Activities glycolipid: sialyltransferase

Glycolipid Sialyltransferase Activities in Mouse and Human Tumor Cells... 

Figure 6. Effect of CMP-NeuAc concentration (V/S), of pH (V/pH), of enzymatic protein concentration (V/protein), and of incubation time (V/t) on the activity of synaptosomal membrane-bound sialyltransferase. Calf brain cortex. Acceptor substrates for sialyltransf erase (if) lactosylceramide ( ) desialylated fetuin (%) endogenous glycoprotein (endogenous glycolipids.

Kinetic Properties of Sialyltransferases. The sialyl-transferase activities with the endogenous glycoprotein and glycolipid acceptors in the standard assays (15) were linear with time for at least 60 min, while those with the exogenously added GMi and DS-fetuin were linear with time only for about 30 min (Figure 1). Activities were directly proportional to the amount of enzyme added up to 0.75 mg protein/assay (Figure 2). 

Whereas the activities of CMP-NeuNAc lactosylceramide sialyltransferase increase postnatally (38), the activities of the CMP-NeuNAc glycoprotein sialyltransferase decline dramatically during early development (17). Separate regulatory controls on the glycoprotein and glycolipid metabolism have also been implicated in a study on the brain tissues of Tay-Sachs disease patients. 

Figure 4. Effects of nonionic detergents on sialyltransferase activities. X. Endogenous glycoproteins A, exogenous DS-fetuin O, endogenous glycolipids O, exogenous GM, (16).

Sialyltransferases. In eukaryotic cells, addition of terminal sialic acid to glycoconjugates is carried out by specific enzymes called sialyltransferases (STs). STs catalyze the transfer of a sialic acid from the activated nucleotide sugar donor CMP-N-acelylneuraminic acid (CMP-NeuSAc) to the terminal nonreducing position of oligosaccharide chains either of glycoproteins or glycolipids. 

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. 

Hematoside and Sialyltransferase in Newborn Rat Kidney Cells. Another transformed cell system that we have investigated has shown a positive correlation between expression of the transforming virus and glycolipid content. Newborn rat kidney cells (NRK) transformed by either the Kirsten isolate of murine sarcoma virus (Ki-MSV) or a temperature-sensitive mutant were analyzed for glycolipid composition. The major sialylglycolipid in NRK cells is Gm3 When the cells were grown at the permissive temperature for transformation (31°C), there was substantially less hematoside in both transformed lines than in the control cells (unpublished observation of P. H. Fishman, S. A. Aaron-son, and R. O. Brady). At the nonpermissive temperature (39 ), the cells transformed by the mutant had more hematoside than the control cells. Lactosylceramide CMP-NANA sialyltransferase activity was deter-... 

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