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J. Biol. Chem., Vol. 277, Issue 1, 169-177, January 4, 2002
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From the 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
Gal
Purification, Characterization, and Subunit Structure of
Rat Core 1
1,3-Galactosyltransferase*
§,
, and§**
W. K. Warren Medical Research Institute,
the Departments of ** Medicine and ¶ Biochemistry and
Molecular Biology, and the § Oklahoma Center for Medical
Glycobiology, University of Oklahoma Health Sciences Center,
Oklahoma City, Oklahoma 73104
1
3GalNAc
1Ser/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--Ser/Thr is UDP-Gal:GalNAc--Ser/Thr 3-galactosyltransferase (core1 3-Gal-T). Core 1 3-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 3-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 Km for UDP-Gal of 630 µM and an apparent Vmax of 206 µmol/mg/h protein using GalNAc1-O-phenyl as the
acceptor. The reaction product was generated using asialo-bovine
submaxillary mucin as an acceptor; treatment with
O-glycosidase generated the expected disaccharide Gal13GalNAc. These studies demonstrate that activity of the core
1 1,3-Gal-T from rat liver is contained within a single, novel,
disulfide-bonded, dimeric enzyme.
*
This work was supported in part by National Institutes of
Health Grant RO1 AI48075-01 (to R. D. C.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence may be addressed: Dept. of Biochemistry
and Molecular Biology, University of Oklahoma Health Sciences Center,
975 N.E. 10th St., BRC Rm. 417, Oklahoma City, OK 73104. Tel.:
405-271-2481; Fax: 405-271-3910; E-mail: richard-cummings@ouhsc.edu.
To whom correspondence may be addressed: Novazyme
Pharmaceuticals, Inc., 800 Research Pkwy., Ste. 200, Oklahoma City, OK
73104. Tel.: 405-271-8144; Fax: 405-271-1030; E-mail: wcanfield@novazyme.com.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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