Lipid isolation from cells

Lipids analysis has benefited from nanoESI with the ESI Chip.24 27 Lipids isolated from cells using liquid-liquid extraction can be quantified and separated using MS-MS scans such as multiple precursor and neutral loss scanning to identify head groups and side chain information.26... 

Lipid (Section 27.1) A naturally occurring substance isolated from cells and tissues by extraction with a nonpolar solvent. Lipids belong to many different structural classes, including fats, terpenes, prostaglandins, and steroids. 

The location of the double bonds in unsaturated glycerophosphocholine (GPC) lipids is made difficult mainly due to the limited amounts of these compounds isolated from cells. When a thin layer of a GPC lipid is exposed to the action of ozone, partial or total ozonization of the unsaturated fatty acid residues may take place, depending on the particular compound being analyzed. The ozonized hpid is dissolved and analyzed by tandem MS. A typical injection into the instrument may be 5 xL containing 10-20 ng of the lipid. An illustrative example is the ozonized l-stearoyl-2-arachidonoyl-GPC, showing the systematic fragmentation pattern in Scheme 21, typical of the ozonized GPC lipids. Thus, the molecular peak appears increased by one nominal unit (M + H, m/e 1002),... 

The lipids isolated from a cell preparation are separated on thin-layer chromatography, and the desired fractions are recovered by the extraction procedure described earlier in this chapter. In this instance, interest is centered on the phosphatidylcholine fraction, and it can be subjected to the protocol outlined in Scheme A (Figure 4-24). 

The lipids isolated from the membranes of 4.74 x 109 human red blood cells were spread as a monolayer with an area of 0.89 m2. Assuming the red blood cell approximates a disk 7 jun in diameter and 1 jun thick, show that the membrane covering the red blood cell must be two lipid molecules thick. 

PhosphattdykhoHne (PC) and phosphatidylethanolamine (PE) are the major phospholipids of cell membranes. Table 6.1 lists the amount of lipid in cell membranes expressed as micrograms of lipid per milligram of membrane-bound protein. The membranes were isolated from cells of adipose tissue from rats that had been raised on a diet containing sunflower oil (100 g oil/kg diet) as the source of lipids. The diet was essentially free of cholesterol and phospholipids because plants do not contain cholesterol and the phospholipids of vegetable oils are removed during the refining process. 

Lipids are the naturally occurring organic molecules isolated from cells and tissues by extraction with nonpolar organic solvents. Note that this definition differs from the sort used for carbohydrates and proteins in that lipids are defined by a physical property solubility) rather than by structure. 

Direct interaction of sHsps other than a-crystallin with membranes has been reported only for Synechocystis Hsp 16.6 (Torok et al., 2001). Purified recombinant protein interacted with lipid vesicles, as monitored by fluorescence anisotropy, to reduce lipid fluidity and also increased the surface pressure of lipid monolayers. There was evidence for both lipid specificity and membrane penetration of the sHsp. Thylakoids isolated from cells deleted for Hsp 16.6 were more fluid membranes than wild-type cells, even in the presence of very little sHsp. Ability to stabilize directly photosynthetic membranes by lipid interaction could explain data supporting a role for sHsps in photosystem II protection, but the specificity of this effect has not been very well documented (Harndahl and Sundby, 2001 Heckathorn et al., 1998 Lee et al., 2000 Nakamoto et al., 2000). 

Five lipids (ceramide IV, sphingosine, sphingosine phosphate, and dihydro- and phytosphingosine) were isolated from cell cultures and analyzed on a silica column (ELSD, drift tube T = 70°, N2 nebulizer gas at 1.6 bar) using a 90/01/1/1 chloroform/ethanol (200 proof)/triethylamine/formic acid mobile phase [782]. Good resolution and peak shapes were achieved and elution was complete in 16 min. A linear response from 1.7 to 17 ig injected was generated and a detection limit of 100 ng injected were reported. 

A large concentration of ornithine-containing lipid was found in a basic membrane material prepared from a mutant strain of Rhodo-spirillum rubrum (22). Studies on the structure of a similar lipid isolated from van Niels strain SI of R. rubrum (heterotrophically or auto-trophically grown) were in agreement with structure (9) (23). In heterotrophically grown cells a second ornithine-containing lipid was found it was stated that the two lipids seem to be quite dissimilar in structure (23). 

