New Techniques in Glycosyltransferase Research
Glycosyltransferases (GLTs) of eukaryotic origin catalyze the transfer of glycose units to the appropriate glycoprotein, ceramide, or glycolipid core structures. These enzymes may recognize a specific terminal glycose unit or the penultimate sugar, in addition to the terminal sugar unit.1–4 GLTs are believed to be localized in the Golgi bodies,5–7 and most of them are membrane-bound. Many have been solubilized using various detergents, and their purification is under way.8–12 We are involved in the purification of glycolipid:glycosyltransferases (GSL:GLTs) and in the study of their kinetic properties.8–13 This article describes relatively easy methods for isolation14,15 of glycolipid substrates for GSL:GLTs and the development of some convenient, relatively inexpensive assay methods.16 Both advances were essential before substantial numbers of fractions obtained during column chromatography could be assayed.
KeywordsEmbryonic Chicken Sodium Metabisulfite Rabbit Bone Marrow Palmitoyl Chloride Buffy Coat Layer
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- 2.S. Basu, M. Basu, J.W..Kyle, and H.C. Chon, Biosynthesis in vitro of gangliosides containing Gg-and Lc-cores, in “Ganglioside Structure and Function” by (eds. R. Ledeen, M. Rapport, K. Suzuki, and R. Yu) Plenum Press, 249 (1984).Google Scholar
- 3.S. Basu and M. Basu, Expression of glycosphingolipid glycosyltransferases in development and transformation, in “The Glycoconjugates” by (ed. M. Horowitz) Academic Press, New York, Vol. 3, 265 (1982).Google Scholar
- 4.T.A. Beyer and R.L. Hill, Glycosylation pathways in the biosynthesis of nonreducing terminal sequences in oligosaccharides of glycoproteins, in “The Glycoconjugates” by (ed. M. Horowitz) Academic Press, New York, Voo. 3, 25 (1982).Google Scholar
- 5.R.L. Hudgin, R.K. Murray, L. Pinteric, H.P. Morris and H. Schachter, The use of nucleotide sugar: glycoprotein glycosyltransferases to asses Golgi apparatus function in Morris Hepatomas, Canad. J. Biochem., 49, 61 (1971).Google Scholar
- 8.S. Basu, M. Basu, J.W. Kyle, T. De, K. Das, and R.J. Schaeper, in “Enzymes of Lipid Metabolism” by (eds. L. Freysz and S. Gatt) (in press) Plenum Press, New York.Google Scholar
- 9.S. Basu, T. De, J.W. Kyle, and M. Basu, Biosynthesis of eukaryotic cell surface glycosphingolipids using solubilized glycosyltransferases, Proc. Int. Symp. Biomol. Struc. Interactions, Suppl. J. Biosci., 8 (in press).Google Scholar
- 10.J.W. Kyle, Characterization of glycolipid galactosyltransferases from embryonic chicken brain, Ph.D. Thesis, University of Notre Dame, Notre Dame, IN (1985).Google Scholar
- 13.H.C. Chon, Solubilization of glycolipid sialyltransferases from embryonic chicken liver and brain. Use of radiolabeled glycolipids in vitro to assay sialyltransferases, M.S. Thesis, University of Notre Dame, Notre Dame, IN (1984).Google Scholar
- 14.M. Basu and S. Basu, Enzymatic synthesis of blood group B specific pentaglycosylceramide by an a-galactosyltransferase from rabbit bone marrow, J. Biol. Chem., 257, 169 (1983).Google Scholar
- 16.M. Basu, T. De, K.K. Das, J.W. Kyle, H.C. Chon, R.J. Schaeper, and S. Basu, Glycosyltransferases involved in glycolipid biosynthesis, Methods in Enzymol (in press).Google Scholar