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Invited Feature Article A Tribute to the Phospholipid Fredric M. Menger,* Mary E. Chlebowski, Ashley L. Galloway, Hao Lu, Victor A. Seredyuk, Jennifer L. Sorrells, and Hailing Zhang Department of Chemistry, Emory University, Atlanta, Georgia 30322 Received April 1, 2005. In Final Form: June 9, 2005 Introduction Proteins and nucleic acids receive so much attention and hype that a third biological building block, the phospholipid, might well be suffering from an inferiority complex. Phospholipids have a trivial structure (Figure 1), and this has certainly not added to their self-esteem. Moreover, phospholipid molecules cannot fold into interesting coils, cannot catalyze reactions, cannot duplicate themselves, and cannot transport oxygen. Nonetheless, one should feel no pity for the seemingly mundane phospholipid. Living systems could not have evolved until their biochemistries had been enclosed within lipid membranes. This is not to relegate the membrane merely to “a sausage casing with the interesting stuff inside”, which would grossly understate the case for phospholipids. Actually, the cell membrane is a remarkable community of molecules, embedded in a structural motif called a bilayer (Figure 2), where multiple types of dynamic events take place. Motions of proteins and nucleic acids might seem rather dull when compared to those within phospholipid self-assemblies, as briefly summarized below. Phospholipid bilayers undergo a melting-like phase transition at a temperature designated Tm.1 Below Tm, saturated lipids in the bilayer exist in the gel or “solid state” with linear all-trans chains. Above Tm, the bilayer exists in the more disordered liquid crystalline or “liquid state” with several gauche C-C bonds in each carbon chain. The transition is accompanied by decreases in membrane thickness,2 NMR line widths, and ESR-based order parameters3 along with enhanced membrane permeability.4 Values of Tm (e.g., 42 °C for DPPC in Figure 1) increase with chain length and decrease with chain unsaturation.5 Relaxation times of chain methylenes near the center of the bilayer are 100 times faster than those near the polar headgroup.6 As a unit, the lipid molecule can rotate, rock, diffuse laterally, flip-flop, and engage in interbilayer migration (Figure 3). Thus, phospholipids rotate around the long molecular axis at 108-109 Hz above Tm and at 2 orders less than this below Tm.7 More importantly, lateral mobility within one of the two bilayer “leaflets” allows a
Figure 1. Structure of dipalmitoylphosphatidylcholine (referred to in the text as DPPC), a typical phospholipid.
lipid molecule to (a) drift to a site of action (e.g., to a lipiddependent protein); (b) accommodate a morphological change (e.g., pseudopod formation or membrane fusion); and (c) assemble, along with other membrane components, into domains or “rafts”.8 Many factors, including cell type, affect lateral diffusion rates, but they are typically quite fast. Thus, lipids can cross an entire cell surface in a few minutes.9 Phospholipids can also flip-flop (a scientific term) whereby molecules jump from one bilayer leaflet to the other.10 It may take days for a molecule to flip-flop unless an enzyme (a “flipase”)11 catalyzes the process or large amounts of cholesterol are present. Similarly, the exchange rate of lipid molecules between two membrane bilayers is slow: spontaneous interbilayer transfer has half-times ranging from 2 to 24 h or more depending upon the particular lipid and temperature.12 One final technical point by way of introduction is the following: phospholipids form water-filled spheres (Figure 2), called vesicles or liposomes, with single-bilayer (“unilamellar”) shells. Vesicle diameters depend on the mode of preparation: (a) sonication gives small unilamellar vesicles (SUVs), 30-50 nm; (b) extrusion through a porous membrane gives large unilamellar vesicles (LUVs), 100200 nm; and (c) electroformation gives giant (cell-sized) unilamellar vesicles (GUVs), 5-200 µm. All three vesicles types have been used in our work. In concluding the section, it is necessary to define what to expect from this article. First, we did not intend to write an exhaustive review but rather to summarize our own work. Thus, what follows resembles a lecture in content. Because we address herein sundry questions and exploit diverse methodologies, there is also a review aspect
