Purification, Characterization, and Subunit Structure of Rat Core 1 β1,3-Galactosyltransferase*
By T. Ju, Richard D. Cummings, W. Canfield. Journal of Biological Chemistry, 2002
103 citations recorded by the source corpus.
The O-linked oligosaccharides (O-glycans) in mammalian glycoproteins are classified according to their core structures. Among the most common is the core 1 disaccharide structure consisting of Galβ1→3GalNAcα1→Ser/Thr, which is also the precursor for many extended O-glycan structures. The key enzyme for biosynthesis of core 1 O-glycan from the precursor GalNAc-α-Ser/Thr is UDP-Gal:GalNAc-α-Ser/Thr β3-galactosyltransferase (core1 β3-Gal-T). Core 1 β3-Gal-T activity, which requires Mn2+, was solubilized from rat liver membranes and purified 71,034-fold to apparent homogeneity (>90% purity) in 5.7% yield by ion exchange chromatography on SP-Sepharose, affinity chromatography on immobilized asialo-bovine submaxillary mucin, and gel filtration chromatography on Superose 12. The purified enzyme is free of contaminating glycosyltransferases. Two peaks of core 1 β3-Gal-T activity were identified in the final step on Superose 12. One peak of activity contained protein bands on non-reducing SDS-PAGE of ∼84- and ∼86-kDa disulfide-linked dimers, whereas the second peak of activity contained monomers of ∼43 kDa. Reducing SDS-PAGE of these proteins gave ∼42- and ∼43-kDa monomers. Both the 84/86-kDa dimers and the 42/43-kDa monomers have the same novel N-terminal sequence. The purified enzyme, which is remarkably stable, has an apparent K m for UDP-Gal of 630 μm and an apparent V max of 206 μmol/mg/h protein using GalNAcα1-O-phenyl as the acceptor. The reaction product was generated using asialo-bovine submaxillary mucin as an acceptor; treatment withO-glycosidase generated the expected disaccharide Galβ1→3GalNAc. These studies demonstrate that activity of the core 1 β1,3-Gal-T from rat liver is contained within a single, novel, disulfide-bonded, dimeric enzyme.
Subfields: Immunology, Molecular Biology, Organic Chemistry
View Paper: https://www.semanticscholar.org/paper/58e8e132ef8a1eacf28ef4b2958ee21384c0623a
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Citations in the corpus
- Organization of Golgi Glycosyltransferases in Membranes: Complexity via Complexes (2004, background): “Both the monomers and dimers of core 1 b1,3GalT, for which the locations of the intersubunit disulfides are not known, are active as well ( Ju, Cummings & Canfield, 2002b ).”