?/?/# '/#

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39 A

FIG. 5. Schematic representation of molecular orientation in phospholipid bilayers below and above the phase transition. For detailed explanation and

references see text. The head-groups of the phospholipid molecules are represented by open circles; and fatty acyl chains by solid lines. Tightly packed (solid) acyl chains showing inhibited motion by NMR are represented by straight lines. Mobile (liquid) acyl chains are represented by curved lines. The dimensions shown in the above scheme relate to the data obtained with dipalmitoyl phosphatidylcholine bilayers,c71.82.144) The change in thickness and molecular packing, as shown by X-ray diffraction, indicates that phosphoJipid molecules would be more mobile and would occupy a larger area at the interface of the m e m b r a n e at temperatures above the T~ (For dipalmitoyl PC, from 48/~2 to 58/~2). These areas correspond to the relative packing of the same molecules at the air-water interface as condensed (21 °C) and expanded (29°C) films at 24 dynes/cm. ~87,8~ A chang~ in volume of 1.4~o (89) 4 ~ocs6~ at the transition temperature of bilayers has been measured by dilatometry, and explained as an increase in frequency of the gauche configuration of the C----C bonds. {89~ The latter authors predicted an increase in permeability of as much as one order of magnitude at the transition temperature, which has been observed experimentally in a recent study. (97) Other techniques which have also been used to study the changes in molecular motion associated with the gel-to-liquid-crystalline phase

156 D. PAPAHADJOPOULOS AND H. K. KIMELBERG transition, include infrared and Raman spectroscopy,(9°'91) thermoturbidimetric analysis,{92' 93) fluorescence polarization and intensity of fluorophore probes, {94-97) the order parameter of electron spin resonance (ESR) probes {98-1°I) and the line width of NMR spectra. {3°'a6) The relationship between the phase transitions observed with biological membranes and the lipids extracted from them will be discussed later in connection with permeability properties.

B. Fluid State: Molecular Mobility and Orientation Considerable insight into the detailed molecular arrangements of the liquid-crystalline phase have been obtained by resonance techniques. Proton magnetic resonance studies by Chapman and his colleaguesC45~ have shown the presence of a high resolution spectrum above T~, indicating a mobile, liquid hydrocarbon interior. More recent studies of ESR with spin-labeled probes have shown that the fluidity of the bilayer varies with the distance from the interface, becoming more fluid toward the middle399~ In studies with ESR probes, it is difficult to establish with certainty to what extent the probe itself disturbs the properties of its immediate environment,t1°2) The same reservation would apply to the even bulkier fluorescent probes, which would tend to act as impurities and interfere with the packing of the phospholipid molecules. In any case, the conclusions of Hubbell and McConnell and other workers using spin-labeled probes {1°3'1°4) appear to be substantially correct. Recent analysis of NMR spectra, {1°5-107, s67 have confirmed that the molecular motion within a fluid bilayer increases substantially along the length of the hydrocarbon chains. The picture that emerges from these studies with lecithin bilayers indicates a relatively rapid molecular motion of the choline groups, relatively hindered motion in the region of the glycerol backbone and the first few carbons into the interior, and an increasingly mobile region extending into the middle of the bilayer (see Fig. 5). Hubbell and McConnell,C99~ and more recently McFarland and McConnell, tl°s) have presented evidence with ESR probes, indicating a decrease in the anisotropy of motion along the fatty acyl chains after

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the seventh carbon atom. In an attempt to account for the difference in fluidity in these two regions of the bilayer, McFarland and McConnell(l°a) have proposed that carbon atoms 1 to 7 of the acyl chains are packed at an average angle of approximately 30° to the plane of the interface, while the rest of the chains are packed, on the average, perpendicular to the same plane, with larger intermolecular distances. Although this is a static representation it is to be understood as average orientation along the chain length, with cooperative motion among many molecules. Evidence for an average orientation with a deviation of 28 ° +_ 5° from an axis perpendicular to the plane of the bilayer has also been obtained with other spin probes. (1°9) Utilizing the observation that the spin-labeled compound TEMPO is excluded from bilayers below T~, a method has been proposed which could give an indication of the percentage of fluid vs. solid regions within a membrane.(1 lO) The biological significance of these studies can be appreciated from comparisons of the spectra of spin-labeled probes embedded in biological membranes, and the bilayer membranes composed of the extracted lipids. These studies indicate a considerable similarity in the motion and solubility of the probes in bilayers and membranes of nerve cells,(t1~-112) Mycoplasma laidlawii,(lla) and mitochondria of neurospora.(114) However, studies with erythrocytes,(~12) mycoplasma (115) and inner mitochondria(116) membranes have shown that the probes are slightly but significantly less mobile in the intact membranes compared to their motion in isolated lipid bilayers. These results could be taken as evidence that, at least in some membranes, the proteins tend to inhibit the motional freedom of the lipids, although the majority of the lipid molecules still conform to the constraints of pure bilayers. Restriction of the molecular haotion of the lipid acyl chains has also been concluded from the broadening of the proton NMR spectra following the recombination of lipids and proteins from erythrocyte ghosts (117) and in intact erythrocyte membrane fragments. (117a) The effect of the double bonds in decreasing the order parameter along the fatty acid chains was studied recently by Eletr and Keith (118) with yeast mutant membranes and synthetic lecithin vesicles. They concluded that the disruptive effect of cis-double bonds (lowering of T~) is more pronounced when the double bonds are located toward

158 D. PAPAHADJOPOULOSAND H. K. KIMELBERG the methylene end of the acyl chains, and that the segment of the chain between the double bond and the polar group is left relatively well ordered. In a series of elegant papers with ESR probes, McConnell and his colleagues have been able to obtain estimates of molecular mobility within lipid bilayers. Kornberg and McConnell(119) have estimated that phospholipid molecules move very rapidly within the plane of the membrane (molecular frequency of the translational step for lateral diffusion, > 103 sec- 1 at 0°C), while the exchange of molecules between the two sides of the bilayer is very slow (~<2 x 10- 4 sec- 1 at 40°C). Although the exact numerical values for such movements involving probe molecules might differ to an unknown degree from the values relating to unlabeled molecules, they provide a valuable insight into the extent of the fluidity and stability of the phospholipid bilayer. The values obtained by direct measurement of lateral diffusion of spinlabeled phospholipid probes in oriented phospholipid multilayers~12°) are in close agreement with those of Tdiuble and Sackman (1°1) obtained with a steroid probe and different technique ( D - 10-Scm2/sec; exchange frequency = 10 7 s e c - 1 ) . Significantly, this value for the diffusion coefficient in phospholipid bilayers is in close agreement with the value (D - 6 x 10 -s cm2/sec) obtained for the lateral diffusion of similar probes in membranes of sarcoplasmic reticulum vesicles. (121) The lateral diffusion of fluorescent labeled antibodies bound to surface antigens of living cellsc122) is considerably slower, but close enough to invite a comparison. (123' 121) The evidence presented above, indicating rapid lateral mobility of both lipid and protein components within artificial and biological membranes, cannot be extrapolated as a general phenomenon, or as involving complete molecular mixing of all the components. It is possible that the fluidity of biological membranes varies considerably in different types of membranes. (124) Furthermore, it is quite possible that different areas within the same membrane might be gel-like or fluid-like depending on the make-up and segregation of individual components. Indeed, there is evidence indicating that a considerable amount of the membrane lipids in E. colit125) and Mycoplasma laidlawii(81, 113,126) behave as a gel-like phase (below TO at the growth temperature for these microorganisms. Oldfield and Chapman (3°) have

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discussed the possibility that in membranes not containing cholesterol (most microorganisms), the coexistence of both gel (solid) and fluid regions might tend to provide a needed stability for the membrane of these cells. It is clear from the above, and other recent studies with fluorescent and ESR probes t2s' 127-129, 34-) as well as studies with NMR spectra of protons and other atoms, O3°-136'56) that these techniques provide promising tools for examining molecular motion and interactions in phospholipid as well as biological membranes.

C. Gross Morphology and Orientation of Polar Groups Direct visualization of the aqueous phospholipid dispersions has been obtained by electron microscopy of osmium-fixed~14' 13,137/138) or negatively stained preparations cls' 16, lS~ and more recently by the freeze-etching technique3139) The circular concentric lamellae, seen under the microscope, were earlier referred to as myelin figures. The osmium tetroxide-fixed preparations have a trilamellar appearance, with the dense lines representing OsO4 deposited at the lipid-water interface3140.141~ Black lipid films stained with uranyl acetate show two dense lines at a distance of 45 A.c142~ When negative stain is included during the preparation and sonication of the vesicles, c16' 18~all the lamellae become visible. However, addition of the negative stain outside the formed vesicles under conditions avoiding osmotic rupture cls~ results in deposition of stain only in the periphery of the particles indicating that the bilayers are normally impermeable. The conclusion of Hauser et al. ~1"~3~ that the multilamellar vesicles are permeable to the stain, in contrast to sonicated single-lamella vesicles, appears to be based on accidental rupture of the large vesicles under the conditions used. A high resolution (6/k) electron density profile has been obtained with oriented Dipalmitoyl PC bilayers at 95 70 relative humidity at temperatures below the T~. It shows a sharp trough in the middle of the bilayer, which has been assigned to the localization of the CH 3 groups, and two high density peaks at a distance of approximately 47A, representing the head-group region with the phosphate-choline dipole lying fiat on the plane of the surface, t144"145) For bilayers above their

160

D. PAPAHADJOPOULOS AND H. K. KIMELBERG

T~,the central trough is more diffuse(83) and the thickness approximately 40/~, a value obtained for bilayer membranes of human brain phospholipids ~12~ and for egg-yolk lecithin,t146'147'83~ From a repeat distance of 64/k for egg-yolk lecithin in excess water (> 45 ~ by weight), Small ~69) has calculated a thickness of 29.6/k for the hydrocarbon layer, 16/k for the two hydrated phosphorylcholine regions, and 18.5~ for the free-water zone. The area per molecule in the above system was calculated to be 67-71/k 2. This area corresponds to the area per phospholipid molecule of lecithin in monomolecular films at the air-water interface compressed to 20-25 dynes/cm/14~ ' 149.73~ The thickness of the lipid layer-will, of course, vary with the acyl chain lengths and temperature, as discussed earlier. The thickness of the water layer also varies with the amount of water and the degree of hydration of the polar groups. (44"1s~ The presence of a net negative or positive charge increases the thickness of the water layer, presumably due to the long-range repulsive forces between lamellae,t21~ On the other hand, divalent metals decrease the thickness by screening the negative charges/11' 1s~ The existence of phases other than the lameUar has been established primarily by X-ray diffraction.(15°' 44~ However, the hexagonal phase has been visualized directly with OsO4-fixed preparations ~141) and also by negative staining) 151' l s2) It appears that PE gives hexagonal phases under conditions (in equilibrium with excess water) where PC and most other phospholipids give only lamellar3 t¢6"t53) Another example of a lamellar phase in excess water is the case of cardiolipin in the presence of divalent metals3 ts4) This phenomenon is not well understood at present but could be related to the limited degree of hydration of these lipids even in the presence of excess water. In any case, the appearance of a hexagonal phase with water channels through the lipid layer could be related to the function of certain lipids in membranes. Freeze-fracture studies have shown that fast freezing can preserve the phase structure of the different lipid dispersions. It39~ The fractures through the middle of liquid-crystalline bilayers appear smooth, ~139. ~55 but those of the same bilayers quick-frozen from below their T~ show regular striations/155) Freeze fractures of several biological membranes (~s6) and reconstituted phospholipid-rhodopsin com-

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plexes(1 sT, 15a} show that the smooth internal fractures are interrupted by a regular array of intramembrane particles. These particles presumably reflect the regions of the membrane where protein is embedded(, 59) and have been shown to undergo reversible aggregation in response to pH. u6°) Aggregation was also observed when mycoplasma membranes were quick-frozen and fractured from a temperature below their T~.(115'1s5) These experiments demonstrate clearly the occurrence of phase separation, with areas of pure (solid) lipid bilayer and areas of aggregated proteins. Such phase separations can be observed also with lipid membranes composed of mixtures of phospholipids with very different transition temperatures (discussed earlier), or with phospholipid-cholesterol mixtures (discussed in § 5A). The orientation of the polar groups at the membrane-water interface is also a subject of some interest, and still not well understood. For example, two conflicting models have been proposed for the orientation of the phosphoryl choline groups of the phosphatidyl choline membranes. ('61-164) Hanai et al. (162) have used the well-known isoelectric behavior of PC vesicles in an electric field t2x~ to argue for the case of a coplanar configuration. According to this argument, if the quaternary ammonium ion was located toward the aqueous phase, and consequently closer to the hydrodynamic-slip-plane, it would contribute to a positive surface potential, which is not the case found experimentally. On the other hand, Shah and Schulman tx64) interpreted their data of Ca 2+ binding to PC monolayers as evidence for a vertical orientation of the phosphorylcholine group, with internal salt linkage between the phosphate and trimethylammonium ions. Statistical-thermodynamic analysis of the intermolecular forces between PC bimolecular leaflets has led to the suggestion~65) that the quaternary ammonium ions act as independent counterions to the phosphate and that the surface behaves as a diffuse charge layer. This suggestion is in accord with the relatively high mobility of the choline group shown by NMR studies (see earlier discussion). Studies based on the reactivity of the amino groups at the surface of phospholipid vesicles hav,e led to the conclusion t~66) that the ionic groups of PE and PS are involved in interand intra-molecular salt linkages, respectively. The reactivity of the amino groups of PE liposomes toward trinitrobenze sulfonic acid (TNBS) and acetic anhydride was shown to be

162 D. PAPAHADJOPOULOS AND H. K. KIMELBERG enhanced by mixing PE with P C (166) and by addition of detergents. ¢167) The subject of the availability of the amino groups in both lipid bilayers and erythrocyte membranes was discussed recently.<8) Binding of biand multivalent metals will be discussed in §4F in relation to permeability. Another important parameter of the phospholipid, as well as biological membrane interface is the structure of the associated water. There is evidence for bound water around the polar groups, which does not freeze at the freezing temperature of the bulk-water. <7~'~6s~ NMR spectra indicate that bound water exhibits restricted anisotropic motion, which was interpreted as immobilization3169) The estimates for bound water vary considerably from four to nine molecules per head-group (strongly immobilized) to many layers (loosely bound). Of course, the configuration of the head-groups, the presence of charge, divalent metals, etc., tend to vary the amount of bound water, with perhaps important changes on the properties of the membrane317o, 171~ D. Effects of Sonication

The physicochemical properties of phospholipid vesicles produced by exposure of the coarse dispersion to ultrasonic radiation are of considerable importance, since these vesicles are extensively used as convenient model membranes. The effect of sonication was studied first by Saunders and his colleagues~x72-174, 5a) by light scattering. These studies described the formation of small aggregates of egg-yolk phosphatidyl choline of an average particle weight of 2-4 x 106 and an axial ratio of 5.3 calculated for a prolate elipsoid. The ionization of the head-groups of sonicated phospholipid dispersions was studied by Abramson <92) and his colleaguestlTs-17a) and more recently by Wills et al. ~179) using titrimetric techniques. The molecular orientation of the phospholipidmolecules within the aggregates was assumed in the early studies to involve a large micelle with all the polar groups exposed to the aqueous medium,t 175,180) The more recent titrimetric study however indicates that not all ionizable phosphate groups are exposed to the aqueous medium, t179) Studies involving electron microscopyC181,18,19j and ion capture <47) demonstrated that ultrasonication ofphospholipid aqueous suspensions produces small unilamellar closed spheroidal vesicles. Furthermore,