* Corresponding author. E-mail:
[email protected]. (1) Nagle, J. F. J. Chem. Phys. 1973, 58, 252. Nagle, J. F. Annu. Rev. Phys. Chem. 1980, 31, 157. (2) Janiak, M. J.; Small, D. M.; Shipley, G. G. Biochemistry 1976, 15, 4575. (3) Seelig, A.; Seelig, J. Biochemistry 1974, 13, 4839. (4) Papahadjopoulos, D.; Jacobson, K.; Nir, S.; Isac, T. Biochim. Biophys. Acta 1973, 311, 330. (5) Barton, P. G.; Gunstone, F. D. J. Biol. Chem. 1975, 250, 4470. (6) McFarland, B. G.; McConnell, H. M. Proc. Natl. Acad, Sci. U.S.A. 1971, 68,
(7) Lasic, D. D. Liposomes: From Physics to Applications; Elsevier: Amsterdam, 1993; p 53. (8) Binder, W. H.; Barragan, V.; Menger, F. M. Angew. Chem., Int. Ed. 2003, 42, 5802. (9) Lee, G. M.; Jacobson, K. Curr. Top. Membr. 1994, 40, 111. (10) DeKruijff, B.; Zoelen, E. J. J. Biochim. Biophys. Acta 1978, 511, 105. (11) Smith, B. D.; Boon, J. M. J. Am. Chem. Soc. 1999, 121, 11924. (12) Roseman, M. A.; Thompson, T. E. Biochemistry 1980, 19, 439. Martin, F. J.; MacDonald, R. C. Biochemistry 1976, 15, 321.
10.1021/la0508691 CCC: $30.25 © 2005 American Chemical Society Published on Web 07/15/2005
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Figure 2. (a) Schematic of phospholipids assembled into a bilayer. (b) A vesicle, shown in cross section, composed of a bilayer surrounding an aqueous domain.
Figure 4. (a) Micelle-forming surfactant with a carbonyl at its C-8. (b) Locations (arrows) at which a carbonyl probe was placed onto vesicle-forming phospholipids.
croemlusions,14 coacervates,15 enzyme catalysis,16 etc.), and eight lessons learned from them now follow. Emphasis will be given to experimental design and bottom-line conclusions; tables of data, literature details, and other accoutrements, if desired, can be found in the cited references. The reader will note that this article deals with basic phospholipid research. If patented material is desired, then we suggest that the reader search elsewhere. Although our basic research has certain practical ramifications, the primary objective here was to become a bit wiser, and for this we make no apologies. Water Penetration17
Figure 3. Schematic of various motions possible in a bilayer.
to the text. (The word “we” in the previous sentence refers, of course, to the students who carried out the experiments and to whom the senior author will always be grateful). Second, we intended to write primarily for “scientists not in the immediate field” and “with the non-specialist in mind.” This task has been made easier by the fact that often in the past we have at least attempted to do just this: write in a style comprehensive to the interested novice. Science is, after all, profitably directed toward the young and open-minded. In summary, our papers on phospholipids were culled from those reporting on other past and present trysts (with gemini surfactants,13 mi-
13 C NMR chemical shifts of ketone carbonyl groups are solvent-sensitive (e.g., +7.5, +5.6, and 0.00 ppm relative shifts in methanol, chloroform, and heptane, respectively). Almost a decade before studying phospholipids, we applied the solvent dependence to micellar systems composed of single-chained surfactants bearing a carbonyl at a specific location (Figure 4). It was found that even a carbonyl at the chain’s inner C-8 experiences a polar environment equivalent to that of an alcohol. Thus, micelle interiors are rather “wet”, and/or internal chain-carbons frequently sample the aqueous micelle surface. In sharp contrast to the situation with micelles, 13C NMR data showed that static water is absent at five locations in bilayers made from phospholipids (Figure 4). Recent molecular dynamics calculations by others affirm this conclusion: the bilayer interior is dry except, possibly, for water molecules in transit.18
(13) Menger, F. M.; Keiper, J. S. Angew. Chem., Int. Ed. 2000, 39, 1906. (14) Menger, F. M.; Elrington, A. R. J. Am. Chem. Soc. 1991, 113, 9621. (15) Menger, F. M.; Peresypkin, A. V.; Caran, K. L.; Apkarian, R. P. Langmuir 2000, 16, 9113. (16) Menger, F. M. Pure Appl. Chem., accepted for publication. (17) Menger, F. M.; Aikens, P.; Wood, M. Chem. Commun. 1988, 180. (18) Tieleman, D. P.; Marrink, S. J.; Berendsen, H. J. C. Biochim. Biophys. Acta 1997, 1331, 235.
