Mucin biosynthesis: Purification and characterization of a mucin β6N-acetylglucosaminyltransferase

P. A. Ropp, M. R. Little, P. W. Cheng

Research output: Contribution to journalArticle

67 Scopus citations

Abstract

We have purified, to apparent homogeneity, a mucin β6N-acetylglucosaminyltransferase (β6GlcNAc transferase) from bovine tracheal epithelium. Golgi membranes were isolated from a 0.25 M sucrose homogenate of epithelial scrapings by discontinuous sucrose gradient centrifugation. The Golgi membranes were solubilized with 1% Triton X-100 in the presence of 1 mM Galβ1-3GalNAcαbenzyl (Bzl) to stabilize the β6GlcNAc transferase. The solubilized enzyme was bound to a UDP-hexanolamine-Actigel-ALD Super-flow affinity column equilibrated with 1 mM Galβ1-3GalNAcαBzl and 5 mM Mn2+. Elution of the enzyme with 0.5 mM UDP-GlcNAc resulted in a 133,800-fold purification with a 1.3% yield and a specific activity of 70 μmol/min/mg protein. Radioiodination of the purified enzyme followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed a single band at 69,000 Da. Kinetic analyses of the β6GlcNAc transferase-catalyzed reaction showed an ordered sequential mechanism in which UDP-GlcNAc binds to the enzyme first and UDP is released last. The K(m) values for UDP-GlcNAc and Galβ1-3GalNAcαBzl were 0.36 and 0.14 mM, respectively. Acceptor competition studies showed that the purified β6GlcNAc transferase can use core 1 and core 3 mucin oligosaccharides as well as GlcNAcβ1-3GalβR as acceptor substrates. Proton NMR analyses of the three products demonstrated that GlcNAc was added in a β1-6 linkage to the penultimate GalNAc or Gal, suggesting that this enzyme is capable of synthesizing all β6GlcNAc structures found in mucin-type oligosaccharides.

Original languageEnglish (US)
Pages (from-to)23863-23871
Number of pages9
JournalJournal of Biological Chemistry
Volume266
Issue number35
StatePublished - 1991

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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