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it was shown that prolonged sonication results in a homogeneous population of these single-shell vesicles. ¢1a) More recent careful studies of the hydrodynamic properties of sonicated egg PC vesicles gave an average particle weight of approximately 2.1 x 106 daltons and an external diameter of approximately 250,~. c19) This indicates the presence of only approximately 2700 phospholipid molecules per vesicle, arranged in a closed bilayer. A number of more recent studies are in substantial agreement with these conclusions (182-184' 2°' 52) (Fig. 1). The appearance of double-shelled vesicles in some electron micrographs t185) has been interpreted as resulting from a view through a crescent-shaped single-shelled vesicle3186, 2o~ The internal volume of a spherical vesicle of 250 or 300 A diameter and 50,~ membrane thickness should be 1.77 × 106 or 4.2 x 106,~3 respectively. This corresponds to an internal volume of 0.4 ~tl. or 1.0 ~tl. per ~t mole of phospholipid respectively. The captured volume of phosphatidyl serine vesicles was estimated recently to be 1.0 ~tl./~tM¢48~and 0.6 ~tl./~tM for 4 ~ PA in PC vesicles, c~85~However, the captured volume measured for phosphatidyl choline vesicles was substantially smaller, 0.1 I~I./I~M.C5¢~ A flattened biconcave spheroid has been proposed by these workers to take into account the smaller than expected captured volume. The absence of surface charge in this latter case might be favoring a relatively collapsed (flattened) spheroid. The limiting size of vesicles obtained by sonication could be a reflection of the strain in molecular packing as the radius of curvature is reduced.C187) Several lines of evidence have led to the conclusion that the production of the limiting size vesicles by sonic.ation is a two-step process, involving first, disintegration of the large particles to small unstable fragments, and second, reaggregation of the small fragments into small vesicles. ¢52)It has been calculated, assuming that the outside and inside monolayer of the vesicle membrane retain similar molecular packing (approximately 70 A2), that the number of molecules participating in the inside monolayer should be only 26-30 ~o of the total, c2°~This asymmetry has been verified experimentally by the broadening of the NMR signal of the choline residues by manganese ions added externally ¢18s, 52~ and also by the localization of a spin-labeled PC probe. ¢54~ The studies with spin-labeled PC vesicles (tempocholine attached

164 D. PAPAHADJOPOULOS AND H. K. KIMELBERG to the phosphatidyl residue) also indicated that the molecules in the internal monolayer of the vesicles were relatively more immobilized3S4) Recent proton NMR studies with sonicated dipalmitoyl PC vesicles have shown that the acyl chains are more disordered in small vesicles compared to larger ones, t56) although another study with egg PC led to the conclusion that the molecular organization of the sonicated vesicles is qualitatively the same as in the unsonicated vesicles352' ss) This latter view is also supported by 13C NMR studies. ~13s) As with multilamellar vesicles (99' 103) the packing of the molecules seems to be tightest (decreased motion) around the glycerol backbone and the first few methylene groups of the acyl chains, while the methylene groups toward the interior and the polar head-groups are more mobile3135' 107) The overall bilayer structure of hollow-sphere particles has recently been substantiated by X-ray diffraction studies ofsonicated vesicles. ~147) Other physicochemical studies with aqueous dispersions of sonicated phospholipid vesicles include measurements of partial specific volume, (189) dielectric constant and conductivity,(19°'191) lightscattering temperature-jump relaxationsJ 19z) light-scattering calculations for hollow spheres J193) ultrasonic attenuation measurements,~194-) estimation of trans-membrane potentials with ESR probes (19s) and studies with fluorescent and spin-labeled probes. (19sa' 196) It can thus be concluded that the sonicated vesicles are a very convenient and well-characterized system of model membranes. Their structure is one of a closed spheroidal particle bounded by a substantially unmodified lipid bilayer, which encloses an interior aqueous space. Similar size vesicles can be produced by sonication of biological membranes (197) and are also found intracellularly as natural components of biological cells (synaptic vesicles, micropinocytotic vesicles, etc.). However, for any functional comparison between phospholipid vesicles and biological membranes, one has to take into consideration the effects of the other membrane components, such as cholesterol and proteins. 4.

PERMEABILITY PROPERTIES

A. Cations One of the most striking characteristics of unmodified phospholipid

PHOSPHOLIPIDVESICLES(LIPOSOMES)

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166

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membranes is their extreme impermeability to cations. Recent studies with phospholipid vesicles (summarized in Table 2) have given values of permeability coefficients in the range of 10-13 to 10- 14 cm/sec for monovalent cations,tl as, 4a, 143)This is two to three orders of magnitude lower than similar values for the passive permeability of red blood cellstX98, 199) and the comparison is even more unfavorable in relation to nerve membranes32°°) The electrical resistance values obtained with black lipid films of phospholipids formed from decane are similarly very high, ranging from 10a to 101° f~-cm2. (201) These results are not at all unexpected, considering that these membranes are a thin (approximately 40 A) hydrocarbon layer stabilized by the phospholipid headgroups. Calculations of the energy involved in the displacement of ions from an aqueous solution to a medium of low dielectric have indicated that this process is extremely unfavorable,t2°2' 2o3) The mechanism for cation-anion diffusion is discussed in § 4E in relation to activation energies. The early studies involving phospholipid vesicles and also bilayers indicated that there was no appreciable discrimination between monovalent cations, although considerable discrimination was found between cations and anions32' 2o4~47) Since most biological membranes exhibit considerable discrimination for Na ÷ and K ÷, it wa~ concluded that the property for monovalent cation selectivity does not reside in the membrane lipids. However, it is well known that monolayers of fatty-acid soaps show considerable specificity among monovalent cations32°s) Some early analysis of the cations extracted with different phospholipid fractions from erythrocytes have indicated preferential binding to either K ÷t2°6) or Na+. t2°7) Furthermore, it was predicted by Eisenman, t2°s~ on the basis of his work with ion-specific glass electrodes, that carboxyl and phosphate groups have the appropriate field strength for Na÷/K + discrimination. A recent re-evaluation of the subject with vesicles formed from purified non-oxidized phospholipids has revealed that the acidic species phosphatidylserine (PS) and phosphatidylglycerol (PG) do indeed discriminate between Na ÷ and K ÷3209) The biological significance of this observation is somewhat uncertain, since the actual permeability coefficients for either cation with PS and PG vesicles are quite low compared to biological membranes, and the discrimination ratio also on the low side, approximately 10-fold

PHOSPHOLIPID VESICLES(LIPOSOMES)

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difference in favor of K + over Na +. The importance of phospholipids in cation transport and discrimination has become more certain through a recent study, which demonstrated a good correlation between the K/Na specificity of the various phospholipids with their ability to activate delipidated preparations of the (Na+ K)-stimulated ATPase. t21°) It appears possible, from the above observations, that the selectivity of biological membranes for Na + and K + is at least partly a function of specific membrane phospholipids, although the mechanism is not at present clear. The above study has indicated that the polar groups are certainly involved in the mechanism of discrimination. For example, a comparison of three phospholipid preparations (all derived from purified egg phosphatidylcholine and containing the same fatty acyl chains, but with different head-groups) showed that phosphatidylcholine exhibited no discrimination, phosphatidic acid only slight discrimination at low pH, and phosphatidylglycerol very good discrimination,t2°9) However, a recent report by Scarpa and DeGier ~6s) has shown that a highly unsaturated PC preparation exhibited some K/Na discrimination, while other less unsaturated preparations did not. It thus appears that the degree of unsaturation might also be important in the expression of monovalent cation selectivity. This effect could be brought about, either by specific dipole interactions with the double bonds, or by the influence of unsaturation on the area per phospholipid molecule (more expanded), and consequently on the packing of the head-groups and/or the structure of water at the interface. It appears from the studies just discussed that unmodified phospholipid membranes are extremely impermeable to cations and charged species in general. However, the ionic permeability can be increased many orders of magnitude and in a very specific fashion by numerous ionophoric compounds ~24'35' 36,9) and also by various proteins (§ 6). The increase in the volume of liposomes after induction of cationic permeability was discussed by Singer and Bangham.t2H~ The exchange of H + with K ÷ across liposomal membranes mediated by antibiotics, with or without uncouplers was described by Henderson et al., ~212) who also discussed the relation to similar actions in erythrocytes and mitochondria.C21a) Oxidation-reduction reactions across liposome membranes have also been studied, t67'2~4'2~5) It appears from these

168 D. PAPAHADJOPOULOS AND H. K. KIMELBERG interesting studies that both a lipophilic electron carrier and proton carrier are needed for maximal reaction rates across the lipid membrane. The creation ofa H ÷ gradient across the membrane could be measured either with a pH electrode after release of protons t67~or by the quenching of a fluorescent probe3 2~5)

B. Anions

Anion diffusion rates measured by isotopic 36C1 fluxes through phosphatidylcholine liposomes were found by Bangham et al32~ to be much higher than that for cations. Some discrimination between different anionic species was also found with similar membranes3216) Comparing the C1/K ratios of diffusion rates, Papahadjopoulos and Watkins t47) reported a ratio of more than 100 for PS vesicles containing 10 ~o stearyl amine, approximately 30 for PS vesicles, and only 0.5 for phosphatidic acid (PA) and phosphatidylinositol (PI) vesicles. A comparisola of the permeability coefficients of CI and K at 36-°C with PS vesicles (Table 2) indicates a ratio of 40 and also of 3300. The apparent activation energy (Arrhenius) for C1- diffusion (approximately 14 kcal/mole) is considerably smaller than that for cations, t4a~ In a study with spherical bilayers composed of PC, Pagano and Thompson~217) obtained a C1/q'qa ratio of 232 and an activation energy for CI- diffusion of approximately 11 kcal/mole. In sharp contrast to the above results, the transferance numbers for the charge-carrying species obtained from various black liigid films indicate a higher permeability for cations. 124) The only black lipid (bilayer) membranes indicating anionic perm-selectivity are the ones carrying net positive charge, t218'219) The work of Pagano and Thompson t21v) has resolved the apparent discrepancy by estimating the diffusional rates isotopically and the transferance numbers conductimetrically with the use of spherical bilayers of large surface area. It was established that, although the observed Na + isotopic flux agrees with the calculated values from conductance, the CI- isotopic fluxes were larger by a factor of 103. It was concluded that the C1- diffusion includes a component which does not contribute to the steady-state current (electrically silent). The possibility was suggested that the CI-

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diffusion is facilitated by a lecithin-heavy-metal complex.C219) More recently, BanghamC9) discussed several other possibilities for the diffusing species, including molecular chlorine and HC1. Studies with spin-labeled phosphatidylcholine vesicles have indicated that HC1 could be the diffusing species. ~195~ For the case of Br- diffusion, studies with bilayer membranes suggest that molecular bromine is the diffusing species. ~22°~ These studies are of considerable biological interest, because they reflect the behavior of certain cell membranes, where the anion diffusion has also been shown to be electrically silent.C221~ A possible relationship exists between C1- flux through vesicles and the rate of flip-flop (inside-outside) motion of phospholipid molecules in the same vesicles. Thus, the outward translocation of spin-labeled PC gives a rate of 1.7 x 10 -14 molecules/cm2 sec at 30°C,~5.~ while equivalent rates in negatively charged vesicles are sloWer. Comparable values for C1- flux through PC vesicles are 0.8 × 10-14mol/cm2sec at 4°C ~143) and through PS vesicles, 0.06 x 10-14 mol/cm 2 sec at 36°C.~.8) The equivalent rates for cation flux are not comparable, being 2 to 3 orders of magnitude slower (Table 2).

C. The Role of Fixed Charges An important consideration in relation to the function of the polar head-groups of phospholipids is whether the presence of fixed charges affects the permeability rates of cations through membranes. In early theoretical considerations of the repulsive forces generated by the presence of ionized head-groups, it was suggested~222) that ionized phospholipids would tend to disrupt the biotnolecular leaflet in favor of micellar aggregates of radially oriented molecules. However, more recent studies with both bilayers and vesicles composed of pure acidic phospholipids at physiological ionic strength have indicated high electrical resistance and very low permeability to ions, (223'24'218'4s) in spite of the presence of one negative charge per mblecule. In general agreement with these experimental findings, Ohki and Aono (224) have calculated the energy requirements for a bilayer-

170

D. PAPAHADJOPOULOSAND H. K. KIMELBERG

micelle transformation, and concluded that even for phospholipids carrying one charge per molecule, the most stable configuration (in 0.1 M NaCI solution) is that of a bilayer. Higher surface charge would tend to favor the formation of a cylindrical aqueous pore type of configuration. In spite of the generally low permeability of acidic phospholipid membranes to cations, it is clear that the surface charge does play a considerable role in defining the permeability coefficients. An examination of Table 2 indicates that negatively charged P G membranes are considerably more permeable to Na + and K ÷ compared to vesicles composed predominantly of neutral PC membranes with similar fatty acyl residues. Generally, the presence of acidic groups increases the permeability to cations and decrease that of the anions, while the presence of excess positive charge has the opposite effect.(2,,7.225) The effect of surface charge on the ionic permeability of phospholipid membranes has also been described in a number of studies with black lipid films, t218"226) Positively charged membranes were found to be anion perm-selective, in contrast to negatively charged ones, where the transferance numbers indicate cation permselectivity.t218.219) The data assembled in Table 2 also gives the permeability coefficients for C1- and glucose which will be discussed in § 4D. Some discrepancies of the permeability coefficients obtained by different groups shown in Table 2 are worth a brief discussion. The values for Na + permeability through PC (0.12 × 10 -13 cm/sec) and PS (0.045 × 10-13 cm/sec) obtained recently by Hauser et al. (14.3) are 10-13 times lower than the values obtained by Johnson and Bangham (ls5) and Papahadjopoulos et al. t4.s) However, the discrepancy is at least partly due to the differences in the temperature used in the above studies. The low values (1.3) were obtained at 4°C, whereas the others were at 36-37°C. The activation energy for the diffusion of cations is very high, approximately 27 kcal/mole for PS vesicles and Na +,(4.s) and thus a difference of more than 10-fold in permeability coefficients would be expected for a temperature difference of 33°C. Earlier values, indicating higher permeabilities and lack of discrimination between N a ÷ and K ÷ for PS vesicles,wT) were explained later as being due to incomplete sonication of the vesicles and effects of auto-oxidation during the preparation of the lipids and vesicles. (186, 209, 4-8,14-3)

PHOSPHOLIPIDVESICLES(LIPOSOMES)

171

D. Water and Non-electrolytes In contrast to the low permeability to ions, phospholipid bilayer membranes exhibit considerable permeability to water, ranging from 5 to 100 × 10 -4 cm/sec.t24) These values are well within the range of water permeability of several biological membranes, t19a~ The coincidence of the osmotic and isotopic water fluxes obtained in several laboratories t227-229) indicates that there are no aqueous pores within the phospholipid membranes, a conclusion consistent with their high electrical resistance. As suggested by Haydon and his coworkers,t23°'231) the water permeability of phospholipid membranes can be explained simply on the basis of solubility and diffusion in bulk liquid-hydrocarbons of the appropriate thickness. This is supported by the temperature dependence of the water permeability coefficient, which gives an apparent Arrhenius activation energy of 12-14 kcal/mole between 10-45°C. t231' 232) This is in fair agreement with the published values of activation energy for diffusion (4 kcal/mole) and partition (Skcal/mole) of water into liquid hydrocarbon, which would be additive for two processes in series, t232~ All the above studies on water permeability were done with black lipid films. However, Bangham et al. t6s) have calculated substantially similar diffusion rates (0.8 to 18 × 10-*cm/sec) from experiments based on the rate of swelling of multilamellar phospholipid vesicles. In the same study, a graded permeability for several non-electrolytes was reported: urea (which was as permeable as water)> glycerol > erythritol ,> manitol. A permeability coefficient for water of 44 x 10-4cm/sec (at 25°C) was obtained by osmotic swelling of large (0.5 to 10 I~) phospholipid vesicles, t233) Glucose permeability coeffÉcients are quite low, and have been estimated with PC bilayer membranes as 2.5 x 10- lo cm/seeC234) and also with unilamellar vesicles, approximately 0.4 x 10- lo at 36°C.t4s~ As shown in Table 2, the activation energy for glucose diffusion through unilamellar vesicles is approximately 19 kcal/mole, a value similar to the activation energies reported for erythitol and glycerolt23s) through multilamellar vesicles. This latter study reported that the activation energies for the diffusion rates of these non-electrolytes was not different with membranes of different degrees of unsaturation or

