Kawashima H, Yamamoto K, Osawa T, Irimura T
Division of Chemical Toxicology and Immunochemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
J Biol Chem. 1993 Dec 25;268(36):27118-26.
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.
UDP-N-乙酰葡糖胺：β-1,4-半乳糖基-β-1,3-N-乙酰葡糖胺转移酶参与多聚N-乙酰乳糖胺生物合成的起始和延伸过程。该酶已从小牛血清中纯化出来，纯化倍数约为125,000倍，产率为0.2%。通过硫酸铵沉淀以及在伴刀豆球蛋白A-琼脂糖、DEAE- Toyopearl、SP- Toyopearl、Sephacryl S-200、AF- Blue- Toyopearl和Mono Q柱上进行层析实现了纯化。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示出一条单一的条带，对应表观分子量为70,000。该组分被4-硫尿苷二磷酸特异性光亲和标记。对反应产物进行外切糖苷酶消化和甲基化分析表明，该酶催化将一个N-乙酰葡糖胺转移至乳糖或N-乙酰乳糖胺末端半乳糖基残基C-3位的β键上。该酶的活性需要Mn2+离子，最适pH约为7.0。乳糖、N-乙酰乳糖胺和UDP-N-乙酰葡糖胺的表观Km值分别为18.2、19.6和0.129 mM。使用几种寡糖测试了受体特异性。结果表明，末端β-1,4-半乳糖基-β-1,4（N-乙酰葡糖胺）序列（II型链）是该酶的优选底物。末端β-1,3-半乳糖基-N-乙酰葡糖胺序列（I型链）、Lewis X三糖（β-1,4-半乳糖基-α-1,3-岩藻糖基-N-乙酰葡糖胺）和单糖（半乳糖）不作为底物。
