Phospholipids, commonly used

Figure 14 Structures of zwitterionic phospholipids commonly used in the formation of liposomes and lipid bilayers. Shown are l-palmitoyl-2-oleoyl-in-glycero-3-phosphocholine (POPC), l,2-ipalmitoyl-in-glycero-3-phosphocholine (DPPC), and 1,2-dioleoyl-iw-glycero-3-phosphoethanolamine (DOPE). As is the case with POPC and DPPC, the acyl chains attached to the polar phosphatidylcholine head group may correspond to various fatty acids, which can be interchanged. DOPE, on the other hand, is comprised of the same acyl chains as DPPC and POPC but differs in the headgroup.

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. 

ITABLE 13.6. Some of the commonly used phospholipids and the attributes of head and fatty acyl (tail) groups... 

Despite the large variety of potential fatty acid components in natural-occurring phosphodiglycerides, only three major fatty acid derivatives of synthetic phospholipids are commonly used in liposome preparation (1) myristic acid (w-tetradecanoic acid containing 14 carbons), (2) palmitic acid (w-hexadecanoic acid containing 16 carbons), and (3) stearic acid (w-octadccanoic acid containing 18 carbons) (Fig. 334). 

Buffered Tetrahydrofuran. In 1973, Tettamanti et al. [19) described an improved procedure for the extraction, separation and purification of brain gangliosides. In this method, the brain tissue was subjected to homogenization and extraction with buffered [potassium phosphate buffer, pH 6.8) tetrahydrofuran. Following centrifugation, diethyl ether was added and the mixture separated into organic and aqueous phase. The gangliosides, recovered exclusively in the aqueous phase, were then freed of residual phospholipids and other minor contaminants [i.e. peptides)by column chromatography on silica gel.This procedure, as shown by the authors,was superior to the commonly used chloroform/methanol... 

Another way to assess ion channel conductance is to use artificial phospholipid vesicles (liposomes) as cell models. These structures (described in more detail in the next chapter) are commonly used to transport vaccines, drugs, enzymes, or other substances to target cells or organs. 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. A liposome model was used to test the ion transport behaviour of the redox-active hydraphile 12.36. The compound transports Na+ and the process can also be monitored using 23Na NMR spectroscopy.26 The presence of the ferrocene-derived group in the central relay allows the ion transport to be redox-controlled - oxidation to ferrocinium completely prevents Na+ transport for electrostatic reasons. Some representative data from a planar bilayer measurement is shown for hydraphile 12.36 in Figure 12.16. 

A typical biological membrane is a complex structure composed primarily of lipids and proteins. The major structural components of the bilayer are various lipids. In eukaryotes, the most common type of lipids are phosphatidylcholines, whereas in prokaryotes (such as Escherichia coli), the main lipids are typically phosphatidylethanolamines (1). One example of a typical eukaryotic neutral (zwitterionic) phospholipid is palmitoyl-oleoy 1-phosphatidylcholine (POPC). The molecular structure of POPC is compared to those of dimyristoylphosphatidyl-choline (DMPC) and the negatively charged dimyristoylphosphatidylglycerol (DMPG), commonly used in membrane mimetics, in Fig. 1. 

Since in all these cases we used oxidants to induce apoptosis, a relatively massive background oxidation of all PnA-labeled phospholipid classes was observed and masked apoptosis-specific PS oxidation. Therefore, we next determined whether a non-oxidant-induced apoptosis model could be used to reveal more explicitly the selective mode of PS oxidation inherent to the apoptosis execution program. To this end, we used agonistic anti-Fas antibody to induce apoptosis in Jurkat B cells according to a commonly used protocol (Fadeel et al., 1999). Importantly, we foundthat anti-Fas-triggered apoptosis in Jurkat cells was characterized by early and selective oxidaiton of cA-PnA-PS (Kagan et al., 2001) without any significant involvement of other classes of phospholipids. 

Custom-made lipids can be produced by de- or reacylation of natural lipids. Commonly used phospholipids with polar heads containing myristoyl (14 0), palmitoyl (16 0), stearoyl (18 0) fatty acids are all classified by four-letter abbreviations, for example, DMPC, where DM stands for the number and type of fatty acids (di-myris-toyl) and PC for the type of polar head (phosphatidylglycero-choline), and similarly DPPC and DSPC (Figure 2). 

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