172 D. PAPAHADJOPOULOS AND H. K. KIMELBERG amounts of cholesterol. However, it w a s s h o w n t235~ that, as the penetration rates markedly decreased from glycol to glycerol to erythitol, the activation energies generally increased (from 15 to 18 to 21 kcal/mole, respectively, with egg PC liposomes). Analogous differences were noted between glucose and sucrose diffusion through unilamellar vesicles, t4s) A summary of~the above results is given in Table 2. Glucose diffusion has also been studied with multilamellar vesicles or liposomes, t236'237) Although it is not possible to calculate permeability coefficients with these vesicles, the reported permeability rates as percent per hour (approximately 20-30 % at 36°C) are comparable to the equivalent values for unilamellar vesicles, t4s~ A comparison of the permeability of small molecular weight non-electrolytes with both model and biological membranes has amply demonstrated that the membrane lipids define, to a large extent, the permeability properties of biological membranes to these s o l u t e s . (238-240' 225' 235~ Similar conclusions concerning the role of membrane lipids as a permeability barrier can be drawn from the correlation of lipid solubility and anesthetic action of numerous narcotic agents, t241-243' 17)

E. Effect of Chain Length, Unsaturation and Phase Transitions

The fatty acyl chains of phospholipids play an important role in determining diffusion rates. This role cannot easily be tested with naturally occurring phospholipids, which contain a variety of fatty acids with varying degrees of unsaturation and chain length. Van Deenen, DeGier and colleagues have reported studies on the permeability of vesicles composed of synthetic phospholipids with welldefined fatty acyl chains. ¢66~ Although these studies were concerned with the permeability rates of small uncharged molecules (glycerol, glycols, etc.), their results are probably generally applicable. Not surprisingly, it was found that the permeability of vesicles generally increased with increasing unsaturation and decreasing chain length. The relative permeability properties of different synthetic phospholipids studied in this report were obtained from the rates of swelling of multilamellar vesicles at different temperatures. The effect of increased

PHOSPHOLIPID VESICLES (LIPOSOMES)

173

unsaturation (resulting in higher permeability rates) was also observed by Klein e t al. (244) in a study of amino acid fluxes through lecithin liposomes. The effect of unsaturation was also observed with Rb ÷ fluxes.(245)

Vesicles of synthetic phospholipids were also studied recently in the authors' laboratory. In this case, the measurements included the diffusion rates of Na ÷ through sonicated vesicles of dipalmitoyl and dioleyl phosphatidylglycerol. The results were in general agreement with the data of DeGier et al. (66) indicating a much lower permeability with the dipalmitoyl vesicles compared to the dioleyl. However, as shown in Fig. 6 a very dramatic increase in permeability of the dipalmitoyl vesicles was observed at the .temperature close to the ,

,

i

,

DOPG DPPG

"~ o 3O n

20

II

iI

/ ~ ~o ÷11 Z P4

o. . . . .

~

,.~

DPPGIChol

__.,._,..,-,---,--,--.---,--,--,--, 20

30 40 T e m p e r a t u r e (°C)

50

FIG. 6. Self-diffusion rates of 22Na+ through synthetic phospholipid vesicles at different temperatures. The vesicles were sonicated at temperatures above their T, under nitrogen, in 0.1 M NaC1 solution buffered at pH 7.4. They were then equilibrated at 22°C, passed through sephadex at that temperature and then dialysed at different temperatures as shown. O ~ ' Dipalmitoylphosphatidylglycerol;Q ..... (~) dioleylphosphatidylglycerol; • • equimolar mixture of dipalmitoylphosphatidylglyeerol and cholesterol. (From Papahadjopoulos et a/.(971).

174

D. PAPAHADJOPOULOS AND H. K. KIMELBERG

expected phase transition. It is remarkable that while these vesicles are considerably less permeable (than the dioleyl PG vesicles) to Na +, both at low and high temperatures, they become more permeable than the dioleyl vesicles at approximately 36-39 °. This unexpected hump in the curve of permeability vs. temperature corresponds to the mid-point of the phase transition as determined by fluorescence polarizationJ 97~ One explanation for this finding is that the anomalous increase in permeability represents the diffusion of ions through discontinuities between liquid and melted domains within the membranes of vesicles undergoing phase transition. The phenomenon is observed both during increasing and decreasing temperatures, and it is much less pronounced with labeled sucrose, indicating no overt rupture or lysis of the vesicles. In contrast to the dipalmitoyl vesicles, the dioleyl vesicles show a gradual increase in Na + permeability throughout the temperature range, giving a linear Arrhenius plot with an activation energy of 24 +_ 2 kcal/moleJ 2461 An interesting report on the role of unsaturation t247J has emphasized the effect of essential fatty acids (linoleic and linolenic) on the diffusion rates of Na ÷, through multilamellar vesicles composed mostly of PC. It was found that the various values of activation energy for Na ÷ diffusion could be correlated with the content oflinoleic plus linolenic, but not with the total unsaturation of the different PC preparations. The activation energy for the PC isolated from normal, control rats (15 kcal/mole) was much higher than the values obtained for PC from essential fatty acid deficient rats (4.5 kcal/mole). However, the value obtained in this study for egg-yolk PC was low (4.5 kcal/mole) compared to the reported value of 15 kcal/molet2~ obtained earlier with similar membranes. More recently, the above study was extended to include the permeability characteristics of PC membranes from different sources to glucoseJ 248} It was reported that the rate of glucose diffusion through liposomes containing PC from essential fatty-acid-deficient rats was considerably lower than that through PC from normal rats. The difference was attributed to the higher amounts of unsaturated fatty acids present in the normal rat PC. However, the same study~24s) indicated that the activation energy for glucose diffusion was only slightly lower with the normal rat PC (26.2 _ 3.9 kcal/mole) compared

pHOSPnOUPID VESICLES(LIPOSOMES)

175

to values with essential-fatty-acid-deficient PC (28.9 _+ 5.8 kcal/mole). DeGier et al. ~235~ have also shown that the activation energies of erythritol and glycerol (20 and 19 kcal/mole, respectively) were substantially similar with either dipalmitoyl, dioleyl or dilinoleyl PC membranes. A comparison of the above studies indicates the following two points. First, increased unsaturation results in increased rates of diffusion for both charged and uncharged solutes through phospholipid membranes. Also, a large increase in permeability coincides with the melting of the acyl chains. These phenomena could be related to the higher frequency of kinks induced by the presence of cis-double bonds and melting of the chains, which results in decreased order within the interior of the bilayer. Tr~iuble t2~9) has proposed that small molecules could be diffusing through a bilayer by following such kinks or paekin 9 defects. Krasne e t a / . t25°) have shown with bilayer membranes that the ionophoric effect of valinomycin, which is presumed to diffuse back and forth across the bilayer as a mobile cation-carrier, is completely inhibited by the freezing of the acyl chains. The effect of cholesterol in lowering the permeability of phospholipid membranes, to be discussed in § 5B, is probably also related to its ability to inhibit the motion of the acyl chains. A correlation between degree of unsaturation and higher glycerol permeability in different species of red blood cells has been pointed out by Van Deenen and DeGier. t251) A similar correlation has been observed more recently t252) for glucoside transport in everted intestinal sacs from normal and essential fatty acid-deficient rats, and also in E. coli K12 mutants, t253~ Although the correlations involving whole biological membranes are more difficult to interpret due to other possible interfering factors, the work with model phospholipid membranes makes a clear case concerning the effect of unsaturation on permeability. Second, the Arrhenius activation energies for the diffusion of cations (Na +) are much higher with membranes containing more polyunsaturated fatty acids, while those of uncharged molecules (glucose) are either similar or slightly lower. These observations, coupled with the fact that DeGier et al. ~66) did not observe any discontinuities for the diffusion rates of small non-electrolytes with the saturated membranes at the phase-transition temperatures, in contrast

176

D. PAPAHADJOPOULOS AND H. K. KIMELBERG

to the results with Na + discussed earlier, indicates that small uncharged molecules diffuse via an entirely different mechanism to the cations. The subject of mechanism of diffusion of electrolytes vs. nonelectrolytes, and the interpretation of the Arrhenius activation energies, was discussed recently by Papahadjopoulos et al. t48~ The discussion was based on the observation that the Arrhenius activation energies for the diffusion of cations through phospholipid vesicles, at temperatures between 30-50°C, was very high (approximately 30 kcal/mole) and the addition of cholesterol decreased this value drastically (to approximately 15 kcal/mole). In contrast, the addition of cholesterol had only a small effect on the activation energy for the diffusion of chloride and glucose, in agreement with De Gier et a/. t235) The conclusion was that cations diffuse via a different pathway to that for anions and nonelectrolytes. Furthermore, it was concludedt4a) that the apparent activation energy obtained with cations is fictitiously high, and the high values result from temperature-dependent structural changes. These structural changes could be associated with the thinning of the bilayers as the temperature increases, which has been observed by X-ray diffraction studies344) Following this argument, it would be expected that membranes with poly-unsaturated fatty acids would give higher apparent activation energies for the diffusion of cations, compared to membranes containing saturated fatty acids (below their T~). or to membranes stabilized by cholesterol. Calculations presented in the above study t4s) indicated that a small, gradual, decrease in the activation energy with increasing temperature would result in much higher apparent activation energies obtained from linearization of Arrhenius plots. It was speculated that diffusion of cations could involve passage through time-average micellar pores through the bilayers, which are lined by the head-groups of the phospholipids. In contrast, uncharged molecules could diffuse through an unmodified bilayer possibly by following the kinks along the acyl chains, t249) Finally, in agreement with previous studies t25'*' 235) it was concluded that the rate-limiting step for the diffusion of solutes through phospholipid membranes, is the dissolution into the membrane matrix, which involves the partition coefficient, and not the diffusion through the interior of the membrane.

PHOSPHOLIPIDVESICLES(LIPOSOMES)

177

The role of unsaturation and phase transitions on the permeability of phospholipid membranes is also reflected in the behavior of various biological membranes. It is well known that Mycoplasma laidlawii stop growing and eventually lyse if the environmental temperature is dropped below the solid-to-liquid-crystalline transition point of the constituent lipids: 255'7a~ More recently, a number of transport and enzymatic processes have been shown to exhibit discontinuities in the Arrhenius plots at temperatures generally corresponding to the phase transition of the membrane lipids, t256-26°'t°°,12s) Such discontinuities have also been studied with reconstituted systems, t21o, 261) where it is clear that the appearance of the kinks in the Arrhenius plots depends on the phospholipids used for reconstitution. The importance of the liquid-expanded state of the lipids on the function of biological membranes is also indicated by the behavior of yeast cells grown anaerobically. Under these conditions, yeast cells cannot synthesize unsaturated fatty acids, but they switch their biosynthetic mechanism in favor of fatty acids of shorter chain length t262) which would tend to increase the molecular area t14s) and thus reduce the solid-to-liquid-crystalline phase transition below ambient temperatures: TM As will be discussed later in § 5A, the presence of cholesterol abolishes the phase transition and thus stabilizes the membranes against temperature fluctuations. This subject will also be discussed later (§ 5B) in relation to lipid-protein interactions.

F. Effects of Bi- and Muiti-valent Metals

The presence of higher valency metals has 15rofound effects on the properties of phospholipid membranes, as is also the case with biological membranes. The early work on the binding of metal ions on aqueous phospholipid dispersions was studied by charge reversal and flocculation. ~26a)More recently, metal binding to purified phospholipid vesicles was determined by measurements of electrophoretic mobility,C21, 73,264, 265)and also by acid-base titrations: 175-17~ Calcium binding on phospholipid monolayers and vesicles has been followed by measurements of surface radioactivity ~264,266-27°) and also by changes in surface potential and surface pressure3271, 149, 163,267, 73)

178

D. PAPAHADJOPOULOS AND H. K. KIMELBERG

As could be expected, the results indicate that anionic phospholipids bind with much higher affinity, and there is evidence indicating that divalent metals do not bind appreciably to purified phosphatidylcholine which is neutral, t272-273) The affinity constants of phosphatidylserine and phosphatidic acid for Ca 2 + have been determined by titrimetric methods, and were found to be of the order of 1 0 4 M - I . t274'177) This value is similar to the affinity constant of the A T P - C a 2+ complex3275) The stoichiometry for the P S - C a 2+ complex was measured as 1 Ca 2+ per 2 PS molecules (267) and it has been proposed that the complex could be arranged as a linear polymer3 TM However, the nature of the bonds involved in the P S - C a 2+ complex is still not clear, and some evidence indicates that a considerable amount of the bound C a 2 + could be accounted for by simple screening of the negative charges, t276) In any case, the screening effect would not account for the differences between Ca 2+ and Mg 2+ observed with both PS monolayers and vesicles, t267' 73,277) The effects of some bi- and trivalent cations on phosphatidylcholine bilayer membranes were reported by Miyamoto and Thompson327s) It was found that Fe 3 + at 10-5 ~ concentration caused a large reduction (by a factor of 103) of the electrical resistance of these membranes. In contrast, other cations such as Ca 2 +, Mn 2 +, Ca 2 +, in concentrations of 10-3 M, increased the resistance of the membranes. The effect of multivalent cations was also studied by Gutknecht and Tosteson t219) who found that, upon addition of ferric and thorium ions to the aqueous phase, sheep red-cell lipid bilayers became anion perm-selective. The effect of Ca 2÷ is of special interest, because of its involvement in the excitation phenomena of nerve membranes. ~279' 39) Through a series of studies with both vesicles and bilayers composed of acidic phospholipids, it was established that Ca 2 ÷ can decrease or increase the membrane permeability, depending on its distribution across the membrane, t2sl-2as) Thus, it was found that the addition of Ca 2+ (1 ham in 100 mM NaC1) to only one side of the membrane produces a decrease in resistance, while, if the Ca 2 ÷ is added to both sides of the membrane, it increases the resistance. Assymetric distribution of C a 2 + across such membranes has also been shown to produce considerable steady-state potential difference and rectification phenomena, t284' 286) It has been proposed (see Fig. 7) that these effects, which are observed

PHOSPHOLIPID VESICLES (LIPOSOMES)

179

o n l y w i t h a c i d i c p h o s p h o l i p i d s c o u l d b e r e l a t e d to t h e m e c h a n i s m o f e x c i t a t i o n o f n e r v e m e m b r a n e s . (283' 39' xs4) S e v e r a l o t h e r w o r k e r s have also proposed that structural changes, involving phospholipids

A '

III)11111111111

PC, PE, Chol.

