Nishikawa Y, Pegg W, Paulsen H, Schachter H
Department of Biochemistry, Hospital for Sick Children, Toronto, Ontario, Canada.
J Biol Chem. 1988 Jun 15;263(17):8270-81.
UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I catalyzes an essential first step in the conversion of high mannose to hybrid and complex N-glycans (Schachter, H. (1986) Biochem. Cell Biol. 64, 163-181; Oppenheimer, C.L., and Hill, R.L. (1981) J. Biol. Chem. 256, 799-804), i.e. the addition of GlcNAc to (Man alpha 1-6(Man alpha 1-3)Man alpha 1-6)(Man alpha 1-3)Man beta 1-4GlcNAc-OR to form (Man alpha 1-6(Man alpha 1-3)Man alpha 1-6)(GlcNAc beta 1-2Man alpha 1- 3)Man beta 1-4GlcNAc-OR. The enzyme has been purified from Triton X-100 extracts of rabbit liver by chromatography on CM-Sephadex, Affi-Gel blue, UDP-hexanolamine-Sepharose, and a novel adsorbent in which UDP-GlcNAc is linked to thiopropyl-Sepharose at the 5-position of uracil. The enzyme exists in crude liver extracts in two molecular weight forms separable on Sephadex G-200. The low molecular weight form was purified 64,000-fold with a specific activity of 19.8 mumol/min/mg. The pure enzyme was free of N-acetylglucosaminyltransferase II-V activities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single major band of Mr 45,000 and two minor bands of Mr 54,000 and 50,000. All three bands showed retarded elution from an affinity column in which the acceptor substrate for the transferase was covalently linked to Sepharose. Kinetic analysis indicated a largely ordered sequential mechanism with UDP-GlcNAc binding to the enzyme first and UDP leaving last. Studies with synthetic analogues of the substrate Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc showed that an unsubstituted equatorial hydroxyl on carbon 4 of the beta-linked Man residue was essential for enzyme activity.
UDP-N-乙酰葡糖胺：α-3-D-甘露糖苷β-1,2-N-乙酰葡糖胺基转移酶I催化了高甘露糖向杂合型和复合型N-聚糖转化过程中的关键第一步（沙克特，H.（1986年）《生物化学与细胞生物学》64卷，163 - 181页；奥本海默，C.L.，和希尔，R.L.（1981年）《生物化学杂志》256卷，799 - 804页），即把GlcNAc添加到(Manα1-6(Manα1-3)Manα1-6)(Manα1-3)Manβ1-4GlcNAc-OR上，形成(Manα1-6(Manα1-3)Manα1-6)(GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAc-OR。该酶已通过在CM-葡聚糖凝胶、Affi-Gel蓝、UDP-己醇胺-琼脂糖以及一种新型吸附剂（其中UDP-GlcNAc在尿嘧啶的5位与硫丙基-琼脂糖相连）上进行层析，从兔肝的Triton X-100提取物中纯化得到。该酶在肝脏粗提物中以两种分子量形式存在，可在葡聚糖凝胶G-200上分离。低分子量形式被纯化了64,000倍，比活性为19.8 μmol/分钟/毫克。纯化后的酶不含N-乙酰葡糖胺基转移酶II - V的活性。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示有一条主要的Mr 45,000条带以及两条次要的Mr 54,000和50,000条带。所有这三条带在亲和柱上的洗脱都延迟，在该亲和柱中转移酶的受体底物与琼脂糖共价连接。动力学分析表明这是一种大致有序的顺序机制，UDP-GlcNAc首先与酶结合，UDP最后离开。对底物Manα1-6(Manα1-3)Manβ1-4GlcNAc的合成类似物的研究表明，β-连接的甘露糖残基碳4上未被取代的赤道羟基对酶活性至关重要。
