N-Asparagine-Linked Oligosaccharides: Processing

Ira Tabas, Stuart Kornfeld

Research output: Contribution to journalArticle

14 Scopus citations

Abstract

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]Glcl1–2Man9GlcNAc. The released glucose is converted to glucose 6-phosphate with hexokinase and ATP and then separated from residual oligosaccharide by anion-exchange chromatography.

Original languageEnglish
Pages (from-to)416-429
Number of pages14
JournalMethods in enzymology
Volume83
Issue numberC
DOIs
StatePublished - Jan 1 1982
Externally publishedYes

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