Sphingomyelin labeled

Fig. 4.5. Sphingomyelin synthesis in Hymenolepis diminuta distribution of label in various intermediates, separated by thin-layer chromatography, after incubation with cytidine-5 -diphospho [methyl-13C]choline. The position of the lipid standards is indicated by the arrows, (a) sphingomyelin (b) dihydrosphingosine (c) sphingosine (d) ketosphingosine (e) ceramide. The origin is at band O. d.p.m., disintegrations/min. (After Bankov Barrett, 1985.)...

Nonetheless, it has been shown to elute immediately after the PC peak. LysoPC, if present, would elute between the PAF and sphingomyelin (Sph) peak. The latter component often shows a double peak, and this can be attributed to the separation of distinct fatty acyl species. It is well to emphasize again that compounds such as PAF and lysoPC have very low levels of unsaturated bonds present, and hence a detector other than an ultraviolet monitor would have to be used. An alternate approach would be to use tritiated PAF or tritiated lysoPC as examples and assay the eluates by liquid scintillation counting. Since the labeled compounds are at tracer dose levels, one could still assay for biological activity associated with the compounds. 

As shown in Table IVA, the cells bind approximately 5% of the total radioactivity added 32% of this eictivity is associated with the cellular lipid fraction and 64% with the nonlipid (presumably protein) fraction. Of the lipid-associated activity, the distribution of I into cellular phospholipids, lysophosphatides, triacylglycerides, and free fatty acids is shown in Table IVB. The iodination reaction labels a variety of lipids of all classes without preference to charge, acid-base properties, or number of fatty acyl constituents, appears not to label intracellular lipids (e.g., cardiolipin, a major mitochondrial component), and labels lipids on both the exterior and interior hydrophilic surfaces of the plasma membrane (i.e., phosphatidylcholine and sphingomyelin as well as phosphatidyleth-anolamine and phosphatidylserine) (Table IVB). ... 

GPC (TAG) increased the amount of radioisotopically labeled macromolecules that was released from the cells from 30-32% up to 55%, and increased the release of labeled lipids from 9-12% up to 19% (Table VA). The indentity of the I-labeled lipid molecules released from the cells after antibody-GPC treatment is shown in Table VB TAG released a significantly higher proportion of sphingomyelin, phosphatidylserine, and... 

Fig. 19.6. STED microscopy discriminates the dynamics of single sphingo- and phospholipid molecules in a living cell membrane dye-labeled sphingomyelin (SM) vs. phosphoethanolamine (PE). Panels (a) and (b) illustrate that freely moving molecules may be transiently trapped in the membrane due to interaction with...

Figure 7.9 Creating a multidomain vesicle by electrofusion of two vesicles with different membrane composition as observed with fluorescence microscopy, (a, b) Images acquired with confocal microscopy scans nearly at the equatorial plane of the fusing vesicles, (a) Vesicle 1 is made of sphingomyelin and cholesterol (7 3) and labeled with one fluorescent dye (green). Vesicle 2 is composed of dioleoylphosphatidylcholine and cholesterol (8 2) and labeled with another...

Two situations must be distinguished, (i) assays of pure enzyme and (ii) assays of cell extracts. When purified enzyme preparations are available, no labeled substrate is required. Natural or synthetic sphingomyelin is prepared, pure or mixed with other lipids, in the form of extruded large unilamellar vesicles (LUV) ca. 100 nm in diameter. When pure sphingomyelin vesicles are used extrusion must take place at a temperature close to or above the gel-fluid transition temperature of the lipid, i.e. often 45-50 °C. LUV and enzyme are mixed in the appropriate assay buffer and aliquots are removed at fixed time intervals. The aliquots are mixed with chloroform-methanol and, after phase separation, phosphorous (from phosphorylcholine) is assayed in the aqueous phase. The procedure has been described in detail by Ruiz-Arguello et al. [91]. 

Pownall et al. (1982) incorporated 1 mol% pyrene-labeled sphingomyelin into lipoprotein complexes and measured its rate of protein stimulated transfer to an excess of acceptor particles. Throughout the transfer reaction, monomer emission represents labeled lipid found in both the donor and acceptor complexes, while eximer emission represents labeled lipid in the donor. The eximer fluorescence can therefore be used as a measure of the pyrene-labeled lipid concentration in the donor complex. 

NMR technique, especially solid state NMR spectrometry, has been frequently used in investigations of the order and mobility of acyl chains within the lipid bilayers. The NMR study of the quadrupole sphtting of deuterons located along the lipid chain permitted to gain information on molecular motions of lipids. For this aim, selectively deuterated lipids have been prepared. 7V-stearoyl sphingomyelin (Figure 7) was deuterium-labelled on different positions of both the sphingosine and the stearoyl chains to capture its motion in membranes. 

Vos JP, Giudici ML, van der Bijl P, et al. Sphingomyelin is synthesized at the plasma membrane of oligodendrocytes and by purified myelin membranes a study with fluorescent- and radio-labelled ceramide analogues. FEES Lett. 1995 368(2) 393-396. 

Dipalmitoylphosphatidylcholine (31) has been quantified in amniotic fluid by FABMS using a d9-labelled standard (97). Such measurements were an alternative to the determination of lecithin-sphingomyelin ratios as a predictive test for lung viability in mid-term and late foetal development. This study is also noteworthy inasmuch as quantification using FABMS has not been widely exemplified. An advantage of the mass spectrometric method is its specificity for the dipalmitoyl derivative (MW 735 daltons). The FAB spectrum also afforded evidence that the mixed palmitoyl oleoyl (MW 761) and tetradecanoyl (MW 707) esters were also present. Homologues were also detected (98) by FABMS of zwitterionic ornithine containing lipids (32) from Thiobacillus thiooxidans and were further discriminated by their CAD spectra. 

A notable feature of the lipid regions of biological membranes is that the different phospholipid types may be asymmetrically distributed across the bilayer. For the erythrocyte membrane for example, it has been demonstrated by surface labelling and phospholipase digestion that the sphingomyelin and phosphatidylcholine are located in the outer half of the bilayer, whereas the phosphatidylethanolamine and phosphatidylserine are localized to the inner half (Zwaal et al., 1973). 

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