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Figure 5. Six phospholipids that had been substituted with a methyl group on the second chain and examined by differential scanning calorimetry for the enthalpy of melting.
Figure 7. (a) Typical archaebacterial lipids with ether (rather than ester) linkages and macrocyclic rings. (b) Synthetic tethered phospholipids (ring size ) 32-44 atoms) studied by calorimetry. Figure 6. ∆H for solid-liquid melting vs position of the methyl group for the six lipids in Figure 5.
Our carbonyl work embodied an uncertainty in common with all other probes that have been used with phospholipid membranes: probes inevitably present a risk that the compounds themselves are altering the local environment in which they are embedded. In our case, however, the ketone carbonyl is small and relatively innocuous (compared to, for example, fluorescent probes and spin labels). Moreover, the fact that a carbonyl reports polarities that differ between micelles and bilayers attests to the reliability of the two messages. Molecular Packing19 We had ready access to many phospholipid derivatives via the power of synthetic organic chemistry. This enabled us to study phospholipids with chain substituents of diverse size (methyl, n-butyl, phenyl), polarity (alkyl, keto), number (one or both chains), and location along the chains. Only one experiment with such compounds will be described here: a differential scanning calorimetric determination of the enthalpy of melting at Tm. The experiment utilized six phospholipids whose second chain had been substituted with a methyl group at carbons 4, 6, 8, 10, 12, or 16 (Figure 5). Because the enthalpy of melting diminishes when a bilayer is disordered, we could determine how disorder caused by a methyl depends on its location. Figure 6 gives a plot of the enthalpy of melting versus the location of the methyl group on the phospholipid chain. Surprisingly perhaps, methyl groups in the middle of the chain disrupt bilayer packing far more effectively than do methyl groups near the headgroup or the chain terminus. The simplest explanation is that methylenes near the headgroup are relatively immobilized, whereas methylenes near the chain terminus have a high degree of motional freedom. Therefore, at both of these sites (and for opposite reasons) methyl substitution has a lesser disruptive effect than at the center of the chain. It is no accident that nature achieves membrane fluidity by (19) Menger, F. M.; Wood, M. G.; Zhou, Q. Z.; Hopkins, H. P.; Fumero, J. J. Am. Chem. Soc. 1988, 110, 6804.
incorporating cis-unsaturation near the middle of her phospholipid chains. Archaebacterial Phospholipid20 Archaebacterial lipids such as those in Figure 7 possess two structural features that are not usually found in the lipids of other organisms: (a) The hydrocarbon chains are connected to the glycerol backbone by means of ether, rather than ester, linkages. (b) The hydrocarbon chains are frequently joined to form a macrocyclic ring. Ether linkages have an obvious advantage over esters in that they impart hydrolytic stability to the lipid at the high temperatures confronted by thermophilic bacteria. Nature knows her chemistry! It was less clear, however, what advantage (if any) is associated with macrocyclic rings. This uncertainty led us to prepare and examine diether lipids containing giant rings (Figure 7). A Glaser oxidation (carried out at an unorthodox 140 °C) connected two acetylene-terminated chains on a lipid into macrocyclic rings; the triple bonds in the resulting ring were then hydrogenated. It was found that chain “tethering” (i.e., joining the two chain termini with a single additional C-C bond) elevates Tm considerably. For example, a lipid with two independent 18-carbon chains has a Tm of 55.6 °C, whereas its macrocyclic analogue has a Tm of 70.1 °C. Why should this be? A simple explanation proposes that localized, disordered microdomains, involving chain motions within individual lipid molecules, precede the main melting transition at Tm. Because tethering can be reasonably expected to impede such microdomain formation, higher temperatures are required to achieve the solid-to-liquid transition. Temperature stability associated with macrocyclic lipids might well have provided thermophilic bacteria with an evolutionary advantage in their hot water environment. In related work, the spiro compound in Figure 8 was synthesized to investigate a phospholipid that is unable to bend and fold.21 Although the compound self-assembles (20) Menger, F. M.; Chen, X. Y.; Brocchini, S.; Hopkins, H. P.; Hamilton, D. J. Am. Chem. Soc. 1993, 115, 6600. (21) Menger, F. M.; Ding, J. Angew. Chem., Int. Ed. Engl. 1996, 35, 2137.