Since it was observed that protein synthesis continues after the cessation of net lipid synthesis, it was of interest to determine whether the synthesis of membrane proteins continues under these circumstances. Membranes isolated from cells deprived of glycerol show an increase in protein content, but no increase in lipid content, indicating that membrane... 

The permease can be measured as a phosphotransferase activity in vitro, as well as a transport system in vivo. Membranes isolated from cells induced for the lac system in the absence of lipid synthesis were prepared, and phosphotransferase activity was measured. It was found that the phosphotransferase activity of membranes from glycerol-deprived cells, when normalized to j -galactosidase activity of the culture, was identical to that of the supplemented cells, indicating that the Enzyme II component of the /flc-transport system was, in fact, synthesized at the same rate with respect to j -galactosidase activity in both the deprived and the supplemented cultures (Fig. 8). The results also indicate that Enzyme II is integrated into the membrane to the same extent, in both deprived and supplemented cultures. 

Lipids are naturally occurring organic molecules that have limited solubility in water and can be isolated from organisms by extraction with nonpolar organic solvents. Fats, oils, waxes, many vitamins and hormones, and most nonprotein cell-meznbrane components are examples. Note that this definition differs from the sort used for carbohydrates and proteins in that lipids are defined by a physical property (solubility) rather than by structure. Of the many kinds of lipids, we ll be concerned in this chapter only with a few triacvlglycerols, eicosanoids, terpenoids, and steroids. 

The actions of proteins isolated from sea anemones, or other coelenterates, involve mechanisms different from those described for saponins. Thus, hemolysins from sea anemone R macrodactylus are capable of forming ion channels directly in membranes (98). The basic protein from S. helianthus also forms channels in black-lipid membranes. These channels are permeable to cations and show rectification (99). This ability of S. helianthus toxin III to form channels depends upon the nature of the host lipid membrane (100). Cytolysin S. helianthus binds to sphingomyelin and this substance may well serve as the binding site in cell membranes (101-106). 

Treatment with iron chelators and a-tocopherol protect against lipid p>eroxidation and hepatocellular injury in iron-overloaded rats (Sharma etal., 1990). When hepatocytes are isolated from rats, which have been pretreated with a-tocopherol, there is a significant reduction in iron-induced lipid peroxidation and improvement in cell viability in vitro (Poli et al., 1985). Similar effects were seen when hepatocytes were incubated with iron chelators (Bacon and Britton, 1990). Treatment of moderately, but not heavily, iron-loaded rats with desferrioxamine in vivo inhibits the pro-oxidant activity of hepatic ultrafiltrates (Britton et al., 1990b). 

Sample preparation used to extract proteins from cells prior to analysis is an important step that can have an effect on the accuracy and reproducibility of the results. Proteins isolated from bacterial cells will have co-extracted contaminants such as lipids, polysaccharides, and nucleic acids. In addition various organic salts, buffers, detergents, surfactants, and preservatives may have been added to aid in protein extraction or to retain enzymatic or biological activity of the proteins. The presence of these extraneous materials can significantly impede or affect the reproducibility of analysis if they are not removed prior to analysis. 

In the organism tissues, fatty acids are continually renewed in order to provide not only for the energy requirements, but also for the synthesis of multicomponent lipids (triacylglycerides, phospholipids, etc.). In the organism cells, fatty acids are resynthetized from simpler compounds through the aid of a supramolecular multienzyme complex referred to as fatty acid synthetase. At the Lynen laboratory, this synthetase was first isolated from yeast and then from the liver of birds and mammals. Since in mammals palmitic acid in this process is a major product, this multienzyme complex is also called palmitate synthetase. 

Proulx [30] summarized the published lipid compositions of BBM isolated from epithelial cells from pig, rabbit, mouse and rat small intestines. Table 3.1 shows the lipid make-up for the rat, averaged from five reported studies [30], On a molar basis, cholesterol accounts for about 50% of the total lipid content (37% on a weight basis). Thus, the cholesterol content in BBM is higher than that found in kidney epithelial (MDCK) and brain endothelial cells (Table 3.1). Slightly different BBM lipid distribution was reported by Alcorn et al. [31] here, the outer (luminal) leaflet of the BBM was seen to be rich in sphingomyelin content, while the inner leaflet (cytosol) was rich in PE and PC. Apical (brush border) and basolateral lipids are different in epithelia. The basolateral membrane content (not reported by... 

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