Phospholipid exchange

The method of introduction of the fluorophore into the membrane is also important. Many probes are introduced into preexisting vesicles, natural membranes, or whole cells by the injection of a small volume of organic solvent containing the fluorophore. For DPH, tetrahydrofuran is commonly used, while methanol is often employed for other probes. The amount of solvent used should be the absolute minimum possible to avoid perturbation of the lipids, since the solvent will also partition into the membrane. With lipid vesicles this potential problem can be avoided by mixing the lipids and fluorophore followed by evaporation of the solvent and codispersing in buffer. For fluorophores attached to phospholipids, this is the only way to get the fluorophore into the bilayer with natural membranes, phospholipid exchange proteins or other techniques may have to be employed. 

D10. DiCorleto, P. E., Warach, J. B., and Zilversmit, D. B., Purification and characterization of two phospholipid exchange proteins from bovine heart. /. Biol. Chem. 254, 7795-7802 (1979). 

H22. Helmkamp, G. M., Jr., Harvey, M. S., Wirtz, K. W. A., and Van Deenen, L. L. M., Phospholipid exchange between membranes. Purification of bovine brain proteins that preferentially catalyze the transfer of phosphotidylinosotol.. Biol. Chem. 249, 6382-6389 (1974). 

T2. Tall, A. R, Abreu, E., and Shuman, J., Separation of a plasma phospholipid transfer protein from cholesterol ester/phospholipid exchange protein. J. Biol. Chem. 258, 2174-2180 (1983). 

W15. Wilson, D. B., Ellsworth, J. L., and Jackson, R. L., Net transfer of phosphatidylcholine from plasma low density lipoproteins to sphingomyelin-apolipoprotein A-II complexes by bovine liver and human plasma phospholipid exchange proteins. Biochim. Biophys. Acta 620, 550-561 (1980). 

Two mechanisms have been proposed to explain the transport of phospholipids from the ER to other cellular membranes protein-mediated transfer and a vesicular process. Several experiments have demonstrated that water-soluble proteins, known as phospholipid exchange proteins, can bind to specific phospholipid molecules and transfer them to another bilayer. Vesicular transport of phospholipids and membrane proteins in structures known as transition vesicles from the ER to the Golgi complex is not clearly understood. However, evidence of transfer of luminal material from the ER to the Golgi cistemae clearly supports vesicular transport. 

Phospholipid translocator proteins, phospholipid exchange proteins, and transition vesicles are involved in the complicated process of membrane synthesis and delivery of membrane components to their cellular destinations. 

DiCorleto and Zilversmit (1977) reported that the phospholipid exchange proteins from either beef heart or beef liver were not able to catalyze the exchange of phosphatidylcholine between phosphatidylcholine multi-lamellar vesicles and small unilamellar vesicles. However, by adding acidic phospholipids to the multilamellar vesicles, protein-enhanced exchange was observed. This assay is versatile and can be easily performed. Both substrates are well-defined particles in which the composition can be readily manipulated. The separation of acceptor and donor membranes by centrifugation is rapid and nearly complete with 98—99% of the multilamellar vesicles being sedimented and with 90% of the small unilamellar vesicles remaining in the supernatant. 

Somerharju et al. (1981) developed an assay for phospholipid exchange activity with the fluorescent phospholipid derivative, l-acyl-2-parinaroyl-phosphatidylcholine. When vesicles of this fluorescent phospholipid are... 

Figure 1. Effect of donor particle concentration on the initial rate of lipid transfer. The computer-drawn curves are based on the kinetic model by Van den Besselaar et al. (1975) for protein-catalyzed phospholipid exchange. The kinetic constants are for, the transfer of phosphatidylcholine from liposomes containing 12 mol% phosphatidic acid to liposomes containing 2 mol% phosphatidic acid. The three curves represent three different acceptor concentrations indicated above the appropriate curves.

Transfer Activity of the Bovine Brain Phospholipid Exchange Protein with Acceptor Vesicles of Varying Composition 1... 

The amount of lipid that is subject to rapid exchange appears to vary with the composition of the multilamellar vesicles and their method of preparation. DiCorleto and Zilversmit (1977) found that multilamellar vesicles of phosphatidylcholine phosphatidylethanolamine cardiolipin (70 25 5, mol%) contained 7% of exchangeable phosphatidylcholine. Wirtz et al. (1979) found that 4.5% of the phosphatidylcholine of egg phosphatidylcholine phosphatidic acid (90 10, mol%) multilamellar vesicles was fully exchangeable. Bozzato and Tinker (1982) found that 8.5 2% of the phospholipid of annealed dipalmitoylphosphatidylcholine dipalmitoylphosphatidylglycerol (95 5, mol%) multilamellar vesicles was exchangeable, whereas the phospholipid exchange of nonannealed vesicles was 11 3%. 

In Table 4 the amino acid composition of purified SCP2 is also compared with the amino acid composition of a protein described as a nonspecific phospholipid exchange protein (CM2) [49]. The amino acid compositions of SCP2 and CM2 nearly identical. The correlation coefficient is 0.992 [21]. 

Furthermore, it is mandatory to specify and standardize the solubilization of oxidized phospholipids for incubation with cells or individual target molecules. Physiologically relevant systems are pure lipid micelles or vesicles depending on the chemical structure of the lipid, complexes with proteins (e.g. albumin) and plasma lipoproteins. Furthermore, it has to be taken into account that oxidized phospholipids exchange between lipid surfaces much faster than regular membrane phospholipids containing two long hydrophobic acyl chains (Li et al.,... 

Phospholipid-Exchange Systems Paul Mazliak and J. C. Kader... 

On the contrary, the microsomes of plant cells proved to contain all the enzymes necessary for the biosynthesis of either polyunsaturated fatty acids (at least linoleic acid (Vijay and Stumpf, 1971 Ben Abdelkader et al., 1973 Kader, 1977a) or the m or phospholipids of plant membranes i.e., phosphatidylcholine and phosphatidylethariolamine (Devor and Mudd, 1971 Moore et al., 1973). Therefore one was forced to the conclusion that there must be a transfer of lipids from microsomes to other intracellular organelles to correlate the limited synthetic capabilities of the various organelles with the uniformity of their lipid composition. We proposed, in 1968 (Mazliak et al., 1968), that an intermembrane phospholipid exchange was implied in this necessary cooperation between microsomes and other cellular organelles. 

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