Vesicles lipid flip-flop

Figure 4.8 Comb-shaped polyacrylate with hydrophobic side-chains integrates vesicle membranes, but hardly disturbs its ordering. Only melting of the membrane and or neutralization of the acrylate carboxylate group produces strong local disturbances. The polymer now promotes lipid flip-flops. ...

Early examples of synthetic flippases were lipidated polymers, which used bilayer distortion to bring about lipid flip-flop. In contrast to these mechanical flippases, synthetic species that apply the principles of molecular recognition to create phospholipid complexes capable of transverse diffusion have been shown to enhance lipid flip-flop in model membrane systems. Boon and Smith generated asymmetric bilayers by adding synthetic NBD phospholipids to the outer leaflet of POPC vesicles and then determined the rate of flip-flop to the inner leaflet... 

Kleinfeld, A.M., Chu, P. and Storch, J. (1997) Flip-flop is slow and rate-limiting for the movement of long chain anthroyloxy fatty acids across lipid vesicles. 

Note, however that the concepts about the lipid membrane as the isotropic, structureless medium are oversimplified. It is well known [19, 190] that the rates and character of the molecular motion in the lateral direction and across the membrane are quite different. This is true for both the molecules inserted in the lipid bilayer and the lipid molecules themselves. Thus, for example, while it still seems possible to characterize the lateral movement of the egg lecithin molecule by the diffusion coefficient D its movement across the membrane seems to be better described by the so-called flip-flop mechanism when two lipid molecules from the inner and outer membrane monolayers of the vesicle synchronously change locations with each other [19]. The value of D, = 1.8 x 10 8 cm2 s 1 [191] corresponds to the time of the lateral diffusion jump of lecithin molecule, Le. about 10 7s. The characteristic time of flip-flop under the same conditions is much longer (about 6.5 hours) [19]. The molecules without long hydrocarbon chains migrate much more rapidly. For example for pyrene D, = 1.4x 10 7 cm2 s1 [192]. 

Covesicles of the cationic nitrobenzoate 6 and DODAC, or corresponding DPP-analogues, e.g. 7, are hydrolysed at pH 8. Nitrophenolate absorption appears at 400 nm. The outer benzoate esters at the outer vesicle surface are hydrolysed within minutes and the same head groups on the inner surface survive for 1-15 hours (Figure 4.9). Detailed kinetics of flip-flop dynamics and OH permeation have been evaluated in these systems. Monolayer lipid membranes made of macrocyclic bolaamphiphiles showed enhanced dynamic stability. ... 

Note that in this experimental set-up, the molar amount of lipids in the vesicle population equals the molar amount of DMPC in the MLs. During the incubation step DMPC and DSTAP, spontaneously percolate between both colloidal particles. However, for thermodynamical reasons, i.e., slow trans-membraneous flip-flop movements, only the outer leaflet of the ML coat and the outer shell of the vesicle membrane are involved in the exchange process (3, 13). As 2/3 of the total lipid contents is present in the outer layer of the vesicles and MLs, an equilibrium will be reached when 1/3 of the lipids has transferred. Thus, if the starting vesicles contain 10% DSTAP, ultimately, 3.33% arrives in the ML population. 

There is independent physical evidence for non-uniform distribution and restriction from transmembrane diffusion of a-Toc in lipid membranes. Differential scanning calorimetry results indicated that it partitioned into the most fluid domains in lipid vesicles. Fluorescence studies showed that a-Toc has a very high lateral diffusion rate in egg lecithin but it does not take part in transbilayer (flip-flop) migration even over many hours . It is not known if this behavior of a-Toc extends to natural biomembranes where actual structures and conditions may dramatically change migration phenomena. 

Reactivity in aggregates may be used to get useful infonnation on mobility in these systems. Vesicles are particularly amenable to these studies because, as mentioned earlier, mobihty in these aggregates is lower than in micelles. For instance, it is estimated that above T, lateral diffusion of the lipids within the plane of the vesicle bilayer is very fast (diffusion coefficient of 10 cm s , in the fluid phase), though three orders of magnitude slower than in an aqueous medium. Accordingly, randomization of a hpid in a leaflet of the bilayer of a 500 A vesicle will occur in milliseconds, whereas the slow transverse (flip-flop) movement from one leaflet to another may take up to several days [1, 7, 60]. 

If step (2) results in an enhanced rate of transbilayer movement of the functional lipid because of the raised temperature, one would expect, upon bringing the pH back to 8 [step 3], to detect a new fast process accounting for the ester lipid that has moved from the interior to the exterior of the vesicles. This is in fact what the authors were able to monitor. By repetition of cycles (l>-(3), all the ester surfactant is eventually cleaved. By variation of the incubation time, a lifetime of the flip-flop process could be determined. The ti/2 data are reported in Table 3. Scrutiny of this table reveals that apart from the above-mentioned temperature of phase transition, the flip-flop process is also affected by the structure of the lipid backbone. For instance, dialkylammonium amphiphiles are much more mobile than those featuring a glycerolUke backbone increasing the length of the chain decreased the rate of... 

Mechanism of Proteolioid Vesicle Penetration into Monolayers. The principle conclusion from the penetration studies at the air-water and oil-water interfaces is that intrinsic membrane protein in vesicles greatly facilitates the transfer of material into monolayers. In marked contrast lipid vesicles do not penetrate monolayers to any appreciable extent although some exchange of lipid between a monolayer and the outer lipid layer of a liposome can occur (48.49). It is established that both glycophorin (50) and the anion transporter (51) increase the rate of "flip-flop" when incorporated into bilayers. Thus in the initial encounter between the proteolipid vesicles and the monolayer the protein-enhanced rate of "flip-flop" between the inner and outer halves of the vesicle bilayer would facilitate lipid transfer to the monolayer. The process of redistribution of lipid between vesicle and monolayer would bring the protein into intimate contact with the monolayer leading to penetration. 

Two different bending elastic moduli exist A f, when the exchange of lipid molecules between the monolayers of the bilayer is free, and when it is blocked. When the exchange is forbidden, the number of the molecules in each monolayer of the bilayer is constant. At free flip-flop, the bending elasticity energy is lower because it has been minimized with respect to the difference between the number of molecules in each monolayer and, consequently, k < kf. For all phenomena related to the out-of-plane fluctuations of membranes, the relevant quantity is k [6-8]. These phenomena include the thermal fluctuations of quasispherical vesicles [9,10], as well as vesicle suction in micropipettes at very low suction pressures [1]. 

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