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Figure 8. Synthetic phospholipid with four rigid spiro units per chain.
Figure 10. Schematic showing a polycation (PEVP) binding to the outside of a phospholipid vesicle and inducing the flipflop of an anionic surfactant (SDS) from the inside to the outside of the bilayer. Figure 9. Structures of SDS and PEVP used in detecting polymer-induced flip-flop by laser electrophoresis. The structure of the lipid component, DPPC, is given in Figure 1.
into vesicular bilayers, it has no detectable phase transition below 110 °C because, we believe, folding into bent (gauche) conformations, as occurs with conventional lipids above Tm, is not an option. Induced Flip-Flop22 The following experiment was drawn from a longstanding collaboration with Professor A. A. Yaroslavov and his group at Moscow State University where this work was carried out. Laser electromicrophoresis provided key data. This method allows one to define the conditions under which the surface of a vesicle bears zero charge and, therefore, ceases to migrate in an electric field. The following runs were carried out with vesicles whose components are given in Figures 1 and 9. These consist of DPPC (a phospholipid that exists in the solid state at 25 °C), SDS (an anionic surfactant absorbed into both leaflets of the bilayer), and PEVP (a polycation). When a DPPC vesicle (an SUV) containing anionic SDS was mixed with the polycation PEVP, vesicle migration in an electric field ceased after exactly half of the SDS had been charged neutralized by PEVP. Apparently, the PEVP adsorbs to the external vesicle surface until the anionic charge on each SDS in the outer leaflet is balanced by a cationic charge on the polymer. In contrast, SDS-bearing vesicles of egg lecithin (a natural phospholipid mixture in the liquid state at 25 °C) behaved quite differently: The entire SDS population of both leaflets was charge neutralized by externally bound polymer before vesicle migration ceased. The conclusion is inescapable: an adsorbed polycation can induce SDS to flip-flop from the inside leaflet to the outside leaflet (Figure 10), but this is possible only with vesicles in the liquid state.
Figure 11. (a) Compounds that show little or no activity in allowing entry and exit of cations from vesicle systems. (b) Highly active flux compound. All three sections of the compound (chain, polyether, and benzyl group) are necessary for ion transport.
One of our early goals was to synthesize non-peptide organic molecules that form channels in phospholipids membranes. These channels would allow ions to pass through in much the same manner that gramicidin, a natural antibiotic, transmits ions when embedded in membrane systems. So potent is gramicidin that a single
channel carries a greater ion current than can an entire 1.0 × 1.0 mm2 gramicidin-free membrane. There now exist, of course, many elegant synthetic channels in the literature;24 our own contribution to the field is, by comparison, rather unpretentious in structure although its activity is remarkable, surpassing that of gramicidin. The discovery of our flux-promoting compounds was, admittedly, fortuitous. Originally, the goal had been to synthesize and test phospholipids bearing a polyether chain (Figure 11) in hopes that this chain would promote ion movement across bilayers. Although the phospholipids proved inert (probably because the polyethers preferred to reside in the bulk water phase rather than in the bilayer), intermediates acquired in the synthesis of the phospholipids, and having the general structure RO(CH2CH2O)nR′, were highly active. Optimal structural requirements for a catalyzed ion flux are (a) R ) a long chain such as dodecanoyl; (b) n ) 5; and (c) R′ ) benzyl (Figure 11). The need for R′ ) benzyl was the most unexpected of the three specifications. We surmise that the benzyl group associates with the phospholipid’s quaternary ammonium group via a well-documented ion-dipole attraction.25 With the benzyl group fixed to the bilayer surface and the long hydrocarbon chain inserted deep into the bilayer, the central polyether units can lie fully extended and parallel to the phospholipids’ chains. When a bundle of these flux-inducing molecules assemble into
(22) Yaroslavov, A. A.; Udalyk, O. Y.; Kabanov, V. A.; Menger, F. M. Chem.sEur. J. 1997, 3, 690. (23) Menger, F. M.; Davis, D. S.; Persichetti, R. A.; Lee, J.-J. J. Am. Chem. Soc. 1990, 112, 2451.