TOP B

OUTSIDE ÷--

b_z ~l

VIEW

+-+-

-

~g

0 ~

-ItxXl,~O~co2*c,Z*c,Z,~+co2*c,Z*c,t* m e ~ + I No+- . + ^ . . ^ . ^ . -No+-No ^. ^ _A - N e e - N o , I . . .,

N////A mK

+-

MOe÷..zxx~,'v, xx',e,,,~-

NN,,',..,,

_IKXXg~q~'~I i~le+

I -'

I,,-

INSIDE

T R A N S - MEMBRANE

VIEW

FIG. 7. Schematic, hypothetical, representation of the localization of phosphatidylserine (PS) domains in a nerve axon membrane. A. Top view showing areas containing PS bilayer surrounded by areas composed of other (neutral) phospholipids, cholesterol and proteins. B. Trans-membrane view showing phospholipids present in a central area of bimolecular thickness, with proteins embedded in the bilayer and also bound at the lipid-water interface. The negative charges of the PS molecules are shown to be neutralized by calcium ions on the outside (extracellular) face of the membrane, and by positive charges of a polypeptide chain on the inside (intracellular) face. The PS bilayer is at a metastable state under these conditions and the eLxonmembrane at the resting state. Removal of calcium ions or of the polypeptide chain (induced by depolarization) could thus trigger an unstable state for the PS bilayer domain, which is characterized by a large increase in ionic permeability. Such unstable state of high permeability can be induced by an asymmetric distribution of charges across PS bilayer membranes which can revert back to an impermeable membrane when the charge distribution is balanced. (From Ohki and Papahadjopoulos, (2B3))

180 D. PAPAHADJOPOULOS AND H. K. KIMELBERG and calcium, are at least partly responsible for the electrochemical events observed during the generation of action potentials across a nerve membrane. C2s7-29 a)

5.

ROLE OF C H O L E S T E R O L

A. Physical Studies The role of cholesterol as a membrane component has been studied rather extensively with phospholipid model membranes. Such studies in well-defined systems (monolayers, bilayers and vesicles) have produced clear-cut evidence relating the physicochemical parameters of cholesterol interactions to the possible biological role of this interesting compound. The first observations on the interactions of cholesterol with phospholipids were obtained with monomolecular films, where it was established that, in mixed films, the area per molecule for lecithin plus cholesterol was considerably smaller than the area occupied by each component in pure films at the same surface pressure.(292, 43, 148) Since cholesterol is a rigid molecule, and the phospholipids showing this effect are in the liquid-expanded state, the phenomenon could be described as a condensation of the area per molecule of the phospholipid affected by the presence of cholesterol. Extensive further work with monolayers of pure synthetic lipids with different chain length and degrees of unsaturation have produced additional evidence indicating the complexity of the system, and several models accounting for the mechanism of the condensation effect were proposed.~163, 294--301) The subject was discussed by Chapman and Wallach 14s) and Phillips. c3°2) In summary it can be stated that the condensing effect of cholesterol is a general phenomenon, but the degree of condensation depends on the state of liquidity of the phospholipid molecules, as defined by the chain length, unsaturation, branching and temperature. Thus, cholesterol will produce condensation with phospholipids, which are neither fully expanded (such as those with short chain lengths or highly unsaturated) nor fully condensed (such as straight chain saturated). Most natural phospholipids extracted from membranes would belong into this intermediate category, although

PHOSPHOLIPID VESICLES(LIPOSOMES)

181

some individual species within a given total mixture would be in one or the other of the extreme cases, t3°3' s04~ Other physical studies have indicated that the condensation effect observed with monolayers can be observed with phospholipid bilayers as well, and have provided further insight into the molecular mechanism of the interaction. Chapman and his colleagues, ~3°5,3°6~ and more recently Lee et al. ~86J and Darke eta/., ~1°7~have shown by high resolution proton NMR that cholesterol restricts the motion of the CH 2 groups of the acyl chains of phospholipids normally in their liquidcrystalline phase. The motion of the choline protons is not affected to a similar degree. Other studies with differential scanning calorimetry and X-ray diffraction indicate that the presence of cholesterol at 1/1 molar ratio with dipalmitoyl phosphatidylcholine completely abolishes the endothermic phase transition, and produces a diffuse 4.45/k high-angle spacing instead of the sharp 4.2/k spacing, indicating a crystalline structure, t3°7~ It thus appears that the addition of cholesterol produces a more condensed state where the hydrocarbon chains of the phospholipid are motionally restricted, but it also prevents the crystalline packing characteristic of the phospholipids below their liquid-crystalline transition temperature (T~). Examination of the low-angle X-ray diffraction spacing, which gives the thickness of the membrane, is in accord with the above considerations. The thickness of the lipid layer of the membrane was found to increase by 3/k t3°8~ in the presence of cholesterol, with an accompanying decrease in the area per molecule of egg lecithin in the plane of the membrane. A more detailed analysis of the electron density profile across the membrane with oriented multilayers of egg lecithin with and without cholesterol ts3~ has verified the increase in thickness (from 37/k to 42/k peak-to-peak distance) and also indicated a sharp localization of a low electron density region in the middle of the membrane. The increase in thickness has been observed also with black lipid film by optical reflectance33°9~ The overall orientation of the cholesterol molecule, compatible with these studies, would be one closely parallel to the orientation of the phospholipid molecules, wiih the hydroxyl group anchored into the lipid-water interface, t31°~ Studies with ESR labels have extended the above observations indicating, not unexpectedly, that cholesterol decreases the fluidity t311~and

182

D. PAPAHADJOPOULOS AND H. K. KIMELBERG

increases the average orientation of the phospholipid acyl chains perpendicular to the plane of the membrane, is°9) Oldfield and Chapman t312) have shown that, when cholesterol is added to egg yolk PC,

A Ledthin i~o~w chain meltin9 temper~lu~

[.~.c~min * Id

Chain carbon number

end

B o

42 A

phospholipid

phospholipid, cholesterol

FIG. 8. Characteristics of phospholipid-cholesterol membranes. A. Diagrammatic representation of variation of fluidity along the PC hydrocarbon chains in bilayers with and without cholesterol. The vertical scale is derived from widths of wide lines in the NMR spectra, but it means to be quantitative only. (1°7) B. Schematic representation of the molecular architecture of a phospholipid bilayer with and without cholesterol. The graphic representation for phospholipid molecules as in Fig. 5. Cholesterol is represented by a small circle (hydroxyl), a larger circular area (the four-ring steroid skeleton) and a line (hydrocarbon tail). The dimensions are taken from the X-ray data of Levine and Wilkins(83) with egg phosphatidylcholine-eholesterol multilayers. For other explanations and references, see text.

PHOSPHOLIPID VESICLES (LIPOSOMES)

183

it decreases the mobility of a nitroxide probe, while when it is added to dipalmitoyl PC (at 20°C, i.e. below the T~) it increases the mobility of the probe. The ESR spectra of both lecithins in the presence of cholesterol become very similar. The orienting effect of cholesterol was also observed with the polarization of a fluorescent probe, t127~ Hubbell and McConnelF 99~ using spin labels at different positions of the fatty acyl chain have observed a decreasing order parameter in lecithin/ cholesterol membranes toward the middle of the chain. More importantly, it was possible to calculate that, while the region from the glycerol residue up to about eight carbon atoms is relatively rigid, the region further into the interior is considerably more fluid. Similar conclusions were arrived at by Darke et al. ~1°7~ by N M R studies, and a comparison of the effect of cholesterol on the motion of different parts of the acyl chains is shown in Fig. 8. They postulate strong van der Waals interactions between cholesterol and the first ten methylene groups of the phospholipid acyl chains, which severely restricts the molecular motion in this region. The rest of the chain toward the terminal methyl groups are relatively free. Evidence also suggests that cholesterol increases the motional freedom of the glycerol backbone, C107~ as well as that of the phosphate, tt33~ Zull et al. °13~ and Colacicco and Rapport TM*~have obtained infrared evidence concerning the hydroxylphosphate interaction. Another significant conclusion reached by Darke et al. Caxs~was that in mixtures of cholesterol/lecithin at less than 1/1 molar ratios, the cholesterol is not statistically distributed, but it forms clusters with lecithin molecules at presumably 1/1 molar ratio. This physical segregation of lipids into domains of separate phases was also observed with mixtures of phospholipids of different acyl chains t77~ and could be of great importance in the physiology of biological membranes. Although most of the above results were obtained with lecithins, the effects of cholesterol have also been studied with brain lipids, t3x61 sphingomyelinO~ 2.317~ and cerebrosides. 017"~ The results indicate that cholesterol has similar effects with different lipids. In summary, it appears that the incorporation of cholesterol into phospholipid membranes produces considerable restriction of molecular motion in the re#on of the first eight to ten carbon atoms of the acyl chain from the lipid-water interface, leaving the rest of the chains

184

D. PAPAHADJOPOULOS AND H. K. KIMELBERG

relatively free. This effect is accompanied by a condensation of the area per phospholipid molecule, a more perpendicular orientation, and a thicker membrane. These conclusions can easily be rationalized in terms of a molecular fit with space filling models. (31s' 318) The degree of condensation depends not only on the structure of the phospholipidacyl chains but also on the stereochemistry of the interacting sterols. The largest effect is observed with sterols containing a 3-13-hydroxyl group, a planar sterol nucleus and an intact side chain. (319-321' 316. 312) Similar stereospecificity is required for a cholesterol effect in natural membranes. (24°) Figure 8b presents a schematic diagram.

B. Permeability Studies In parallel to the physical studies discussed above, the effect of cholesterol as a membrane component has been investigated in terms of permeability and enzyme activity levels. The results obtained so far from these biologically oriented studies are in general agreement with the physical studies. Taken together, they provide the framework for an evaluation of the possible physiological role of cholesterol. The first reports of the effect of cholesterol on the permeability properties of phospholipid membranes indicated that cholesterol considerably reduced the efflux of CI- through vesicles (47) and the rate of water diffusion through black lipid films3322) Further studies have extended these results and established that incorporation of cholesterol tends to decrease the permeability of phospholipid vesicles to glucose and other non-electrolytes,(237' 66,323) and to monovalent cations.(245,321,48) Cholestero 1 also increases the electrical resistance and capacitance of phospholipid bilayers3324' 32s. 48) As discussed in connection with the physical studies (§ 5A), it appears that the ability of cholesterol to decrease the permeability of lecithin liposomes to glucose and erythitol, similarly depends on the degree of unsaturation. In a study with several pure synthetic lecithins, no decrease in permeability was observed when cholesterol was added (1/1 molar ratios) to highly unsaturated (16:0, 22:6 or 18:2, 18:2 or 18:3, 18:3) lecithins, (3°1) in contrast to the results obtained with other less unsaturated lecithins.

PHOSPHOLIPIDVESICLES(LIPOSOMES)

185

Addition of cholesterol to saturated phospholipids abolishes the abrupt increase in permeability normally observed in the region of the phase transition (97) (see Fig. 6). The results discussed above have established that cholesterol has a general effect on the permeability of phospholipid membranes, producing a decrease for anions, cations, as well as non-electrolyte solutes and water. The general decrease in permeability is in full agreement with the condensation effect and decrease in molecular motion within the membranes. However, the decrease in permeability could result from either a decrease in the distribution coefficient of the solutes between water and membrane matrix and/or a decrease in the diffusion rates through the membrane matrix. Some of the evidence obtained to date indicates that the first step, that is, the dissolution into the membrane (which includes the partition coefticien0, is primarily affected.(235, 48) In spite of the very general effect of cholesterol in decreasing the diffusion rates of all solutes tested, temperature studies have revealed that this effect is very different for the cations compared to anions and non-electrolytes. The enthalpies (or Arrhenius activation energies) for the diffusion of non-electrolytes were found not to be appreciably altered by the presence of cholesterol(235) or by the degree of unsaturation. The conclusion from these results was that the energy of activation for the diffusion process was primarily determined by the step involving the passage of the solute across the water-lipid interface (dissolution) and not by the diffusion through the hydrocarbon interior. On the other hand, it was observed that the enthalpies for the diffusion of the cations (Na +, K +, Rb +) were drastically reduced by the presence of cholesterol, while the enthalpies for CI- and glucose were slightly increased. (48) These seemingly contradictory results were rationalized in terms of temperature-dependent structural changes within the membrane. These changes, such as expansion of the area per molecule of phospholipid with temperature and thinning of the membrane, would be inhibited by cholesterol, with the net result of a decrease in the apparent.activation energy.(48) Non-electrolytes, which are probably diffusing through the bilayer via a different mechanism than the cations (§ 4E), are not affected by such structural modifications to the same degree. The above analysis points out that caution should be exercised

186

D. PAPAHADJOPOULOS AND H. K. KIMELBERG

in the interpretation of the values for activation energy obtained by simple linearization of the Arrhenius plots. All the studies discussed up to this point refer to the effect of cholesterol on pure phospholipid membranes. However, the actual decrease in permeability produced by cholesterol (2-5-fold depending on the solute and the temperature) is not very large, and the resulting membranes are very impermeable compared to most biological membranes. For a more physiologically relevant appraisal of the role of cholesterol, the effect of interacting proteins should be taken into consideration. Recent studies involving several proteins and peptides have produced enough evidence for an initial evaluation of the cholesterol effect. The earliest evidence on the possible role of cholesterol on facilitated transport was reported by Szabo et al. ~326) They observed that t h e presence of cholesterol diminished the effect of the macrotetralide actins, which normally produce a large increase in the conductivity of black lipid films. A similar effect was shown with another ionophoric antibiotic, valinomycin, on the rate of Rb + efflux through vesicles.~2¢s~ These antibiotics are believed to act as cation shuttle carriers and the effect of cholesterol could be rationalized as resulting from an increase in internal viscosity of the membrane. This explanation is supported by the observations that increased unsaturation of the phospholipid increases the valinomycin effect,c2.5) and freezing of the acyl chain below the T~ greatly diminishes the usual increase in conductivity produced by valinomycin.¢2s°) More recently, similar effects have been observed with several proteins. As will be described in the following section, a number of purified proteins, either soluble or extracted from membranes, greatly increase the permeability of phospholipid membranes. ~32v-329) When cholesterol is incorporated into the phospholipid membranes, it greatly inhibits the ability of the proteins to increase the diffusion rates for Na+. ¢2¢6~ This inhibitory effect was observed with several proteins including cytochrome c, lysosyme, albumin, the basic protein from myelin (A1), and also with the ionophoric peptide gramicidin. A comparison of the relative diffusion rates of the same phospholipid with / and without cholesterol and the same amount of protein indicates differences of 1 to 2 orders of magnitude due, presumably, to the presence of cholesterol (Table 3).

PHOSPHOLIPID VESICLES(LIPOSOMES)

187

TABLE3. EFFECTOFVARIOUSPROTEINSON"Na+ PERMEABILITYOFPS VESICLESWITHAND WITHOUTCHOLESTEROL Proteint

22Na+ sel~diffusion rate %per hour PS

PS + chol.

Increase in permeability PS

Ratio:~

PS + chol.

