Lecithin monolayers

An essential component of cell membranes are the lipids, lecithins, or phosphatidylcholines (PC). The typical ir-a behavior shown in Fig. XV-6 is similar to that for the simple fatty-acid monolayers (see Fig. IV-16) and has been modeled theoretically [36]. Branched hydrocarbons tails tend to expand the mono-layer [38], but generally the phase behavior is described by a fluid-gel transition at the plateau [39] and a semicrystalline phase at low a. As illustrated in Fig. XV-7, the areas of the dense phase may initially be highly branched, but they anneal to a circular shape on recompression [40]. The theoretical evaluation of these shape transitions is discussed in Section IV-4F. 

The few examples of deliberate investigation of dynamic processes as reflected by compression/expansion hysteresis have involved monolayers of fatty acids (Munden and Swarbrick, 1973 Munden et al., 1969), lecithins (Bienkowski and Skolnick, 1974 Cook and Webb, 1966), polymer films (Townsend and Buck, 1988) and monolayers of fatty acids and their sodium sulfate salts on aqueous subphases of alkanolamines (Rosano et al., 1971). A few of these studies determined the amount of hysteresis as a function of the rate of compression and expansion. However, no quantitative analysis of the results was attempted. Historically, dynamic surface tension has been used to study the dynamic response of lung phosphatidylcholine surfactant monolayers to a sinusoidal compression/expansion rate in order to mimic the mechanical contraction and expansion of the lungs. 

The polarization properties of the evanescent wave(93) can be used to excite selected orientations of fluorophores, for example, fluorescent-labeled phosphatidylethanolamine embedded in lecithin monolayers on hydrophobic glass. When interpreted according to an approximate theory, the total fluorescence gathered by a high-aperture objective for different evanescent polarizations gives a measure of the probe s orientational order. The polarization properties of the emission field itself, expressed in a properly normalized theory,(94) can also be used to determine features of the orientational distribution of fluorophores near a surface. 

L-2 cells (ATCC HTB-149) have been isolated by clonal culture techniques from the adult rat lung. These cells appear to retain differentiated functions that are present in ATII cells of intact rat lungs. L-2 cells are diploid, epithelial cells. They contain osmiophilic lamellar bodies in their cytoplasm and synthesise lecithin by the same de novo pathways as in a whole lung [78], It is not known if L-2 cells are capable to form confluent and electrically tight monolayers. L-2 cells have not been systematically investigated regarding their suitability as a model for absorption studies. 

Our present ideas about the nature of biological membranes, which are so fundamental to all biochemical processes, are based on the Singer-Nicholson mosaic model. This model of the membrane is based on a phospholipid bilayer that is, however, asymmetrical. In the outside monolayer, phosphatidylcholine (lecithin) predominates, whereas the inner monolayer on the cytoplasmic side is rich in a mixture of phos-phatidylethanolamine, phosphatidylserine, and phosphatidylinositol. Cholesterol molecules are also inserted into the bilayer, with their 3-hydroxyl group pointed toward the aqueous side. The hydrophobic fatty acid tails and the steran skeleton of cholesterol... 

Dawson and I (Biochim. Biophys. Acta 59, 103, 1961) studied the interaction of two phospholipases with their common substrate, lecithin, and concluded that the enzyme must extend an active site ( masculine enzyme) into the planar lecithin monolayer or bilayer, respectively, in order to reach the ester bond. The phospholipase A2 from penicillin notaton required a net negative charge at the substrate surface, the a toxin from Cl. welchii, a positive one, to facilitate penetration. 

The huge variety of emulsions used as food, medicinal, cosmetic, and other industrial products make these colloids important practical systems in which the surface monolayers exert considerable influence. We have already discussed the use of lecithin to control the viscosity and the texture of chocolate in Vignette IV in Chapter 4. 

This lamellar phase is formed of alternate sheets of lipid and water. The lipidic sheets containing the lecithin and the cholesterol are made of two superposed layers of oriented molecules. Each of these two monolayers is mixed and consists of lecithin and cholesterol molecules arranged side by side with their paraffinic ends turned toward the inside of the sheet and their polar groups (phosphatidyl choline group for lecithin and hydroxyl group for the cholesterol) outward—i.e., toward the adjacent sheet of water. This constitution of each of the two mono-layers forming the lipidic sheet is in conformity with the conclusion arising from the study of mixed monolayers of cholesterol and lecithin spread on the free surface of water (1). 

Figure 5. Mixed monolayers of cholesterol-1,2-dimyristoyl-3-lecithin system at 23°C., pH 6, and 5 dynes per cm.

Table II. Thermodynamic Excess Functions for an Equimolar Mixed Monolayer of Trilaurin—Dimyristoyl Lecithin...

Discontinuities are seen in the relationship between increase in film pressure, An, and lipid composition following the injection of globulin under monolayers of lecithin-dihydro-ceramide lactoside and lecithin-cholesterol mixtures. The breaks occur at 80 mole % C 16-dihydrocaramide lactoside and 50 mole % cholesterol. Between 0 and 80 mole % lactoside and between 0 and 50 mole % cholesterol the mixed films behave as pure lecithin. Two possible explanations are the formation of complexes, having molar ratios of lecithin-lactoside 1 to 4 and lecithin-cholesterol 1 to 1 and/or the effect of monolayer configurations (surface micelles). In this model, lecithin is at the periphery of the surface micelle and shields the other lipid from interaction with globulin. 

