Lipid requirements

While recent attention has been largely on proteins, it should be borne in mind that membrane fusion ultimately involves the merger of phospholipid bilayers. However, little is known about the specific membrane lipid requirements. When membranes fuse, energetically unfavorable transition states are generated that may require specific lipids and lipid domains for stabilization. Although there is some evidence for a specific influence of lipids on exocytosis, it is still unclear whether specific lipid metabolites are needed or even generated at the site of membrane merger. 

Lipid Screening. The problems of lipid analysis in the newborn is difficult because of the fact that most methods for analysis for lipids require substantial amounts of serum, yet a total lipid determination is very important in various types of disease. This problem can be solved by thin-layer chromatography (59). Figure 38 shows a typical pattern obtained when an extract 7rom 10 microliters of serum is subjected to thin-layer chromatography. If these specimens are scanned, and an internal standard is run, one can obtain a rough approximation of the distribution of the various lipids in the serum. This is shown in Figure 39, in which a normal specimen is run in an adult. 

Opekarova, M. and Tanner, W. Specific lipid requirements of membrane proteins— a putative bottleneck in heterologous expression. Biochim Biophys Acta 1610 11— 22, 2003. 

Basic Protocol 4 Extraction of Lipids Requiring Acid Digestion Dl.l. 7... 

For extraction of lipids, using all four protocols, approximately 35 to 40 hr is required. The time required for the Soxhlet method (see Basic Protocol 1) is up to 12 hr, the chloro-form/methanol/water extraction (Basic Protocol 2), up to 12 hr, n-propanol/water extraction (see Basic Protocol 3), 5 to 6 hr, and extraction of lipids requiring acid digestion (see Basic Protocol 4), 5 to 8 hr. 

In LC-MS analysis, identification of lipids requires the use of accurate mass measurements for the determination of elemental composition, combined with MS" experiments to get information of the molecular fragments. In addition, in LC-MS, one metabolite may produce multiple peaks due to the presence of isotopes, adducts, and neutral loss fragments. Therefore, ion annotation is crucial in order to recognize a group of ions likely to originate from the same... 

Individual mitochondrial enzymes such as the lipid-requiring enzyme cytochrome C oxidase exhibit interspecific variation in ABTs similar to that seen for mitochondrial oxygen consumption (O Brien et al., 1991 Dahlhoff and Somero, 1993b). Thus, the impairment of mitochondrial respiration at high temperatures may be due to loss of activity of enzymes in ATP-generating pathways. 

Vitamin E, like neutral lipids, requires apoB lipoproteins at every stage of its transport (Fig. 27-2). Dietary vitamin E becomes emulsified in micelles produced during the digestive phase of lipid absorption and permeates the intestinal epithelium, similar to fatty acids and cholesterol. Uptake of vitamin E by enterocytes appears to be concentration dependent. Within intestinal cells, vitamin E is packaged into chylomicrons and secreted into lymph. During blood circulation of chylomicrons, some vitamin E may be released to the tissues as a consequence of partial lipolysis of these particles by endothelial cell-anchored lipoprotein lipase. The rest remains associated with chylomicron remnants. Remnant particles are mainly endocy-tosed by the liver and degraded, resulting in the release of fat-soluble vitamins. 

Cereal starches contain low levels of lipids (0.5-1%), which are generally polar lipids requiring polar solvents such as methanol-water for extraction. Lipid content increases with amylose content, and unless the granule integrity is disrupted, the lipids remain inaccessible to normal fat solvents, suggesting that they are present as an amylose inclusion complex. Noncereal starches contain essentially no lipids. 

There is little evidence indicating that rumen protozoa are capable or involved in BH (Harfoot and Hazlewood, 1997 Williams and Coleman, 1988), and it is arguable that protozoa could satisfy their lipid requirements through the ingestion of rumen bacteria, chloroplasts, and other plant lipids. 

Wheeler, C. J., Sukbu L., Yang G., Tsai Y., Bustamente C Feigner P. L Norman, J., and Manthorpe, M. (1996) Converting an alcohol to an amine in a cationic lipid dramatically alters the co-lipid requirement, cellular transfection activity and the ultrastructure of DNA-cytofectm complexes. Biochim. Biophys. Acta 1280, 1-11. 

This indicates that some de novo FA biosynthesis occurs in the apicoplast. It may occur in a discrete compartment or be related to a specific but crucial type of lipid (predominantly C-10 to C-14, as shown by Surolia and Surolia, 2001). This pathway is probably quantitatively minor but appears to be critical to parasite survival. It could be involved in the synthesis of lipids required by the apicoplast or a specific lipid that may be provided to the parasite. A major interest resides in the fact that FAS II is structurally and functionally distinct from the equivalent pathway in the vertebrate host, and would explain the susceptibility of the malarial parasite to herbicides targeting the enzyme. It is considered today as a validated target that still needs to be developed into a practical antimalarial. 

Apart from GPC with Bio-Beads, there are other automated clean-up approaches. Although GPC has several advantages, Focant and co-workers claimed that for the high-fat-content samples (4 g lipids) required in dioxin analysis, GPC suffered from several practical limitations and therefore could not be performed 100% automatically (59). Consequently, new high-capacity disposable silica (HCDS) columns were developed for the Power-Prep system. The combination of PLE and Power-Prep clean-up allowed 10 dioxin samples to be analyzed in 48 hrs. 

Of course, if only one single diffraction maximum is observed, a strict assignment of a certain lattice type cannot be made. The assignment of triclinic, orthorhombic, monoclinic or hexagonal lattices, as they are frequently found in lipids, requires the indexing of several reflections, which in practice, however, are often rather obscure. The analysis can be aided considerably by the comparison to the diffraction from single crystals... 

A specific phospholipid requirement has been determined for optimum in vitro reconstitution of function for more than 50 membrane proteins. If one considers specific lipid requirements for membrane association and activation of amphitropic proteins, the number is in hundreds. Integral membrane proteins fold and exist in a very complex environment and have three modes of interaction with their environment. The extramembrane domains are exposed to the water milieu, where they interact with water, solutes, ions, and water-soluble proteins. Part of the protein is exposed to the hydrophobic-aqueous interface region (Fig. 9). The remainder of the protein is buried within the approximately 30-A thick hydrophobic interior of the membrane. Amphitropic proteins may spend part of their time completely in the cytosol and are recruited to the membrane surface, or even partially inserted into the membrane, in response to various signals. 

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