0.06(36°C) 0.02(36°C) 0:02 (24°C) 0.01(24°C) 49.9 136 C) 0.4 (3°C)

780

20

39

0.9 (24°C)

2030

90

22.5

1.8 (36°C)

600

60

10

4.9 (24°C)

765

490

Cytochrome c (10 mg/ml) A~ (basic myelinprotein' 40.6 (24°C) (0.6 mg/ml) 36.0 (36°C) Hemoglobin (0.18 mg/ml) N 2 (proteolipid protein) 15.3 (24°C) (0.36 mg/ml)

1.5

t Protein was added to the preformed vesicles at final concentration as shown in parenthesis. The final concentration of lipid (phosphatidylserine) in these mixtures was approximately 1 #mole/ml. Cholesterol was added before the formation of the vesicles at equimolar amounts to PS. :~ The numbers in this column refer to the ratio of the increase in permeability (PS over PS + cholesterol) obtained with each protein. Data taken from Papahadjopoulos et al. (2+6~ The ability of proteins to increase the permeability of phospholipids was shown to be correlated with the degree of p e n e t r a t i o n into the lipid bilayer. (32s) It would thus seem reasonable to interpret the above effect of cholesterol as an inhibition of the protein penetration into the bilayer. Evidence supporting this interpretation is provided by m o n o layer penetration studies, which show a substantial inhibition of the expansion of phosphatidylserine m o n o l a y e r p r o d u c e d by cytoc h r o m e c, when the m o l a r ratio of cholesterol to phosphatidylserine in the m o n o l a y e r reaches a value of 1/1.(246) This effect seems again t o be related to the increased viscosity of the m e m b r a n e s in the presence of cholesterol, since inhibition of the protein-induced increase in permeability of vesicles can be obtained by reducing the temperature of dipalmitoylphosphatidylglycerol ( D P P G ) below the To3246>The above phospholipid has been shown to activate a delipidated preparation of (Na + K ) - A T P a s e at temperatures above the Te c21°> The ability of the D P P G to activate this enzyme can be drastically inhibited either by lowering the temperature below the T~ or by addition of cholesterol. It

188

D. PAPAHADJOPOULOS AND H. K. KIMELBERG

is thus clear that both protein penetration into phospholipid bilayers and membrane enzyme activity can be inhibited by cholesterol, presumably as a result of the decreased fluidity. The term viscotropy has been proposed to characterize this phenomenon. (21°) Summarizing the above studies, it can be said that cholesterol produces a significant decrease in the permeability rates of most solutes through phospholipid membranes. This is in accord with the physical studies, indicating an inhibition of molecular motion when cholesterol is added to the phospholipids at temperatures above the T~. In addition, the presence of cholesterol diminishes the penetration of several proteins into the phospholipid bilayer. The result of this last effect is a drastic inhibition of protein (or-polypeptide)-induced ion transport and also inhibition of some enzymatic activities.

C. Biological Implications: Aging and Atherosclerosis Although cholesterol is a major constituent of many biological membranes, its physiological role is still not well understood. Because cholesterol and its esters are found in large amounts in the lipid deposits offatty streaks and plaques associated with atherosclerosis, its metabolism has been studied in considerable detail. (For a review see Portman. (33°)) However, its role as a membrane component has been difficult to define clearly. Some evidence indicates that partial removal of cholesterol from erythrocyte membranes increases the osmotic fragility and glycerol permeability. (331) On the other hand, guinea-pig erythrocytes with increased cholesterol-to-phospholipid ratio show decreased permeability to non-electrolytes, active and passive sodium transport. (332) Similarly, increased cholesterol in the membranes of Acholesplasma laidlawii produces a decrease in the permeability rate of glycerol. (24°) However, other studies have produced conflicting evidence indicating that partial removal of cholesterol has no appreciable effect on the hydraulic conductivity (333) or non-electrolyte permeability (334) of erythrocytes. The studies discussed earlier (§ 5B), involving phospholipid vesicles and other model membranes, strongly indicate that cholesterol could regulate the permeability of biological membranes by affecting the

PHOSPHOLIPIDVESICLES(LIPOSOMES) 189 internal viscosity and molecular motion of the lipids within the membrane. The degree of cohesion between the lipid molecules will in turn determine the motional freedom and localization of membrane enzymes, carrier and gating systems involved in transport and other membrane functions. The role of cholesterol can thus be considered as a stabilizing or damping mechanism, inhibiting structural changes in the membrane due to thermal, mechanical, etc., stresses. It is probably not coincidental that the most stable and metabolically inactive membrane (myelin) has the highest (one to one) cholesterol to phospholipid ratio. 03s. B36~Other plasma membranes vary from a ratio of approximately 0.9 for various species of erythrocytes~33v' 338) to ratios of 0.6 to 0.7 for the more metabolically active plasma membranes of liverO39, 340) and non-myelinated nerve. ~34t~ Intracellular membranes have much lower ratios ~34°) compared to plasma membranes. It is reasonable to suggest that, while cholesterol is needed in order to provide a generally stable membrane framework, it is excluded from the areas of membranes involved in active transport and other processes requiring structural fluctuations and rapid molecular motion. Following the same argument, it can also be suggested that increased cholesterol to phospholipid ratio, along with decreased unsaturation (which would tend to enhance the cholesterol condensing effect), could be detrimental to the function of the plasma membrane, and indirectly to the cellular metabolism. Increased cholesterol content, and also decreased unsaturation have been observed to correlate with aging and the process of atherosclerosis3342-3.*) Moreover, a rise in the free cholesterol content is one of the earlier events involving changes of aortic lipid composition in the pre-atherotic lesions, the fatty streaks.~3,5,346) The above evidence, combined with the studi~s on model membranes discussed earlier (§ 5B), and the well-known atherooenic effect of cholesterol-feeding in animals,°4v~ strongly indicates the possibility that increased cholesterol content in the membranes of aortic cells could be involved as an initiator of the process of atherogenesis. A hypothesis to this effect has been advanced recently328°~ Briefly, the hypothesis suggests that increased incorporation of cholesterol into the plasma of arterial intima cells (induced by high levels of circulating plasma ~-lipoproteins and/or endothelial injury) could have a critical

190 D. PAPAHADJOPOULOSAND H. K. KIMELBERG inhibitory effect on several important membrane enzymes. Inhibition of regulatory enzymes, such as the (Na + K)-stimulated ATPase and the adenyl cyclase (which regulate intracellular K + and cyclic AMP, respectively), would result in alterations of the metabolic state of the cells involved, initiating a complex sequence of events. Inhibition of the pump ATPase will result in loss of intracellular K +, and, consequently, to inhibition of protein synthesis and other intracellular enzyme systems, followed by lysis, due to osmotic imbalance and eventual cell death. Impairment of the adenyl cyclase and the resulting lower cyclic AMP levels, will produce inhibition of several intracellular enzyme systems and secretory mechanisms, and would also induce cell proliferation. Many of the early and subsequent events ih the development of atherosclerosis could be accounted for by the initial inhibitory effects of increased membrane cholesterol. Further experimentation, both with model and biological membranes is, of course, needed before the complete role of cholesterol in cell biology and pathology can be fully elucidated.

6,

INTERACTIONS

WITH

PROTEINS

A. Binding Studies The previous sections of this review have described the various resemblances and differences between phospholipid membranes and biological membranes. Although there are many similarities, there are also several important and informative aspects in which they differ. The most obvious difference is the presence of varying, and often considerable, amounts of protein in biomembranes. Thus, it is important to study the functional and structural effects of adding proteins to phospholipid membranes. This approach is, of course, not novel. Pioneers in the field of model membranes had modified their systems by adding proteins. ¢348) Binding experiments are perhaps the simplest method for studying protein-vesicle interactions. In a recent article proposing some interesting new ideas on biological membrane structure, Vanderkooi and Green °49~ emphasized that, although ionic interactions can occur

PHOSPHOLIPID VESICLES (LIPOSOMES)

191

between proteins and biological membranes, using the extensively studied cytochrome c molecule as an example of this, the dominant interactions within lipoprotein membranes must be through hydrophobic forces. Similarly, Singer and Nicolson (123) have suggested that, since more than 70 9/0 of the proteins of most membranes show nonpolar bonding, these proteins are undoubtedly the most important in determining the structure of the membrane. In the case of the most extensively studied membrane, the erythrocyte membrane, approximately 50 9/0 of the proteins have been found to be water soluble, however, indicating some type of polar bonding. (35°-3s 3) A further 25 9/o of the total membrane protein, as well as 5 0 ~ of the total membrane lipid, can be solubilized in 8 i urea. Thus, it would seen premature to discount the large proportion of proteins, which are chiefly bound to the membrane by polar interactions, such as electrostatic bonds or Ca 2+ bridges, as important determinants of membrane structure. It is quite possible that electrostatic interactions between charged groups on proteins and the charged polar head-groups of the phospholipids could determine some aspects of membrane structure. Thus, they could determine, in part, the surface topography by grouping together phospholipids of similar charge or head-group. Also neutralization of charges on the polar head-groups may well influence the behaviour of the fatty acyl chains to some degree. (35.) In the light of the above findings from the red blood cell membrane, it is of interest that the binding of soluble, as well as membrane proteins, to liposomes both show hydrophobic and polar interactions. Soluble proteins have been extensively studied for some time, because of their ready availability and purity. (35s) The work of Reich and Wainio, (3s6) Green and Fleischer, (357) Oas and Crane (35s) and Oas et al. (359) emphasized the importance of electrostatic interactions between cytochrome c and phospholipids. Later work by [-/art eta/. (360) and Kimelberg e t a / . (361) substantiated the predominant electrostatic nature of the interaction between cytochrome c and phospholipid liposomes (multilamellar vesicles). Calculations o27) indicated that, for pure phosphatidylserine liposomes at low ionic strength, the maximum amount of cytochrome c bound could cover each polar interface with a monolayer of protein. Binding of basic proteins to the polar surface.of the lipid bilayers of liposomes is also suggested by physical techniques,

192 D. PAPAHADJOPOULOS AND H. K. KIMELBERG such as electron microscope s t u d i e s (362'361) and X-ray diffraction studies.(a63,364) These studies will be dealt with more fully in § 6C. In this regard, it is of interest that Lenaz eta/. (365) find that the interaction of asolectin liposomes with basic proteins, such as cytochrome c, inhibits phospholipase c hydrolysis, whereas binding of lipid-depleted submitochondrial particles to these liposomes has no effect. This is to be compared with the findings of Glaser et a/. (366) that phospholipase c will hydrolyse 60-70 ~ of red blood cell membrane lipids. Later work, however, using a purer phospholipase c preparation, has indicated that this may be strictly true only for red blood cell ghosts,(367) and not for the original intact cells. Gulik-Krzywicki eta/. (364) using a combination of binding experiments and X-ray diffraction studies concluded that cytochrome c bound to the polar head-groups of aqueous dispersions of phospholipid membranes without perturbing the structure of the phospholipid bilayers. Lysozyme, however, appeared to show both polar and nonpolar binding, with a thinning of the bilayer membrane and a consequent increase in the area per lipid molecule. A similar pattern of binding had been reported by Sweet and Zull for serum albumin. (36s) Marinetti and Pettit (369) studied the binding of ?-globulin and lipids. They reported that this system also showed both electrostatic and hydrophobic interactions, and also indicated that some covalent bonding might be occurring. Hammes and Schullery(37°) made the very interesting observations that polylysine changed from a random coil to an or-helix conformation upon interaction with phosphatidylserine vesicles and that the ---CH2 groups of the phospholipid fatty acyl chains were relatively immobilized, indicating penetration of the bilayer by the or-helical polylysine. The effect of polylysine was also illustrated by electron micrographs showing structural disorganization of the liposomes. Calissano e t a / . (371) have reported that hemoglobin binds to sonicated 75 ~ P S - 2 5 ~ PC vesicles with a mole ratio of protein to phospholipid of 1:3000, at neutral pH. Studies on proteins derived from membranes have indicated that for such proteins either electrostatic or hydrophobic forces or both can be involved. Cytochrome c, a membrane-associated protein, is bound to the inner mitochondrial membrane by electrostatic forces. (372) Zwaal and Van Deenen (373) have shown that the apoprotein from red

PHOSPHOLIPID VESICLES (LIPOSOMES)

193

blood cells interacts with total membrane lipids by electrostatic interactions followed by hydrophobic interactions. A purified protein from red blood cells, spectrin, has been shown to bind to both positively and negatively charged liposomes at neutral pH. C37.) The Tenenbaum and Folch (375) proteolipid was found to form precipitating complexes with anionic phospholipids, which were further stabilized by divalent cations. (376) Lenaz eta/. (377) stressed the hydrophobic nature of the interactions oflipids with the mixture of membrane proteins present in lipid-depleted mitochondria. They found that the binding of mitochondrial lipids or asolectin to lipid-depleted mitochondria was not inhibited by 1 i NaC1. Although not within the scope of this review, studies on the effects of proteins on phospholipid monolayers or planar bilayers have added, and are currently adding, important information to our knowledge of protein-membrane interactions. Such studies date as far back as 1949 with the work of Schulman and his colleagues on protein monolayer interactions,{378,379)and Mueller and Rudin (as°) for bilayers. Reviews on some aspects of the interaction of proteins with monolayers can be found in articles by Dawson, °sl) Jackson, (382) Dawson and. Quinn (383) and Colacicco.t384) Interactions of proteins with planar bilayers are briefly covered in parts of reviews by Henn and Thomson C385)and Goldup eta/. (42) More recent work will be found in experimental papers by Jain, eta/. (386) and Montal. (387) In addition, studies using these systems will be mentioned when they are particularly relevant. B. Effects of Proteins on Permeability of Vesicles

Binding experiments are important in establishing that stable bonds are actually being formed between protein and phospholipid, and are useful in obtaining values for the actual stoichiometries involved. An equally, if not more important aspect, however, is to establish the functional effects of these interactions. One readily available method of doing this is to measure changes in vesicle permeability. Vesicles prepared under conditions which minimize oxidation of unsaturated fatty acyl chains, and which have been exhaustively dialysed, show very low permeabilities to cations, such as ZZNa+. Permeability coeffici-

194 D. PAPAHADJOPOULOS AND H. K. KIMELBERG ents of the order of 10-13 cm sec-1 have been reported3 ls5' ,8) These are to be compared with values of 10-10 cm sec- 1 or less for biological membranes, such as the red blood cell.(tgs) The permeability of liposomes towards anions or uncharged molecules, such as glucose, is appreciably greater. Values of 4 x 10-11 cm sec- 1 have been reported for glucose and 7 x 10 -12 cm sec -1 for C1-. ¢4s) An increasing number of proteins have been shown to increase the cation permeability of vesicles or liposomes to values found for biological membranes. Thus, soluble basic proteins, such as lysozyme or cytochrome c, but not ribonuclease, have been reported to increase the 22Na+ permeability of sonicated, unilamellar vesicles formed from PS by several orders of magnitude, at neutral pH and low ionic strength.iS27) These effects were later correlated with the ability of such proteins to penetrate or expand monolayers of the same phospholipid. <328) Initial electrostatic attraction between protein and phospholipid were required for these interactions. The permeability effects were attributed to subsequent hydrophobic interactions, as indicated by the ability of these proteins to penetrate monolayers at the air-water interface. Furthermore, although a high initial ionic strength inhibited these interactions, increasing the ionic strength subsequent to an initial interaction at low ionic strength had no effect on the protein-induced permeability increase. The permeability increases in the above studies did not appear to be due to a lytic effect, except in the case of polylysine. Hammes and Schullery(37°) also studied the interaction of polylysine with vesicles, as discussed in § 6A. Similar patterns of electrostatic interactions, followed by hydrophobic binding, which in turn lead to large increases in permeability, have been reported for the action of other proteins on vesicles and for different permeant species. Sweet and Zull (236) found that albumin increased the ~4C glucose permeability of lecithin-cholesterol-dicetyl phosphate liposomes at pH values below the isoelectric point of albumin. Since albumin undergoes a general unfolding at these pH values, the effects can be attributed both to electrostatic interactions and the increased availability of sites on the protein for hydrophobic bonding. Large permeability increases for the CrO2,- anion, caused by the lytic cationic polypeptide mellitinJ ass) have also been reported to be due to electroztatic plus subsequent hydrophobic interactions. Disordering

