Glycolipids cell surface labelling

Further studies using NeuSAc have been related to the metabolism of extracellular CMP-NeuSAc and the detection of ectosialyltransferase activity. On the basis of a lag period observed for NeuSAc uptake and incorporation into glycoconjugates, and the absence of such a lag for CMP-NeuSAc incorporation, two routes have been proposed. The uptake of NeuSAc and metabolism as described above has been studied in hamster and mouse fibroblasts, and cell surface labelling of glycoprotein and glycolipid demonstrated (Datta 1974). The breakdown of CMP-NeuSAc was shown, and incorporation due to NeuSAc uptake rather than direct CMP-NeuSAc transfer proposed (Hirschberg et al. 1976). The uptake of CMP-NeuSAc into the cells (NIL, BHK and 3T3 fibroblasts) could be ruled out, and the K , for NeuSAc uptake was estimated to be 10 mM. Other experiments with CMP-NeuSAc and intact cell cultures (Painter and White 1976, Cerven 1977, see section III.9) pointed to surface sialyltransferase. Further studies by Fan and Datta (1980) provided evidence that both transfer and transport occur, by localization of acceptors within the cell and on the cell surface (plasma membrane), and direct demonstration of the presence of a plasma membrane sialyltransferase. The sialylation due to NeuSAc uptake occurs (at least initially) with different acceptors in comparison with CMP-NeuSAc plasma membrane sialylation. 

Lectins, or proteins with specific binding sites for carbohydrates, can be used as targeting molecules to localize particular glycoconjugates such as glycoproteins or glycolipids on cell surfaces (Fig. 373). Labeled with gold particles, lectins are important probes for detection of cell surface components and intracellular receptors and in immunological or biochemical assay procedures (Bog-Hansen et al., 1978 Kimura et al., 1979 Nicolson, 1978 Roth, 1983 Benhamou et al., 1988 Nakajima et al., 1988). 

In order to obtain some idea about the nature of gly-coconjugates and their gross topographical orientation on the cell surfaces, we measured the binding of 125j labeled lectins and toxin to human neuroblastoma IMR-32 and mouse neuroblastoma N1E-115, NS-20, and N-18 clones (Tables VII and VIII). The "5% TCA Wash" column (Table VII) represents 1251-iabeled lectin or toxin bound to both glycoprotein and glycolipid. The "5%... 

Fig. 6.3. Micelles, liposomes and cell membranes. Micelles are collections of lipid molecules that are relatively nonpolar internally and polar externally. This arrangement allows relative water-solubility of the micelle as a whole. Liposomes contain lipid molecules in a bilayer. They may be used as artificial vehicles for trapping and delivery of drugs to specific tissues. They are also useful as models of cell surfiice function. A real cell membrane is not only a lipid bilaycr, but also includes proteins, glycoproteins, glycolipids, and lipoprotein molecules. The glyco attachments on the outer surface may be important in labeling cells with specific cell-surfece properties.

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