N-Asparagine-Linked Oligosaccharides: Processing

This chapter presents procedure for isolation of oligosaccharide intermediates and assays of the processing enzymes. In isolation of processing intermediates from tissue culture cells tissue culture cells are incubated with radioactive mannose and galactose to label selectively the mannose and glucose residues, respectively, of the oligosaccharides. In some instances it is advisable to use virus-infected cells to limit the types of oligosaccharides that are being synthesized. To prepare the Man₅GlcNAc₂ intermediate in large amounts, a mutant line of Chinese hamster ovary cells that lacks Nacetylglucosaminyltransferase I is used. The enzyme is assayed by measuring the release of [³H]glucose from [³H]Glcl_1–2Man₉GlcNAc. The released glucose is converted to glucose 6-phosphate with hexokinase and ATP and then separated from residual oligosaccharide by anion-exchange chromatography.