Vesicles transport across bilayer

In this and the next section, standard techniques to study transport across bilayer and bulk membranes are briefly introduced. This section covers U-tube and planar bilayer conductance experiments, while Section 3 describes vesicle-based methods. 

Much interest for ion transport has its origin in the field of crown ether chemistry. Therefore, most model studies of ion channels have been more or less based on crown ether chemistries. Pioneering work has been undertaken by Fylcs, who not only synthesized varieties of gigantic molecules starting from crown ethers, " but established a method of the rate assay for ion transport across lipid bilayer membranes, a pH sCat technique. Vesicles having different inside and outside... 

The Ln(III) cations can be used in another fashion in the study of vesicles as pointed out by Bystrov62. The cations can be contained either only inside or only outside the bilayer as they cannot cross the membrane. Using a shift probe, say Pr(III), on the inside of the vesicle the resonances of the head-group of the inside lipids are shifted relative to those on the outside. It then becomes possible to follow differentially the inside and outside lipid signals on making changes in the bathing solution. This has permitted numerous studies of transport across the lipid bilayer. 

Electron transport across organised bilayers is an integral part of biological energy storage systems such as photosynthesis and provides a means of controlling back electron transfer and of separation of the products of redox reactions. Esters of 2,l,3-benzothiadiazole-4,7-dicarboxylic acid (481) have been used to study the transfer of electrons from 2-(morpholino)ethanesulfonic acid (MES) to 1,5-anthraquinone disulfonate in micelles or across vesicle bilayers. The esters absorb the light, accept an electron from MES and transfer it to the... 

In a subsequent study, van Hal et al. [40] reported that a decrease in cholesterol content in liquid state bilayers, which increases bilayer fluidity, resulted in an increase in estradiol transport across SC. With confocal laser scanning microscopy, Meuwissen et al. examined the diffusion depth of gel- vs. liquid-state liposomes labeled with fluorescein-dipalmitoylphosphatidylethanolamine (fluorescein-DPPE) with human skin in vitro [41] (Figure 3) and rat skin in vivo [42] and found that the lipophilic label when applied in liquid-state bilayers onto the skin penetrated deeper into the skin than when applied in gel-state liposomes. Recently, Fresta and Puglisi [43] reported that corticosteroid dermal delivery with skin-lipid liposomes was more effective than delivery with phospholipid vesicles, both with respect to higher drug concentrations in deeper skin layers and therapeutic effectiveness. This is a very surprising result, because skin lipid liposomes are rigid and form stacks of lamellae on the surface of the skin [44]. From the previously mentioned studies it seems clear that the thermodynamic state of the bilayer plays a crucial role in the effect of vesicles on dmg transport rate across skin in vitro. 

Artificial phospholipid vesicles (liposomes) are used to transport vaccines, drugs, enzymes or other substances to target cells or organs. They also make excellent model systems for studying biological ion transport across cell membranes. The vesicles, which are several hundred nanometres in diameter, do not suffer from interference from residual natural ion-channel peptides or ionophores, unlike purified natural cells. For example, the synthetic heptapeptide 5.23 forms pores that promote chloride efflux in vesicle models. Similarly, the ion-pair receptor 2.108 can ferry NaCl from vesicles as an ion-pair ionophore (see Chapter 2, Section 2.6.2), while the hydraphile 5.24 has been shown to transport Na using Na NMR spectroscopy through the bilayer walls of a vesicle model system. 

Specific l-thio- S-D-glucopyranosides to have been synthesised by normal routes are those bearing the 5-substituents (119), (120), (121) and (122). The antibiotic amphotericin mimic (123) has, on the other hand, been made by addition of the thiolate anion to the corresponding maleic acid compound. It affects ion transport across vesicle bilayers. ... 

The volume change can therefore be used as a reporter of urea concentration in the vesicle and so determine its permeability coefficient for lipid vesicle membranes of varying composition. Preliminary experiments indicate that urea has a permeability coefficient of 6.8 x 10 cms in SOPC, and 1.4 x 10 cms in DOPC at 25 C. These values compare extremely well with the earlier reported values of urea permeability (4.0 x 10" cms" at 25°C in eggPC [33], or 1.4 x 10 cms at 25°C in DOPC [87]). These experiments now lay the founding protocols for experiments aimed at quantifying drug transport across giant vesicle bilayers. 

In an investigation of a possible way of delivering nucleoside drugs across cell membranes, the glucosyl phospholipid (115) of thymidine was prepared. This was found to interact with large unilamellar vesicles so as to place the nucleotide derivative Inside the vesicles, suggesting transport across the lipid bilayer. i Similar mannose phosphate derivatives of AZT, ddT and FDU were... 

Artificial membranes. Much work on these is being carried out some of it to obtain excitable membranes (e.g. Mueller and Rudin 1976) some to study the permeability of ions (e.g. Bangham Standish and Weissman 1965). A favoured procedure is to ultrasonicate a mixture of lecithin and water which produces a suspension of vesicles with a bilayer structure (Huangs 1969). These vesicles lend themselves to studies of transport across natural membranes (Papakadjopoulos Nir and Ohki, 1972). 

Figure 13.9. Membrane permeability coefficience of solutes. Solute permeabilities across typical lipid bilayers of liposomes or lipid vesicles are presented as their respective coefficients in cm/s. In the absence of other transport processes, it would require 10 s to move Na+ across 1 cm distance. When there is a concentration difference across a membrane, multiplying the concentration difference (mole/ml equivalent to mole/cm ) by the permeability coefficient (cm/s) allows estimation of flow rate (mole/s-cm ). For example, a concentration difference of 1Q- mole/cm Na (or 1 x 10" M Na ) would provide a flow of 10 mole/cm x 10" cm/s = IQ- mole/s through 1 cm or 0.006 mole/s through 1 pm of a membrane bilayer.

While reconstitution of the calcium-dependent ATPase from the lipid deprived enzyme can easily be achieved, attempts to reconstitute simultaneously the abolished accumulation of calcium had no success55,70. Yet, in a number of reports the reconstitution of calcium transport from the enzyme after purification and/or after lipid exchange has been described160,, 70 172) jn these experiments it was attempted to reconstitute vesicles which could retain calcium ions which were transported into the vesicular space by the transport protein across the lipid bilayer. Different lipid pro-... 

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