PHOSPHOLIPID VESICLES(LIPOSOMES)

195

of the hydrocarbon region of lipid bilayers by melittin has been shown by ESR techniques33 s9) Kaplan~39o) has very recently reported increases in the permeability of the CrO2,- anion in negatively charged vesicles caused by lysozyme. Calissano et al3371) found that hemoglobin at neutral pH, high ionic strength and very low concentrations (see § 6A), caused large increases in the permeability of 75 9/0 PS-25 ~ PC vesicles to S6Rb +. No effect was seen on glucose diffusion, and other proteins had little or no effect at the same concentrations and under the same conditions. Apart from the case of hemoglobin, most of the preceding work has indicated that soluble proteins initially interact with liposomes through electrostatic forces. This is often followed by hydrophobic interactions, causing distortion of the hydrocarbon region of the lipid bilayer. It is these latter events which we postulate lead to the observed permeability increases. Neglecting the complications of colloidal theory, and its application to charged surfaces,~39x' 392) we can simply consider that electrostatic interactions are relatively strong forces, being inversely proportional to the distance, while hydrophobic forces, such as London-van der Waals dispersion forces, are inversely proportional to much higher orders of the distance, t72) Thus, initially, electrostatic forces could stabilize the protein at the lipid interfacet72) allowing relatively weak hydrophobic bonds to be formed. These in turn result in distortion of the bilayer interior and subsequent permeability increases. If the number of such hydrophobic interactions is large, they can become the dominant attractive forces after the initial electrostatic interaction. It has also been suggestedt32s) that initial electrostatic interactions might induce conformational changes in the protein exposing nonpolar sites, thus increasing the potential for hydrophobic interactions. The effect of proteins in increasing vesicle permeability, which we have ascribed to hydrophobic interactions resulting in distortion of the bilayer, also appears to require fluid fatty acyl chains. Thus, the presence of cholesterol or a lowering of the temperature below the transition point for the phospholipid (such as DPPG) abolishes the cytochrome c-induced permeability increase, as well as partially inhibiting protein-induced expansion of monolayer filmsc246) (see also § 5 on cholesterol). Strong interactions can also be obtained between negatively

196 D. PAPAHADJOPOULOSAND H. K. KIMELBERG charged proteins and negatively charged vesicles in the presence of divalent cations. Juliano et a/. C3291 found that Ca 2÷ increased the effect of very low concentrations of serum albumin on PS vesicles at pH 5.5 and 4.5 and the red blood cell membrane protein, spectrin, at neutral pH. Calissano and Bangham ~393) have reported that S-100, a soluble protein specific to the nervous system, causes permeability increases in PS/PC vesicles at neutral pH in the presence of Ca 2÷ or Mg 2+. It has also been reported ~32s) that at high concentrations albumin can increase the permeability of acidic PS vesicles at neutral pH, in the absence of divalent cations. Two of the proteins mentioned above are associated with biological membranes, namely, cytochrome c and spectrin. These have been considered to be peripheral proteins t1231 as distinct from the more hydrophobic integral proteins. Such peripheral proteins are, of course, popular, because they are easily separated from membranes by manipulations of ionic strength or cation composition. In the case of cytochrome c or spectrin, the conditions which are optimal for interaction with liposomes are similar to those which bind the proteins to their appropriate biological membranes3361, 329) Sweet and Zull (374) reported that spectrin, at a concentration two orders of magnitude higher than used in the above study, was equally effective in increasing the glucose permeability of negatively or positively charged liposomes at neutral pH. Other membrane proteins may well show little or no electrostatic component and interact with phospholipid membranes solely through non-polar binding. Recent unpublished work in this laboratory indicates that the basic AI protein from myelin interacts with PS vesicles more effectively at high ionic strength than low ionic strength, but does not interact with PC (neutral) vesicles. In contrast, however, myelin proteolipid interacts equally well with PC or PS vesicles. Recently, Gould and London t394) have reported that the AI protein increases the glucose permeability of liposomes composed of central nervous system phospholipids. Kinsky and his colleagues have incorporated a number of antigens into multilamellar vesicles or liposomes and ~Ytensively studied the effects of antigen-antibody interactions in increasing the permeability of such liposomes, in the presence of complement. This important aspect of protein-liposome interactions has been recently reviewed by Kinsky33s)

PHOSPHOLIPID VESICLES (LIPOSOMES)

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198 D. PAPAHADJOPOULOS AND H. K. KIMELBERG Thus, we can discern a pattern of interactions ranging from pure electrostatic interactions to pure hydrophobic interactions based on permeability studies and the requirements for electrostatic attractive forces, the effects of ionic strength, interaction of proteins above their isoelectric point, with negatively charged liposomes, and whether neutral (PC) liposomes are affected. Another criterion, which has been used, is the u~e of positively charged liposomes. The effects of some of these conditions on the ability of a variety of proteins to cause permebility changes in liposomes are summarized in Table 4. From consideration of Table 4 there appears to be a continuum of interactions of proteins from those showing only electrostatic interactions to those showing only hydrophobic or non-polar interactions. The latter type is not confined to membrane proteins, moreover, since a soluble protein, hemoglobin, also shows a hydrophobic interaction. Recent studies in this laboratory show, somewhat surprisingly, that hemoglobin interacts with both PS and PC vesicles to give several orders of magnitude increase in 22Na+ permeability. ~2.6) We can identify at least one protein, ribonuclease, which shows only electrostatic interactions ta2v) and no hydrophobic interactions, as indicated by the absence of a permeability effect and limited penetration of a monolayer. °28) Some proteins, which cause permeability effects at low ionic strength, show much less effect at high ionic strength, i.e. lysozyme and cytochrome c. On this basis, we identify an electrostatic-mediated non-polar interaction, the latter involving penetration of the bilayer interior. The interaction of other proteins, such as the basic AI protein, which is derived from the myelin membrane, is not affected by ionic strength, but do require negative charges on the phospholipid surface. The myelin proteolipid, which is extracted by organic solvents, and rendered water-soluble after prolonged dialysis,t375 ' 395)appears to require no electrostatic forces to interact with liposomes. Possible ways in which we think these interactions might occur are shown in Fig. 9. If the preceding mechanism for protein-induced permeability effects is correct, some relationship should exist between the non-polarity of the protein and its effect on increasing liposome permeability. BigelOW 1396) has calculated a parameter, which he terms average hydrophobicity (Hd? av.) for a number of proteins, which is a measure of the non-polarity of a protein. In Table 5, we compare these values for

PHOSPHOLIPID VESICLES (LIPOSOMES)

I

2

3

199

4

FIG. 9. Schematic representation of several types of phospholipid bilayerprotein interactions. (1) Electrostatic binding at the lipid-water interface without hydrophobic associations (e.g. ribonuelease). (2) and (3) Electrostatic binding followed by hydrophobic associations. In case number (2), the hydrophobic contacts between protein and lipid occur at the interface, resulting in an increase in the area per phospholipid molecule, deformation in the acyl chain packing, and thinning of the bilayer. In case number (3), the hydrophobic contacts occur in the interior of the bilayer through penetration of a portion of the protein (e.g. cytochrome c, lysozyme, basic myelin protein). (4) Hydrophobic association not mediated by electrostatic binding (e.g. proteolipid myelin protein, hemoglobin). It is to be noted that all four cases discussed above represent extreme examples, where one type of interaction predominates. Any actual interaction may, of course, involve elements of more than one type. The term hydrophobic associations as used above and in the text is meant to involve all types of polar and non-polar associations excluding charge interactions.

various proteins with the effects on permeability, together with pertinent d a t a on the conditions used. T h e r e is some qualitative correlation, b u t the overall impression is that this c o m p a r i s o n is not rigidly applicable. T h e correlation is very g o o d for ribonuclease, which is least effective in increasing vesicle permeability a n d also is least h y d r o p h o b i c . O n the other hand, a l t h o u g h c y t o c h r o m e c has a relatively high h y d r o p h o b i c i t y index, it is only m o d e r a t e l y effective in increasing vesicle permeability and at higher c o n c e n t r a t i o n s than the other proteins. Thus, it is necessary to postulate a requirement for other factors. O n e possibility is that the spatial distribution of the p o l a r and n o n - p o l a r a m i n o acid residue~ on the protein surface involves segregation of such groups. T h e flexibility of the protein might also be significant, since a m o r e flexible molecule should allow c o n f o r m a t i o n a l changes, which expose h y d r o p h o b i c residues of the protein, after interaction with membranes. Changes from a r a n d o m

200

D. PAPAHADJOPOULOS AND H. K. KIMELBERG

TABLE 5. COMPARISON OF HYDROPHOBICITIES OF PROTEINS AND THEIR EFFECTIVENESS IN INCREASING LIPSOME PERMEABILITY

Protein

Hydrophobicity Liposome leak (n~bav.) to 22Na÷ or

pH

Concentration of protein mg/ml

Refs.

7.4 7.4 7.4 7.5 7.4 4.5 7.4 3.5 7.4

-12.6 5.5 0.5 25.4 0.3 0.3 0.09 0.12

1 1 1 3 1 4 4 2 2

86Rb+ total/hour Ribonuclease (bovine) Lysozyme Hemoglobyn Cytochrome c Serum albumin (bovine) Serum albumin (bovine) Insulin (bovine) Insulin (bovine)

870 970 1090 1110 1120 1120 1180 1180

0.05 0.20 40.0 39.0t 30.0 75.0 2.5 8.8 0.08

t 60 mM KCI, 86Rb + leakage, remainder are 10 mM NaC1, 22Na+ leakage. All liposomes have a negative charge at indicated pH values. References

(1) Kimelberg and Papahadjopoulos.1327~ (2) Kimelberg and Papahadjopoulos (unpublished results, pure PS vesicles, 10 mM NaCI). (3) Calissano et al. ~3711 (4) Kimelberg and Papahadjopoulos.t328~

coil to an a-helix have been demonstrated for polylysineC37°) after interaction with PS liposomes, and for the basic myelin protein after interaction with SDS micelles. ~397) The fact that cytochrome c appears to be such a good example of a protein oil-drop structure, 139s) makes its penetration of phospholipid monolayers~383) and effect on permeability quite surprising, and requires the postulation of some confirmational changes after binding,~3zs) although even cytochrome c possesses some non-polar groups on its surface. (398) Indeed, changes in the CD spectrum ~36a) and mid-point redox potential c214) have been reported for cytochrome c after binding to liposomes. It has been calculated recently that a sample of nineteen membrane proteins has significantly more non-polar amino-acid residues than a much larger sample (205) of soluble proteins3399) It may be, however, that the properties of inteoral membrane proteins are also determined by the topography of their surface polar and non-polar groups. As compared to the classical oil-drop structure of soluble proteins, as recently confirmed for cytochrome ¢,(398) it has been suggested that membrane proteins may be amphipathic in the same sense as phospho-

PHOSPHOLIPID VESICLES(LIPOSOMES)

201

lipids.~,oo, ,ox, 3,~9)This has been shown to be the case for the red-blood cell sialoprotein, ~*°2) microsomal cytochrome bs, t4°3'*°*) and the reduced nicotinamide adenine dinucleotide-cytochrome b s reductase.{*os)

C. Structure of Protein-Vesicle Complexes

The physical and functional properties of unmodified vesicles, and the large number of techniques available for measuring these properties, have been discussed in previo~!s sections of this review. All are potentially capable of studying some aspect of how proteins interact with phospholipid membranes, yielding complementary information. Thus, X-ray diffraction and electron microscopy can be used to investigate how proteins modify the structure of phospholipid membranes. Both techniques will yield information on the overall width of the lipid and adsorbed protein, or the long-range order of the system. In addition, X-ray diffraction can be used to determine (by means of the wide-angle spacings) the liquidity or crystallinity of the fatty acyl chains, tsl'4x°) Differential scanning calorimetry (DSC), already applied successfully to aqueous dispersions of phospholipids and membranes t27) and N M R can measure how proteins modify the fluidity of the hydrocarbon chains. Fluorescent t129) and spin-label probes taa) will yield information on the degree to which the molecular motion of the probe is hindered in its environment, and also, in the case of fluorescent probes, the polarity of the environment. The electron microscope was perhaps the first technique used for studying the structure of protein-liposome complexes. Stoeckenius ~14) and Stoeckenius et al. t36z) studied the complexes formed by the interaction of globin, formed from beef hemoglobin, with brain phospholipid extracts at pH 4.0 by electron microscopy. In the absence of protein, a typical pattern of alternating dark and light areas was obtained in the multilamellar liposomes, or myelin figures as they were termed at that time. According to Stoeckenius et al., °62) the dark or electron dense areas represent the polar head-groups of the phospholipids, and the light areas the fatty acid chains. The addition of protein increased the width of the outermost dark line, indicating adsorption

202

D. PAPAHADJOPOULOS AND H. K. KIMELBERG

of protein to the outer polar head-groups. Increased interperiod spacing for lipid-cytochrome c complexes were found by Sun and Crane. t4°6) Kimelberg et al. t361) obtained an increase in width of all the electron dense areas in complexes of cytochrome c and cardiolipin plus PC vesicles. The average center-to-center distance increase from 45 to 88,~ was consistent with an adsorption of a single layer of cytochrome c between each bilayer. Freeze-fracture electron microscopy has recently been used to study the interaction of rhodopsin with phospholipids, t157) Electron microscope studies are not very accurate as regards the actual increases in width or the relative increases in the hydrophobic fatty acid areas and the polar, head-group areas. Such studies do have the advantage, however, of showing whether a closed vesicular structure for the liposomes is preserved. The addition of protein also seemed to decrease the number of lamellae per vesicle, when added to the multilamellar liposomes, forming a large number of bilamellar or even unilamellar vesicles.~18"361) Early X-ray diffraction studies on phospholipid-histone complexes, ~4°7) and later studies on complexes formed from cytochrome c and acidic phospholipids, showed an increase in the long spacings consistent with one or two layers of protein being intercalated between each phospholipid bilayer. Papahadjopoulos and Miller tl 8) and Shipley eta/. t363) found increased long spacings for PS liposomes from 56/~ to 86 or 115/~, after the addition of cytochrome c. Blaurock t4°8) found an electron dense area on either side of an electron density profile of a bilayer, due to cytochrome c interacting with asolectin. The spacing of 84/~ found in the presence of cytochrome c, was consistent with one layer of cytochrome c separating each bilayer, and in excellent agreement with the 88/~ repeat distance found in the electron microscope studies previously mentioned3361) The detailed work of Gulik-Krzywicki et al. ~364) o n a variety of proteins showed both electrostatic and hydrophobic interactions for lysozyme-cardiolipin complexes. They were able to delineate, from considerations of partial specific volumes, the relative widths of both the protein and lipid phases. In the phase which they termed hydrophobic, they found a decrease in the width of the lipid bilayer and a consequent increase in the area per molecule, assuming no interdigitation of protein into the bilayer. They postulated the presence

PHOSPHOLIPIDVESICLES(LIPOSOMES)

