Incorporation into lipid vesicle membranes

Incorporation of Alamethicin into Lipid Vesicle Membranes and Generation of Donnan Potential Gradient across the Membrane. Alamethicin from Tricho-derma viride was purchased from Sigma and used without further purification. 

Purified membrane proteins or enzymes can be incorporated into these vesicles in order to assess what factors (eg, specific lipids or ancillary proteins) the proteins require to reconstitute their function. Investigations of purified proteins, eg, the Ca " ATPase of the sarcoplasmic reticulum, have in certain cases suggested that only a single protein and a single lipid are required to reconstitute an ion pump. 

Vesicular proteins and lipids that are destined for the plasma membrane leave the TGN sorting station continuously. Incorporation into the plasma membrane is typically targeted to a particular membrane domain (dendrite, axon, presynaptic, postsynaptic membrane, etc.) but may or may not be triggered by extracellular stimuli. Exocytosis is the eukaryotic cellular process defined as the fusion of the vesicular membrane with the plasma membrane, leading to continuity between the intravesicular space and the extracellular space. Exocytosis carries out two main functions it provides membrane proteins and lipids from the vesicle membrane to the plasma membrane and releases the soluble contents of the lumen (proteins, peptides, etc.) to the extracellular milieu. Historically, exocytosis has been subdivided into constitutive and regulated (Fig. 9-6), where release of classical neurotransmitters at the synaptic terminal is a special case of regulated secretion [54]. 

Using N-terminus modified polylysine, we developed a synthesis for an amphiphilic polychelator, A,a-(DTPA-polylysyl)glutaryl phosphatidyl ethanolamine (DTPA-PL-NGPE). This polychelator was incorporated into the liposomal membrane and micelle core during liposome or micelle preparation. This system sharply increased the number of chelated Gd atoms attached to a single lipid anchor. This increased the number of bound reporter metal atoms per vesicle and decreased the dosage of an administered... 

We assume that the affinities of these specific IgG molecules for spin-labeled lipid haptens such as (V), (IX), or (X) are of the same order of magnitude. Figure 10 illustrates the effect of antibody binding on the paramagnetic resonance spectra of (X) incorporated into lipid membrane vesicles. 

Diacyllipid-polyethyleneoxide conjugates have been introduced into the controlled drug delivery area as polymeric surface modiLers for liposomes (Klibanov et al., 1990). Being incorporated into the liposome membrane by insertion of their lipidic anchor into the bilayer, such molecules can ster-ically stabilize the liposome against interaction with certain plasma proteins in the blood that results in signiLcant prolongation of the vesicle circulation time. The diacyllipid-PEO molecule itself represents a characteristic amphiphilic polymer with a bulky hydrophilic (PEO) portion and a very short but extremely hydrophobic diacyllipid part. Typically, for other PEO-containing amphiphilic block... 

Lipid analysis is typically performed either by incorporating a nonexchangeable radiolabel marker, such as (14C) or (3H) cholesteryl hexadecyl ether (CHE), into the vesicle membrane, or by analyzing the phosphate content and extrapolating the result according to the original composition of the vesicles. Both approaches assume that the label concentration and vesicle composition do not change on vesicle preparation or subsequent manipulation. The phosphate assay is carried out as follows ... 

Over 40 years since it what found that phospholipids can form closed bilayered structures in aqueous systems, liposomes have made a long way to become a popular pharmaceutical carrier for numerous practical applications. Liposomes are phospholipid vesicles, produced by various methods from lipid dispersions in water. Liposome preparation, their physicochemical properties and possible biomedical application have already been discussed in several monographs. Many different methods exist to prepare liposomes of different sizes, structure and size distribution. The most frequently used methods include ultrasonication, reverse phase evaporation and detergent removal from mixed lipid-detergent micelles by dialysis or gel-filtration. To increase liposome stability towards the physiological environment, cholesterol is incorporated into the liposomal membrane (up to 50% mol). The size of liposomes depends on their composition and preparation method and can vary from... 

The composition of a vesicle membrane can be manipulated in a wide range using different lipid classes and different lipid chain lengths. A prerequisite is that the lipids form well hydrated lamellar phases and that the vesicles are stable against aggregation and ftision. When these prerequisites are fulfilled, even membrane proteins can be incorporated into the synthetic membranes using appropriate procedures, such as detergent dialysis. Therefore, most physico-chemical studies of membrane properties of bilayers of defined composition have been performed with liposomes of the multilamellar or unilamellar type. 

Fig. 9 Surface modification of cells with ssDNA-PEG-lipid. (a) Real-time monitoring of PEG-lipid incorporation into a supported lipid membrane by SPR. (r) A suspension of small unilamellar vesicles (SUV) of egg yolk lecithin (70 pg/mL) was applied to a CH3-SAM surface. A PEG-lipid solution (100 pg/mL) was then applied, (ii) Three types of PEG-lipids were compared PEG-DMPE (C14), PEG-DPPE (C16), and PEG-DSPE (C18) with acyl chains of 14, 16, and 18 carbons, respectively, (b) Confocal laser scanning microscopic image of an CCRF-CEM cell displays immobilized FITC-oligo(dA)2o hybridized to membrane-incorporated oligo(dT)20-PEG-lipid. (c) SPR sensorigrams of interaction between oligo(dA)2o-urokinase and the oligo (dT)2o-PEG-lipid incorporated into the cell surface, (i) BSA solution was applied to block nonspecific sites on the oligo(dT)20-incorporated substrate, (ii) Oligo(dA)20-urokinase (solid line) or oligo(dT)20-urokinase (dotted line) was applied...

Lipid-protein interactions are of major importance in the structural and dynamic properties of biological membranes. Fluorescent probes can provide much information on these interactions. For example, van Paridon et al.a) used a synthetic derivative of phosphatidylinositol (PI) with a ris-parinaric acid (see formula in Figure 8.4) covalently linked on the sn-2 position for probing phospholipid vesicles and biological membranes. The emission anisotropy decays of this 2-parinaroyl-phosphatidylinositol (PPI) probe incorporated into vesicles consisting of phosphatidylcholine (PC) (with a fraction of 5 mol % of PI) and into acetylcholine receptor rich membranes from Torpedo marmorata are shown in Figure B8.3.1. 

Measurements of the quantities of glycolipids inserted into the membrane have also been reported by a technique based on the use of C-labeled lipid anchors. In this method, the carbohydrate (a-o-Man) was covalently coupled to the anchor at the surface of a pre-formed vesicle. Indeed, the liposome structure was shown to remain intact in the treatment. Nevertheless, the measurement of the incorporated mannose was performed after separation of bound and unbound material by centrifugation. The yields of coupling were shown to increase with the increase of the initial mannose/ C-anchor ratio, but non covalent insertions were displayed at high initial mannose concentrations. Therefore, the aforementioned method was not as accurate as could have been expected for the use of radioactive materials [142]. Radiolabeled phospholipids were also used for such determinations thus the amounts of glycosphingolipids incorporated into liposomes were quantified by the use of H-phospholipids whereas the amounts of glycolipids were determined by a sphingosine assay [143]. 

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