Purification and characterization of UDP-GlcNAc:Gal beta 1-4Glc(NAc) beta-1,3-N-acetylglucosaminyltransferase (poly-N-acetyllactosamine extension enzyme) from calf serum.
UDP-GlcNAc:Gal beta 1-4Glc(NAc) beta-1,3-N-acetylglucosaminyltransferase is involved in the initiation and the extension of poly-N-acetyllactosamine biosynthesis. This enzyme has been purified to about 125,000-fold with a 0.2% yield from calf serum. The purification was achieved by ammonium sulfate precipitation and chromatography on concanavalin A-Sepharose, DEAE-Toyopearl, SP-Toyopearl, Sephacryl S-200, AF-Blue-Toyopearl, and Mono Q columns. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band corresponding to an apparent M(r) of 70,000. This component was specifically photoaffinity-labeled with 4-thiouridine diphosphate. Exoglycosidase digestion and methylation analysis of the reaction products demonstrated that the enzyme catalyzed the transfer of one N-acetylglucosamine to position C-3 of the terminal galactosyl residue of lactose or N-acetyllactosamine in the beta linkage. The enzyme required Mn2+ ions for its activity and showed a broad pH optimum around 7.0. Apparent Km values for lactose, N-acetyllactosamine, and UDP-GlcNAc were 18.2, 19.6, and 0.129 mM, respectively. Acceptor specificity was tested using several oligosaccharides. The results indicated that terminal Gal beta 1-4Glc(NAc) sequences (type II chains) were preferred substrates for the enzyme. Terminal Gal beta 1-3GlcNAc sequences (type I chains), Lewis X trisaccharides (Gal beta 1-4(Fuc alpha 1-3)GlcNAc), and monosaccharides (galactose) did not serve as substrates.
Kawashima
H
H
Division of Chemical Toxicology and Immunochemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Yamamoto
K
K
Osawa
T
T
Irimura
T
T
D000818:Animals; D002240:Carbohydrate Sequence; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000097:blood / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
D017351:N-Acetylglucosaminyltransferases; C042261:N-acetyllactosaminide beta-1,3-N-acetylglucosaminyltransferase