Lipid transfer activity vesicles

Several functions of MTP have been identified all have been implicated in coordinating successful lipoprotein assembly (Fig. 27-1). MTP transfers lipids between vesicles in vitro, and this activity is likely to be its major function. MTP can pick up lipids from membrane (step A) or vesicles and droplets (step B) and transfer them to the nascent apoB. In addition, the lipid transfer activity of MTP has been implicated in the accretion of neutral lipids from the cytosol into the ER lumen (step C). Compounds that inhibit in vitro transfer activity of MTP decrease apoB secretion by cells, indicating that this activity is essential for apoB lipoprotein secretion. Apart from transferring lipids, MTP has been shown to interact physically with apoB (step D). This activity... 

The transfer of radiolabeled phospholipids between vesicles and erythrocyte membranes could be used to assay lipid transfer activity. Intact erythrocytes are not an ideal substrate for routine measurements of transfer activity because some transfer proteins do not readily accelerate the transfer of phospholipids from these membranes. Van Meer et al. (1980) found that a very high concentration of the phosphatidylcholine-specific transfer protein was necessary to exchange the phosphatidylcholine of intact red blood cells. Erythrocyte ghosts are a more active substrate for this protein (Bloj and Zilversmit, 1976). However, the nonspecific transfer protein from bovine liver accelerates the exchange of phospholipid between intact erythrocytes and phosphatidylcholine vesicles (Crain and Zilversmit, 1980c). 

Helmkamp (1980a) studied the effect of the fatty acid composition of the acceptor lipid on the stimulation of phosphatidylinositol transfer from rat liver microsomes to phosphatidylcholine vesicles by bovine brain exchange protein. Acceptor vesicles containing egg phosphatidylcholine or dioleoyl phosphatidylcholine gave approximately the same transfer activity, whereas dielaidoyl phosphatidylcholine or dimyristoyl phosphatidylcholine vesicles produced lower transfer rates. Zborowski and Demel (1982) used the same protein and measured the rate of transfer of phosphatidylinositol from a monolayer to phosphatidylcholine vesicles. Vesicles of egg, dioleoyl, dielaidoyl, and dipalmitoyl phosphatidylcholine, even below its phase transition temperature, all gave equivalent transfer rates. However, a reduced rate was found when dimyristoyl and dilin-oleoyl phosphatidylcholine, and other phosphatidylcholines with two polyunsaturated fatty acids, were used. Table IV shows a comparison of the transfer activities measured in the two assays. The transfer rates are expressed as a percent of the transfer rate obtained with egg phosphatidylcholine acceptor vesicles. 

Other systems like electroporation have no lipids that might help in membrane sealing or fusion for direct transfer of the nucleic acid across membranes they have to generate transient pores, a process where efficiency is usually directly correlated with membrane destruction and cytotoxicity. Alternatively, like for the majority of polymer-based polyplexes, cellular uptake proceeds by clathrin- or caveolin-dependent and related endocytic pathways [152-156]. The polyplexes end up inside endosomes, and the membrane disruption happens in intracellular vesicles. It is noteworthy that several observed uptake processes may not be functional in delivery of bioactive material. Subsequent intracellular obstacles may render a specific pathway into a dead end [151, 154, 156]. With time, endosomal vesicles become slightly acidic (pH 5-6) and finally fuse with and mature into lysosomes. Therefore, polyplexes have to escape into the cytosol to avoid the nucleic acid-degrading lysosomal environment, and to deliver the therapeutic nucleic acid to the active site. Either the carrier polymer or a conjugated endosomolytic domain has to mediate this process [157], which involves local lipid membrane perturbation. Such a lipid membrane interaction could be a toxic event if occurring at the cell surface or mitochondrial membrane. Thus, polymers that show an endosome-specific membrane activity are favorable. 

The results indicate that the initial rate of transport of PE is rapid and proceeds without a lag (Fig. 8). The transport process is insensitive to metabolic poisons that disrupt vesicle transport and cytoskeletal structure. The rapid transport kinetics occur at rates consistent with a soluble carrier-mediated process or transfer at zones of apposition between membranes. Analysis of the kinetics of the process is complicated since only PE at the outer leaflet of the plasma membrane is measured, and the basal scramblase activity or the leakage of the ATP-dependent aminophospholipid transporter activity within the plasma membrane may be a step required for the lipid to arrive at this location. Despite these complications, the results clearly indicate that the initial rate of arrival of PE at the plasma membrane occurs on a timescale that clearly distinguishes it from well-characterized vesicle transport phenomena, and is independent of processes involved in protein transport to the cell surface. 

As a new type of electron relay, which is able to penetrate lipid membranes, we tested menaquinone (MQ, Fig. 7). Compounds of this type were not utilized earlier for artificial vesicle-based systems. However, these mimick the functioning of the Z-scheme of natural plant photosynthesis (see Figs 9 and 12). Indeed, the activity of MQ in the redox processes in a lipid bilayer membrane was revealed. However, the quantum yield of the transmembrane electron transfer from a CdS nanoparticle in the inner cavity to a CdS nanoparticle on the outer membrane surface with the participation of MQ appeared to be very low and did not exceed 0.2-0.4%. 

Two light-activated cyclic electron transfer systems have been reincorporated into lipid vesicles in such a way that proton pumping across the membranes may be observed under appropriate conditions. The first of these has been constructed from mammalian cytochrome bc] complex and reaction centres isolated from Rhodopseudomonas sphaeroides (RCbc vesicles), a combination used previously by Packham et al. (1980) for single turnover studies in solution. In order to maintain adequate multiple turnover electron flux under our conditions, it was necessary to add both cytochrome c and ubiquinone-2. In the presence of valinomycin, light activation caused the translocation of four protons outwards across the vesicles for each pair of electrons completing a cycle, although this ratio appeared to fall to two after a significant ApH had built up. 

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