203

of both hydrophobic and electrostatic bonding. Similar results were described by Rand t4°9) for a serum albumin-lecithin--cardiolipin system. In contrast Rand and Sen Gupta, t4°9a) from X-ray diffraction studies of insulin-cardiolipin + PC complexes at pH 3.0, concluded that insulin only showed electrostatic binding. More recent work by Dupont et al. ~41°~ using X-ray diffraction plus a new type of nonphotographic detector, has enabled the kinetics of order-disorder transitions in a lysozyme-PA complex to be determined. These transitions occur with a half-life of minutes. Various spectroscopic resonance techniques have been recently used in studying the structure of protein-phospholipid complexes. The use of such techniques has already been discussed (§ 3A) in reference to pure liposome systems. Hammes and Schullery C37o)examined P S-polylysine complexes by N M R and found line broadening indicating a degree of immobilization of the ---CH 2 groups of the fatty acyl chain. This result was somewhat surprising, in view of the highly charged nature of the polypeptide. This work on model membranes is in agreement with the work of Kamat et al., t i ~7~ who showed that, compared to the dispersion of extracted erythrocyte membrane lipids, the total membranes and reconstituted apoprotein plus lipids showed considerable broadening of the ~ C H 2 line indicating relative immobilization. These authors considered that this evidence indicated non-polar interactions of parts of the lipid chains with membrane protein. There have been a limited number of studies using the technique of ESR. As discussed earlier (§ 5A), the work of McConnell and his colleagues using covalently bound spin labels has led, not only to estimates of the fluidity of the total fatty acyl chains, but also the fluidity of different parts of the chains, an increasing fluidity having been found as one proceeds away from the polar head-groups. This technique has been utilized for the interaction of liposomes with spin-labeled proteins,t41 l) and in a study of the effects of mellitin on lipid bilayers, t3s9} Studies by Berger et al. t412~ on the recombination of red-blood cells apoprotein with red-blood cell total lipid using spir~-labeled stearic acid, showed that recombination at pH 4.0 led to extensive hydrophobic interactions, as indicated by increases in the correlation times. Recently, a spin-labeled phosphatidyl-choline has been used to study the interaction of purified rhodopsin with PC. txsT} Recombination was effected

204 D. PAPAHADJOPOULOSAND H. K. KIMELBERG by mixing the protein and phospholipid together in the. presence of detergent, and removing the detergent by dialysis. The supporting studies, including freeze-fracture electron microscopy, indicated that the protein was penetrating into phospholipid bilayers, and that vesicular structures had been formed. The presence of rhodopsin was found to induce ordering and partial immobilization of the hydrocarbon chains, similar to that found with cholesterol. Gulik-Krzywicki e t al. C412a~ have used free-fluorescent probes and Schechter e t al. c413~ have used fluorescent probes covalently bound to both protein and lipid to investigate the interactions of proteins with liposomes. In the case of the protein used, lysozyme, both electrostatic and hydrophobic interactions were identified by this technique, thus confirming the previous results of the same authors using X-ray diffraction. Azzi e t al. ~414~ reported that cytochrome c-phospholipid complexes enhanced the fluorescence of 8-anilino-l-napthalene sulfonic acid (ANS). A further increase in fluorescence intensity was obtained when cytochrome c reductants were added. This data has, however, recently been criticized. ~129' 415~ Studies quoted in a review by Radda and Vanderkooi ~129~ on fluorescent probes and membranes indicated that quenching of fluorescent probes could be used to study the interactions of cytochrome c with acidic lipids34~ 6~Other workers have shown quenching of fluorescent probe-labeled cytochrome c,~4~7~ or decreased rotation rates for dansylated lysozyme~418~after interaction with phospholipids. Spectroscopic techniques, such as circular dichroism, have been applied to aqueous protein-liposome complexes. In the case of cytochrome c,~364~ the changes found were ascribed to heine-heine interactions. In the case of a polylysine-PS complex, (37°~ the changes were very clearly due to a random coil to 0t-helix transition for the polylysine. The use of differential scanning calorimetry has had limited application in investigating protein-vesicle interactions. The only study to date known to the authors is that of Chapman and Urbina, c354~ who found that the transition temperature of a cytochrome c-phospholipid complex was around 30°C, as compared to 40°C for the phospholipids alone. The cytochrome c thus appears to make the phospholipids more fluid. The fact that proteins might increase the fluidity of bilayers is consistent with their effect in increasing permeability, since increasing

PHOSPHOLIPIDVESICLES(LIPOSOMES) 205 fluidity of the hydrocarbon interior ofbilayers usually leads to increased permeability. As discussed above, however, it has been observed that some proteins decrease the mobility of CH 2 groups asdetermined by N M R C37°'117~ and EPR3157~ This is similar to the effect of cholesterol, which, as has been described in § 5, decreases the permeability of liposomes to a variety of permeants. Clearly, more data is needed on this subject. A systematic study using a variety of techniques on the interaction of several proteins, showing different types of interactions (see Table 4), would help to clarify this relationship between the structural and functional effect of proteins.

D. Interactions of Enzymes with Vesicles This section and the next two sections will cover the more functional aspects of protein-vesicle interactions. Since we are concerned with the interactions of proteins with vesicles or liposomes, the extensive body of work concerned with lipid-dependent enzymes is clearly relevant. This is due to the fact that phospholipids are usually added to lipid-depleted enzymes in the form of water dispersions, i.e. as vesicles prepared either by sonication or dialysis of a butanol-cholate mixture against water, ¢4xgJ and also, of course, because lipid-dependent enzymes represent an important component of biomembranes. For other recent reviews on the subject of the involvement of lipids in membrane enzyme function, and to supplement this necessarily incomplete coverage of a wide area, the reader's attention is drawn to reviews by Rothfield and Romeo t42°) and Kagawa, t421~ who discuss reconstitution of mitochondrial enzyme activity and oxidative phosphorylation. The review by Razin 422 covers methods of solubilization of membrane proteins and reconstitution of enzyme activity and membrane structure. (See also Steck and Fox ~423~and Triggle. ~424~) Much of the earlier studies on the subject of lipid-dependent enzymes utilized proteins obtained from mitochondria. The proteins of the mitochondrial membrane are especially convenient in that many of them, in addition to showing phospholipid-dependent enzyme activity, are also easily characterized spectrophotometrically. Thus,

206 D. PAPAHADJOPOULOS AND H. K. KIMELBERG their purity can be conveniently measured during isolation and their reassociation with phospholipids measured by the increase in their activity. The activity of most of the mitochondrial enzymes was found to be inhibited after extraction with organic solvents, such as a 90 ~o acetone/10~o water mixture3357} The phospholipids have also been removed from mitochondrial enzymes, such as cytochrome oxidase, by the use of detergents. A variety of detergents and conditions have been used, including 3 ~o potassium cholate at 30°C, {425) 2 ~o sodium deoxycholate ~3s6) or triton x-100 and x-114. ~426) The specificity of the phospholipid requirement for reactivation has been found to vary with the different enzymes. D(-)[3-hydroxybutyrate dehydrogenase was the only mitochondrial enzyme showing an absolute specificity, a requirement for PC. Furthermore, synthetic lecithins containing saturated fatty acyl groups were much less effective than egg lecithin, which has considerable unsaturation in its fatty groups, t427~ The authors suggested that this may be due to the fact that, at temperatures below To phospholipids do not form aqueous dispersions. Early work indicated that cytochrome oxidase showed a specificity for certain phospholipids. {3s6,42s) Awasthi e t al. t429) have recently implicated cardiolipin as intimately involved in cytochrome oxidase activity; obtaining a mole ratio of 1:1 of tightly bound cardiolipin to cytochrome oxidase that is essential for enzyme activity. An isooctane-extracted cytochrome c-phospholipid complex was considered to be slightly more enzymatically active than free soluble cytochrome c towards lipid-free cytochrome oxidase, t426) In the latter case, t4°6) phospholipids were added during the assay. Other mitochondrial enzymes, including succinic dehydrogenase t43°-432) and mitochondrial ATPase (adenosine triphosphatase),C4a3, 434~ have also been shown to be phospholipiddependent. More recently, a wide variety of other membrane-bound enzymes have been shown to become lipid, and more specifically, phospholipiddependent, after extraction and partial purification. Such extraction of membrane proteins, usually employing detergents, such as deoxycholate, cholate or triton, has been shown, under certain conditions, to result in the removal of considerable phospholipid343s' 436~ In the case of sodium deoxycholate, it has been shown that proteins and phospholipids are solubilized independently, with the phospholipids forming

PHOSPHOLIPIDVESICLES(LIPOSOMES) 207 mixed detergent-phospholipid micelles343~'4aa) (Also s e e R a z i n t422) for references to other studies of detergent action on membranes.) Many of these enzymes also seem to require specific phospholipids, implying that the phospholipids may be performing a specific catalytic role, as well as the non-specific role of providing an environment of low dielectric constant. The most extensively studied of the enzymes that seem to have specific phospholipid requirements is the (Na + K) ATPase, This is a vital enzyme localized in the plasma membrane, which is responsible for maintaining cell volume and a constant internal ionic environment.t439, 440) Early work t441) indicated that PS was most effective in restoring activity to the extracted enzyme. Later reports, however, showed that other phospholipids, and also cholesterol, were also effective. c442-449) A specific requirement for PS, however, has also been confirmed3450, 4sl, 2~0) Goldman and Albers t45z) reported that PS was necessary for formation of the phosphorylated reaction intermediate. PS has also been specifically implicated for the reactivation of the partially purified adenyl cyclase from cat heart that had been solubilized with Lubrol-PX. t453) PS was found to increase adenyl cyclase basal activity and restore glucagon responsiveness, but not the stimulation by nor-epinephrine. R6thy e t al. ~4s4) have found that PS would partially restore hormonal and F - responsiveness to extracted, inactivated adenyl cyclase from rat liver plasma membranes, while PI restored the basal activity. Diphosphatidylglycerol and other acidic phospholipids, such as phosphatidylserine and phosphatidylinositol, were most effective in reactivating cholate-solubilized mitochondrial ATPase. t434) PG has been shown to be most effective in activating the phosphoenolpyrurate-dependent phosphototransferase enzyme from bacteria t455), and has also been recently shown by Kimelberg and Papahadjopoulost2 ~o) to be as equally effective as PS in restor.ing activity to detergenttreated, delipidized (Na + K) ATPase. Acidic phospholipids are most effective in stimulating the activity of solubilized rat liver liposomal lipase, t456) PC has been shown to stimulate the DPNH-cytochrome c reductase activityof acetone extracted microsomes, ~457)and the Ca 2+ ATPase from sacroplasmic reticulum,t45s)A varietyof enzyme activities have been shown to be inhibitedby phospholipase treatment,indicating a phospholipid requirement. These include several E. coli membrane

K*

208 D. PAP~,HADJOPOULOS AND H. K. KIMELBERG enzymes459 and mammalian plasma membrane enzymes,t43s) such as (Na + K) ATPase. Phospholipids have been shown to be required in the functioning of a variety of bacterial membrane enzymes. These include glycosyl transferase enzymes from gram-negative bacteria t46°-~62) and the E. coli phosphotransferase enzyme mentioned above, t4sS) Phospholipids also appear to be required for the functioning of enzymes involved in cell wall synthesis,t46a-464) Thus, there is a continuously growing list of both bacterial and mammalian membrane-bound e n z y n 3 c s , (4"22'424) which have been found to be phospholipid-dependent after extraction, and there would appear to be growing evidence that the majority of membrane-bound enzymes require lipid or phospholipid as structural and/or functional components. An attempt at a rationale for the specificity of such phospholipid requirements has recently been made in the case of the (Na + K) ATPase. The specificity for PS and PG shown for this enzyme(21°) correlates with a recent observation by Papahadjopoulos ~2°9~ that these phospholipids are unique in showing up to a 10-fold discrimination for K + permeability over Na + permeability (see Fig. 10). Thus, it is tempting to speculate that the PS head-groups may function as the recognition site or a K +-specific pore. In the case ofa PS-lined channel, the small binding preference (K+/Na + = 1.3 at pH 7.4) found,~2°9) could result in a large preference for K + being shown as a final flux through such a channel. The finding that sulfatide can discriminate in favor of Na, C2°1) together with evidence that sulfatide may be specifically associated with (Na + + K +) ATPase in some tissue,t46S)indicates that sulfatidc might function as a Na+-specific site. The effects of the fluidity of the fatty acyl chain on (Na + K) ATPase activity were examined using PG with different fatty acyl chains. It was found that below Tc of dipalmitoyl or distearoyl PG, where the fatty acyl chains are in a frozen state (sce previous section), the (Na + K) ATPase was inhibited, causing discontinuities in the resultant Arrhcnius plot. No discontinuity was obtained with dioleoyl PG, where a phase transition should occur at ~ - 10°C. These data are shown in Fig. 11. The presence of cholesterol, in a 1:1 ratio with DPPG, inhibited (Na + K) ATPasc activity at all temperatures t21°) (see previous section). Priestland and Whittam ~466) have also reported effects of

PHOSPHOLIPIDVESICLES(LIPOSOMES)

209

10

9 8 7

6 5

4 3

.....

2

iiii

1

n --

PC

PE

il

P$

LIt PG

PI

PA

.gulf.

FIG. 10. Comparison of the 42K +/22Na+ permeability selectivity ratios for different lipids and their reactivation of (Na÷+ K ÷) ATPase. Stippled columns: 42K÷/22Na÷ ratio of diffusion rates through sonicated vesicles. Open columns: activation of (Na ÷ + K ÷) ATPase expressed as the ratio of activity with lipid added, over activity without lipid. No lipid control: 0.8 laMP/mg protein/hour. Preparation from frozen rabbit kidney cortex. Protein was estimated by the buret method. Abbreviations: PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PA, phosphatidic acid; sulf., sulfatide. (From Kimelberg and Papahadjopoulos. ram)

temperature on the activation of detergent-treated (Na + K) ATPase preparations by PS with a discontinuity at around 15~C, also shown in Fig. 11. G a r l a n d and Cori ~436) have reported that lipid-depleted glucose6-phosphatase is only fully reactivated with PC which contains unsaturated fatty acyl chains. In the case of the (Na + K) ATPase, the model system closely resembles some biological systems, since temperature effects and biphasic Arrhenius plots of (Na + K) ATPase activity(467, 46s) and active sodium-potassium transport ~469) have been found in m e m b r a n e preparations from a variety of sources with discontinuities occurring at or around 20°C.