Although glycosphingolipids are the specific lipid components in the antigen-antibody complex, their activity is markedly enhanced by other (auxiliary) lipids such as lecithin and lecithin-cholesterol mixtures (15). The present study deals with the effect of lipid composition on the penetration of lactoside—cholesterol and lactoside—lecithin monolayers by rabbit y-globulin. We also investigated the lecithin-cholesterol system. Furthemore, since criteria for the existence of lipid-lipid complexes in monolayers are still few (8, 17), we have used infrared spectroscopy to examine lipid mixtures for the presence of complexes. 

On the one hand, the cholesterol-lactoside system did not show film contraction (unpublished data). This was expected since the monolayers of dihydroceramide lactosides (7) and of cholesterol are not compressible. On the other hand, lecithin-lactoside and lecithin-cholesterol systems did show contraction, which could have been predicted since the lecithin monolayer is of the expanded type and is very compressible. (The area per molecule of lecithin at 2 dynes per cm. is large, 110 sq. A., as opposed to 52 sq. A. for Ci -dihydroceramide lactoside and 40 sq. A. for cholesterol.)... 

Surface Potential. Shah and Schulman have proposed that interaction between dipoles of uncharged lipids in mixed monolayers should result in a change in surface potential, AV. Linearity of the relation of AV to composition of the lecithin-cholesterol monolayer was taken to indicate absence of interaction (17). We do not agree with Shah and Schulman, since surface potential does not appear to be a valid criterion for assaying interaction between dipoles of uncharged lipids. Except for the speculations of Shah and Schulman (17, 18), there is neither theoretical nor experimental evidence that dipole-dipole interactions have... 

The infrared evidence for hydrogen bonding between cholesterol and lecithin in chloroform solution is no evidence of a similar complex in the monolayer but suggests such a possibility. It does not exclude the hydrophobic bonding suggested by Chapman from NMR studies of the aqueous suspensions of equimolar mixtures of cholesterol and lecithin (3). 

The second observation which does not support the unfolded protein model is that when phospholipase A (N. naja venom) was injected into the subphase under the lipid monolayer at equilibrium with globulin, lecithin was readily attacked, as indicated by the rapid fall of surface potential (4, 5, 6). If the penetrated protein were to cover entirely the polar groups of the lipid facing the aqueous subphase (as postulated in the unfolded protein model), the lipid molecules should not be accessible to the lipolytic enzyme. 

A model that is consistent with these observations of the action of trypsin and phospholipase A and with the discontinuities in the All-composition curves (Figures 2 and 3) is one in which the lipid monolayer is not a continuous palisade of uniformly oriented lipid molecules but rather an assembly of surface micelles. In this model, proposed by Colacicco (4, 5), the protein first comes into contact with the lipid molecules at the periphery of the surface micelles and then inserts itself as a unit between them. This is the basis for the generalized nonspecific interaction between lipids and proteins which results in increase of surface pressure. One may thus explain the identical All values obtained with films of lecithin and 80 mole % lactoside by picturing the lecithin molecules outside and the lactoside molecules inside the surface micelles. In this model lecithin prevents the bound lactoside from interacting nonspecifically with globulin and produces the same increase in pressure as with a film of pure lecithin. In the mixed micelle the lactose moiety of the lactoside protrudes into the aqueous subphase. Contact of the protein with these or other nonperipheral regions of the surface micelle would not increase the surface pressure. 

The role of lecithin as an auxiliary lipid in the specific interaction of lactosides with globulin in monolayers is related to two processes complex formation between 3 or 4 molecules of lactoside and each lecithin molecule, and the protection of the lactoside molecules in surface micelles from nonspecific interaction. The location of lecithin at the periphery of the surface micelle would explain why the mixed micelle behaves as lecithin in nonspecific interaction. Lactoside molecules, located in the center of the surface micelle, would be in a position to interact specifically with antibody in the aqueous subphase (5). 

The nature of the lecithin-cholesterol association is probably also that of a complex (1 to 1), in which the OH group of cholesterol is involved through hydrogen bonding. It is not known, however, whether the monolayer consists of a uniform population of bimolecular complexes or of configurations (surface micelles) in which cholesterol molecules are surrounded by an equal number of lecithin molecules. 

Figure 5. P-A curves for synthetic lecithin monolayer and AV-A curve for beef brain lipid at pH 7.8. (O), [Ca2+] =...

Lecithin Monolayers. We have shown from surface pressure—area and surface potential-area curves of various lecithins that the molecular area increases and the interaction with metal ions decreases with increasing unsaturation of the fatty acyl chains (41, 43). 

On the basis of the interaction of metal ions and AV-pH and AV-log C plots (41, 43), we propose ionic structures for dioleoyl, egg, and di-palmitoyl lecithin monolayers represented in Figure 1. Schematically shown in Figure 1A is the internal salt linkage between the phosphate and trimethylammonium groups in dioleoyl lecithin, preventing the inter-... 

Figure I. Schematic of interaction of calcium ion with dioleoyl, egg, and di-palmitoyl lecithins, and of egg lecithin-cholesterol monolayers...

B Egg lecithin-cholesterol monolayers. Increased spacing between phosphate groups results in strong internal salt linkage preventing binding of Ca2+... 

Phosphatidic Acid Monolayers. Phosphatidic acid, prepared from egg lecithin by the action of phospholipase D, forms considerably more expanded monolayers than egg lecithin, presumably because of ionic repulsion between the phosphate groups in the phosphatidic acid mono-layers (42). Phosphatidic acid monolayers showed about four times more increase in surface potential when CaCl2 is substituted for NaCl in the subsolution than did egg lecithin monolayers (43). This again supports the conclusion that the trimethylammonium group competes with Ca2+ for the anionic phosphate group in egg lecithin monolayers (Figure 1A). 

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