(24) Sanchez-Quesada, J.; Ghadiri, M. R.; Bayley, H.; Braha, O. J. Am. Chem. Soc. 2000, 112, 11757. (25) Stauffer, D. A.; Dougherty, D. A. Tetrahedron Lett. 1988, 29, 6039.
Ion Transport23
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Figure 13. (a) Vesicle cross-linking compound possessing a steroid at one end and a chemically reactive maleimide at the other. (b) Schematic showing how a steroid (black squares) embeds into the vesicle bilayer, leaving the malemides to reside in the external water where pairs of them become cross linked by the dithiol. Figure 12. (a) Steroidal nucleophile and (b) steroidal ester that were incorporated into two different populations of vesicles and then reacted with each other.
a domain, a hydrophilic channel is produced through which cations can pass rapidly. Vesicle/Vesicle Reactivity26 Inspired by the fact that membrane/membrane reactivity in biology is a widespread occurrence, we asked, can one phospholipid vesicle chemically attack another vesicle at a measurable rate? Toward this end, we prepared two separate vesicle populations: (a) One set of vesicles had a steroidal p-nitrophenyl ester (Figure 12) anchored to the bilayer. (b) The other set of vesicles had a steroidal nucleophile (a hydroxamate) bound to the bilayer (Figure 12). Because hydroxamates are known to be excellent catalysts for the hydrolysis of p-nitrophenyl esters, we had the possibility of observing and measuring a vesicle/ vesicle chemical reaction. A catalyzed hydrolysis involving the two vesicle populations was indeed observed (the first instance of vesicle/vesicle reactivity to our knowledge), although the reaction was very slow (t1/2 ) 120 min compared to 0.35 min for the corresponding reaction with soluble reactants in bulk solution). In contrast, an extremely fast reaction occurred when the two steroidal reactants were present in one and the same vesicle. In fact, the intravesicular reaction was too fast to measure under our standard conditions, so the pH had to be lowered to retard the hydrolysis rate by protonating the nucleophile. This is an interesting case in which the confinement of two reactants to a 2D space (i.e., the bilayer) leads to enzyme-like reactivity. Soft Materials from Phospholipids27 The question arose as to whether phospholipid vesicles can be linked together into a network and, if so, whether a useful new material could be thereby produced. Toward this end, we synthesized a compound functionalized with a cholesterol unit on one end and a maleimide on the other (Figure 13). Because phospholipid vesicles have an affinity for cholesterol, we could reasonably expect our compound to incorporate into vesicle bilayers, leaving the maleimides to “wave in the breeze” outside the vesicles. And because thiols rapidly Michael-add to maleimides, the addition of a water-soluble dithiol (dithiothreitol) can interconnect (26) Menger, F. M.; Azov, V. A. J. Am. Chem. Soc. 2000, 122, 6492. (27) Menger, F. M.; Bian, J.; Seredyuk, V. A. Angew. Chem., Int. Ed. 2004, 43, 1265.
Figure 14. (a) Phase-contrast photomicrograph (bar ) 40 µm) of a soft-material smear showing a high particle density. (b) Picture of a single particle shown by electron microscopy to be a hollow sphere (bar ) 50 µm).
the vesicles to each other as in Figure 13. Each SUV bilayer was provided with roughly 5-6% cross-linking sites relative to its phospholipid content. The material obtained after addition of the dithiol resembled shaving cream in consistency. Examination by phase-contrast light microscopy showed round, densely packed particles ranging from 5 to 150 µm in diameter (Figure 14). Detailed photos, one of which is given in Figure 14, revealed an unusual “blistered” texture of the particle surfaces. These blisters, which disappear and reappear upon freezing and thawing of the particles, are clearly integral to the particle surface. High-resolution scanning electron microscopy proved that the particles are hollow with ca. 100-nm-thick shells. The shells in turn are composites of small vesicles and lamellar phases bonded into a film by the cross-linking agent. Our new soft material is cheap, composed of natural constituents, and endowed with a particularly high capture volume, which are all components of a potentially useful delivery system. Cytomimetic Phospholipid Systems28 Giant phospholipid vesicles (GUVs) vary in diameter from 10 to 200 µm, and therein lies their most unique property: visibility under the light microscope. Being cellsized, GUVs introduce no annoying curvature issues as do the much smaller and more widely studied SUVs and LUVs, and GUVs undergo a variety of “cytomimetic” events29 including fission, fusion, budding, endocytosis, and birthing (i.e., the ejection of a small vesicle from the (28) Seredyuk, V. A.; Menger, F. M. J. Am. Chem. Soc. 2004, 126, 12256. (29) Menger, F. M.; Gabrielson, K. D. Angew. Chem., Int. Ed. Engl. 1995, 34, 2091.