210

D. PAPAHADJOPOULOS AND H. K. KIMELBERG 50

.50

40

30

20

10

,

O °C

T

4O

3ol

A 0

22o

©PG

ZlO

W _°00

DPPG

PS

-10

DSPG •

33

I

3.2

,I

I

I

33

34

3,5

,

I

36 +xlO 3

FIG. 11. Arrhenius plots of PS and PG-activated (Na + + K +) ATPase, with varying fatty acyl chain substitutions for PG. Phospholipid-depleted (Na + + K +) ATPase was prepared from frozen rabbit kidney outer medulla by a combination of published methods. (497'~*°) Activity at 40* was 100-120 ~tM Pi/mg protein/hour in the presence of PS or PG. Reaction medium was 100 mM NaCI, 10 mM KCI, 0.1 mM MEDTA, 2 na~ MgC12 and 50 mrd Trisacetate pH 7.1. (Na + + K +) ATPase was measured as the amount of Pi appearing during a 20-minute incubation period + 1 mM ouabain after the reaction was terminated with trichloroacetic acid. For experimental details see Kimelberg and Papahadjopoulos. (2~°) The amount of enzyme protein present was 0.028--0.056 nag in a total volume of 1.5 ml. The abbreviations and lamoles of sonicated phospholipid vesicles added are as follows: DOPG, dioleoylphosphatidylglycerol, 0.18 ~tM; DMPG, dimyristylphosphatidylglycerol, 0.24 laM; DPPG, dipalmitoylphosphatidylglycerol, 0.59 laM; DSPG, distearoylphosphatidylglycerol, 0.64 laM; PS, beef brain phosphatidylserine, 0.64 laM. (Kimelberg and Papahadjopoulos, J. Biol. Chem. in press). A variety of mitochondrial enzyme activities have been shown to e x h i b i t b i p h a s i c A r r h e n i u s p l o t s w i t h t h e d i s c o n t i n u i t i e s in t h e c u r v e a l s o o c c u r r i n g at a r o u n d 20°C347°) S t r u c t u r a l c h a n g e s h a v e b e e n

PHOSPHOLIPID VESICLES

(LIPOSOMES)

211

detected in the same temperature range for rnitochondria using E P R techniques.{100,47 i) Blazyk and Steim,c4v2)however, using diffcrential scanning calorimetry, have found a broad endothermic transitionfor rat liver mitochondria centered at 0°C. Thus, if the temperaturedependent discontinuitiesin enzyme activityare due to phospholipid phase transitions,it is necessary to postulate a phospholipid microenvironment for the enzymes in which the enzymatically relevant transitionsare occurring. It is of interestthat such a rnicroenvironment has recentlybeen described for the (Na + K) ATPase by Grisham and Barnett~4va) using E P R techniques. The use of bacterial mutants has shown that temperature-dependent discontinuitiesin enzyme activity or function vary with the phase-transition temperature of different fattyacids incorporated into thc microorganisms. This has been shown for cytochrome oxidase in yeast{2s9) and several bacterial transport systems.(2sa,2s6,12s)Thus, all the enzymes studied to date, namely; (Na + K) ATPase, mitochondrial and microsomal enzymes and bacterial transport enzymes seem to require a fluid hydrocarbon region for optimal activity.The term viscotropic~21°) has been suggested to describe the control of enzyme activity by membrane fluidity. It has already been mentioned that since the phospholipids are added in the form of aqueous dispersions, the above enzymes are in fact interacting with liposomes. The question arises of whether the liposomc structure is retained after such interactions. At least two possibilitiesfor perturbation exist.One, the ezyme protein can interact with and deform the bilayer,but the vesicularstructureof the liposomes (multilamellar,unsonicated) or vesicles(unilamellar,sonicated) is still retained. Second, that residual detergent or enzyme plus detergent might result in structuralbreakdown of the liposomes or vesicles.The occurrence of specificpermeability properties.invesiclesreconstituted with mitochondrial enzymes, ~4v4) or the vesicular structures obtained with other solubilized membranes after removal of detergent~422~ suggests that this is not an important problem. Estimates of residual detergent remaining, such as deoxycholate, show that it can be quite low; 9 0 - 1 0 0 ~ removal ~43s) or 40 Ixg/mg protein(47, s) after 24-hour dialysis. The use of XAD-4 columns can reduce the residual deoxycholate to 0.05-0.07 ~.{436) The large amount of phospholipid required

212

D. PAPAHADJOPOULOSAND H. K. KIMELBERG

in most cases, in order to obtain maximum reactivation, might imply that the enzymes are indeed interacting with intact vesicles. Thus, it is quite possible that some of these membrane proteins are reconstituting their normal trans-membrane functions. In the case of the (Na + K) ATPase, this would involve the active exchange of Na + and K +. Such oriented interactions have been demonstrated in planar bilayers for the (Na + K) ATPase3386) The reconstituted bilayer exhibited zero-volt current in the presence of cations and ATP, which was inhibited by ouabain. Since membrane fragments were used, and the results were very variable, it seems likely that the (Na + K) ATPase containing membrane fragments were incorporated as membrane units into the bilayer. Hong and Hubbell ~157~ made the important point that true recombination of membrane proteins with phospholipids should involve, as a minimal condition, both removal of detergent and removal of native phospholipid. Romeo et a/3461-462~ demonstrated the reconstitution of an active phospholipid-dependent glycosyltransferase-lipopolysaccharide-phospholipid enzyme complex from gram negative bacteria under a monolayer of phospholipid at the air-water interface. Apparently, the interaction at such a monolayer facilitated the formation of an active complex.

E. Encapsulation of Proteins and Enzymes as Models for Cellular Organelles It has been mentioned in a recent review on the subject of reconstitution of biological membranes that the "successful reconstitution of a vesicular biomembrane" would be highly desirable3422) Although a complete reconstitution of a biomembrane in the sense of a red-blood cell membrane or other cell membrane is not yet within the scope of the "liposomist's" art, it is gratifying to see how much progress has been made in reconstituting portions of certain biomembranes. This has been most successful in the case of the inner mitochondrial membrane, undoubtedly due to the availability of the protein components in purified and partially purified forms. Cytochrome c, as noted previously, has been extensively used in studies on protein-phospholipid interactions. It has several practical

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213

and biological advantages. It is quite soluble and can be obtained commercially in a highly purified form. Its concentration can be rapidly estimated spectrophotometrically. In the mitochondria it is bound to the membrane mainly by electrostatic interactions.(a72) It is reduced by the dehydrogenase portion of the electron transport chain and oxidized by cytochrome oxidaseJ 476~ Thu~, it is a soluble, membraneassociated protein, which is enzymatically active. It is also quite basic with an isoelectric point at 10.6, and, consequently, has often been used as a model for the interaction of basic proteins with acidic phospholipids (see §§ 6A-C). Early work on cytochrome c indicated that, when bound to the mitochondrial membrane, it showed different properties from that of the extracted soluble cytochrome c.t477~ Later work indicated further that mitochondrial cytochrome c showed different properties from cytochrome c in submitochondrial particles, t47s-479) Many of these differences appeared to be due to the inverted topography of the vesicles, as compared to the mitochondria,t4s°) The cytochrome c, which is on the outside of the inner mitochondrial membrane, is on the inside surface of the submitochondrial vesicles. A simplified model system for submitochondrial vesicles using diPG/PC liposomes has been described, with cytochrome c trapped inside the diPG/PC vesicles.¢4st'361,2t4) In this form, it shows many of the properties of cytochrome c in submitochondrial vesicles, including lack of extraction by 0.15 lvi KCI,!47s) lack of reduction by ascorbate except in the presence of lipid-soluble redox dyes, such as PMS (phenazine methosulfate) or TMPD ( N , N , N 1 , N l - t e t r a m e t h y l - p - p h e n y l e n e diamine),(*s2~ and a more negative mid-point oxidoreduction potential, t-s3) More recently, the functional cytochrome c-cytochrome oxidase segment of the mitochondrial membrane has been successfully reconstituted from its constituents and phospholipids in the presence of cholate, and shows some of the energy-linked functions of mitochondriaj4S,, 485,474~ The uncoupler-sensitive ejection of protons and uptake of K + in the presence of valinomycin, shown by these reconstituted vesicles is identical to the behavior of mitochondria under the same conditions,t4s4) Jasaitia et al. c4s5) showed electron transportdependent uptake of an anionic dye, which.they ascribed to the formation of a membrane potential. They also reconstituted mitochondrial

214

D. PAPAHADJOPOULOS AND H. K. KIMELBERG

ATPase containing vesicles, showing ATP-dependent dye uptake. These reconstitutions of membrane-dependent energy-conserving reactions, using only the redox components of the electron transport chain or ATPase plus phospholipid liposomes, constitutes promising preliminary steps in the reconstitution of other functions of the mitochondrion. A recent review of work on reconstitution of oxidative phosphorylation, using purified ATPase, submitochondrial particles, coupling factors and phospholipids has been given by Kagawa3 .21) Sessa and Weissmann t*ssa) have been successful in reconstituting one of the properties of lysosomes. They incorporated lysozyme, a positively charged soluble enzyme found inside lysosomes, inside positively charged liposomes. This is an example of incorporation, which is not facilitated by charge attraction between protein and phospholipid. They found that the lysozyme showed latency, a property characteristic of the enzyme inside lysosomes, hydrolysing its substrate only after the membrane barriers were ruptured. Using similar techniques, Gregoriadis et al. ~486) have incorporated albumin and amyloglucosidase into phospholipid vesicles. They have emphasized the therapeutic implications of such techniques, since a therapeutically important agent can thus be masked, both with regard to its own effectiveness and against enzymic attack. The occurrence of an immunological response while in the bloodstream can also be avoided until the agent reaches the preferred site of action. Bangham (487) discusses an early description in 1932 of a lipid emulsion containing strophantin, which was suggested as a good system for obtaining gradual release of this and other drugs after injection. There are, however, important practical problems involving both lipolytic attack on the liposomes in undesired areas, and PoSsible toxic effects of the phospholipids and phospholipid metabolic end-products themselves. Gregoriadis and Ryman t4ss~ have reported, however, that 131I-labeled albumin and (3H) amyloglucosidase entrapped in liposomes showed no leakage into the circulation when injected into rats, and concentrated preferentially i n liver and, to a lesser extent, in spleen cells. Within the liver it was found principally in the mitochondrial-lysosomal fraction. Thus, this technique shows some promise for therapeutic use although it remains to be seen whether it is possible to alter conditions so that the liposomes can be directed to other sites.

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F. Biological Significance of Protein-Vesicle Interactions In § 6A--C we nave compared the interactions of soluble proteins, such as lysozyme and albumin, and the interactions of membrane-associated proteins such as cytochrome c and spectrin, with phospholipid vesicles. The interactions of the latter two proteins with phospholipid membranes appears to mimic their normal binding to their respective cellular membranes. For these membrane proteins at least, their interactions with phospholipid bilayers are good models for their interactionswith biological membranes. Soluble proteins, such as !ysozyme and albumin, are as effective, or even more effective than cytochrome c and spectrin, in perturbing and interacting with liposomes. It is debatable, however, whether under normal conditions these proteins would be able to interact with and perturb preformed cellular membranes in situ. On the other hand, polylysine has been reported to cause selective increases in permeability to small molecules, such as K + or Na +, in Ehrlich ascites tumor cells, ~489~ and plant tissue349°~ Membrane and permeability effects have been noted for polylysine with other cellular membrane systems3491-494~ The general conclusions that interaction of soluble proteins with liposomes requires initial electrostatic interactions followed by hydrophobic associations may apply to many membrane proteins, and has been shown to occur in the recombination of extracted erythrocyte membrane apoprotein with an erythrocyte membrane lipid extract3373~ Studies with several soluble proteins have established the important point that the ability of a protein to increase the permeability of a lipid membrane is related to its ability to expand and/or penetrate into the lipid bilayer332s~ The interaction of hydrophobic proteins from cell membranes with liposomes is the next logical step in the study of protein-liposome interactions. The large amount of effort now being • expended to characterize and purify these proteins should hasten work in this direction. Much work has already been done on partially purified lipid-dependent membrane enzymes, such as the (Na + K) ATPase, as well as total erythrocyte apoprotein and myelin proteolipid, which has been discussed in ~ 6B and 6D. It is of interest that the myelin proteolipid seems to show exclusively hydrophobic interactions with liposomes.

216

D. PAPAI-IADJOPOULOS A N D I-I.K. K I M E L B E R G

W o r k on the (Na + K) ATPase and other membrane enzymes has led to conclusions as to the importance of both the head-groups and the fluidity of the fatty acyl chains, even though the impurity of the enzyme preparations and the presence of residual detergent has been an unknown factor in this work. The findings of a requirement for fluidfattyacyl chains has been mirrored in work on whole membranes. The requirement for a growth temperature above the transition point of the fatty acids in mycoplasma, c78)and increasing data on biphasic changes in Arrhenius plots of enzyme activity(§6D), can be rationalized most simply in terms of temperature-dependent phase transitions of the fattyacyl chains of phospholipids (§ 3A) affectingthe functioning of membrane enzymes. These phospholipid phase transitions have been used to show association of specificphospholipids with certain enzyme activities. Thus Esfahani et al. t12s) showed that discontinuities in the Arrhenius plots of proline transport reflected the unsaturation of the supplemental fatty acids, whereas succinic dehydrogenase activity did not. Lenaz e t a / . ¢47°) have shown that different mitochondrial enzymes show in their Arrhenius plots discontinuities at different temperatures. Thus, the work to date on protein-liposome interactions shows the following points of resemblance with protein-phospholipid interactions in biological membrane : (1) Interactions with most proteins show both ionic and hydrophobic binding. (2) The incorporation of protein increases permeability and decreases electrical resistance of phospholipid bilayers to the levels found in cellular membranes. (3) A significant proportion of phospholipids in both systems appears to be in the bilayer form with fluid fatty acyl chains at temperature > T~. (4) Hydrophobic interactions of proteins with liposomes a n d incorporation of enzymes into membranes both require fluid fatty acyl chains. (5) Enzyme activity of membrane proteins is dependent on the fluidity of the fatty acyl chains in both liposomes and biological membranes. (6) Many enzymes in addition, show a preference for specific phospholipid head-groups when reacting with liposomes. Such

PI4OSI'HOLIPIDVEsicLEs (LIPOSOMES) 217 specificity, although indicated, has not been unequivocally shown in biological membranes. (7) Latency of enzymes towards substrates, characteristic of some enzymes associated with subcellular organelles, can be mimicked by enclosing such enzymes inside liposomes. It is reasonable to expect that much future work will follow on the interaction of purified membrane proteins and enzymes with phospholipid vesicles. Such studies should prove an important experimental approach in dissecting out the details of lipid-protein interactions in biological membranes.

POSTSCRIPT The reader who has managed to remain with us to the end with some degree of comprehension and, we trust, some enlightment, deserves both congratulations and an apology. This formidable compendium of published work is still only a limited review of the field of phospholipid membranes, concentrating primarily on subjects of personal interest, and tinted by past experience and prejudice. It is a fair testimony to the explosive pace at which new and interesting ideas in science are now exploited. Certainly the current degree of interest in this area cannot be considered to antedate the work of Mueller and Rudin and their associates in the early 1960s and that of Bangham and his colleagues in the mid-1960s. In fact, the pace did not pick up until almost 1968. It might appear now that a formidable degree of expertise and instrumentation is required to make any contribution in this area. We hope, however, that we have indicated that the future of this area to a large extent lies in its applications to more biological problems. In this context, the sentiments of David Keilin seem quite appropriate: "The great complexity of living cells and organisms leaves ample opportunity for those not provided with the most modern facilities to make fundamental contributions to the subject by postulating new problems and discovering new paths of approach. There will always be room for such individual workers, however highly organized research becomes. ''~49s~

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D. PAPAHADJOPOULOSAND H. K. KIMELBERG

ABBREVIATIONS A T P a s e for adenosine triphosphatase; D N P H or N A D H for nicotinamide adenine dinucleotide, reduced; D P P G for dipalmitoylphosphatidylglycerol; D S C for differential scanning calorimetry; E D T A for ethylene diamine tetracetic acid; E P R for electron p a r a m a g n e t i c (spin) resonance; N M R for nuclear magnetic resonance; P A for phosphatidic acid; P C (lecithin) for phosphatidylcholine; P E for p h o s p h a t i d y l e t h a n o l a m i n e ; P G for phosphatidylglycerol; P I for phosphatidylinositol; PS for phosphatidylserine; Stare for stearyla m i n e ; Tc for critical temperature (melting of the fatty acid chains); T N B S for trinitrobenzene sulfonic acid; X A D for xanthin-adenine dinucleotide.

ACKNOWLEDGEMENTS Some of the w o r k reported here was supported by grants from the N a t i o n a l Institutes of Health No. G M - 18921-01 and from the American H e a r t Association (in part by the M i d - H u d s o n H e a r t Association) No. 69730. We thank Mr. T. Isac and Mrs. R. L a z o for their expert technical assistance, and Miss Shirley M a t y a s for typing the manuscript. We are also indebted to Mrs R. L a z o for invaluable editorial assistance.

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