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Figure 15. Growth of a protein-containing giant vesicle seen by phase-contrast microscopy (bar ) 25 µm). The negatively charged GUV had been “fed” positively charged SUVs and expanded 25% in volume after 45 min.
interior of a larger one). With the aid of a micropipet, one can inject into GUVs substances such as enzymes, DNA, probes, and reactants (or remove the same if they are internalized within a GUV). Our work with phospholipid GUVs began in earnest after the appearance of the Angelova electroformation method30 for making GUVs. In brief, platinum wires are coated with a film of phospholipid and placed in a water-filled cell where low voltages (100 mV-1.0 V) of alternating current (3-10 Hz) are applied. This causes beautiful giant vesicles to form on the Pt wires (reminiscent of pigeons sitting on a telephone line). A single GUV is often moved with a holding pipet to the center of the cell where it can be perturbed with various physical/chemical stimuli. The response to the stimuli as a function of bilayer composition provides useful information on membrane behavior. We will now describe a single GUV experiment drawn, rather arbitrarily, from a much larger body of published work. A 60 µm GUV was formed from three components (given in mol %): (a) 93% neutral phospholipid; (b) 5% anionic phospholipid; and (c) 2% zein, a plant protein selected for its hydrophobicity and stability. The GUV was then immersed into a population of 100 nm surfactantweakened SUVs made entirely from a cationic lipid. Thereupon, the GUV “consumed” the much smaller SUVs while expanding its girth in the process (Figure 15). Growth was observed only in the presence of protein and only when the GUV and SUVs were of opposite charge. We believe that the GUV and SUVs associate electrostatically, after which the SUVs fuse with the GUV at defects present at the protein/lipid interface (Figure 16). One is reminded here of “fusion proteins” of viruses that reconfigure host membranes prior to viral entry. Five distinct components were used in the preceding experiment: three phospholipids (neutral, anionic, and cationic), a surfactant, and a protein. Each of the components performs a necessary function at a specific site. Collectively and cooperatively, the quintet engages in a biorelevant process, namely, growth. We have called (30) Menger, F. M.; Angelova, M. I. Acc. Chem. Res. 1998, 31, 789.
Figure 16. Giant vesicle, with a protein (shaded ovals) embedded within, treated with a population of SUVs. The diagram is not to scale (with the GUV being, in reality, 500 times larger than the SUVs, with both the GUV and SUV having the same membrane thickness). “Electrostatics seemingly serves a dish of SDS-tenderized small vesicles to a giant vesicle, but the feast does not begin unless protein is around to ring the dinner bell. Afterwards, to continue the metaphor, the giant vesicles loosen their belts.”28
such a structurally diverse and functional assembly of interactive compounds a “chemical system.”31 Mayonnaise, shampoo, paint, batteries, and concrete are everyday examples of chemical systems. However, host-guest complexes, dendrimers, carbon nanotubes, micelles, block copolymers, and so forth are not systems because, by our definition, they are generally not composed of many different components acting cooperatively. If chemists are to better understand the most wonderful system of all, the cell, then they will have to continue to expand their journey from small-molecule chemistry to supramolecular chemistry to systems chemistry. Complexity will, no doubt, be a main obstacle and challenge encountered along the way as scientists continue to study heterogeneously distributed membrane lipids (“rafts”), communication between cells via membrane-to-membrane contact, cell control of membrane permeability and morphology, disruption of cell membranes during viral entry, transport of drugs across cell membranes, the role of membranes in cell differentiation, and membrane participation in hormone release, to name a few. And speaking of journeys, this highly personal visit to the land of phospholipids, populated by those small but resourceful biomolecules, comes to a close. It remains only to thank the National Institutes of Health for their support of our program. LA0508691 (31) Menger, F. M. Angew. Chem., Int. Ed. Engl. 1991, 30, 1086.
