Purification and characterization of human lymphoblast N-acetylglucosamine-1-phosphotransferase.
Purification and characterization of human lymphoblast N-acetylglucosamine-1-phosphotransferase.
复制标题
人淋巴母细胞 N-乙酰氨基葡萄糖-1-磷酸转移酶的纯化和表征。
DOI：
10.1093/glycob/2.2.119
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发表时间：
1992
期刊：
影响因子：
4.3
通讯作者：
Miller,AL
中科院分区：
文献类型：
--
作者：
Zhao,KW;Yeh,R;Miller,AL
N-乙酰氨基葡萄糖-1-磷酸转移酶(GlcNAcPTase)用2%泰吉醇NP-10从培养的人淋巴母细胞中溶解，经扁豆凝集素-Sepharose4B、DEAe-Sephacel和Sephacryl S-400层析纯化，纯化倍数为3840倍，回收率为14%。部分纯化的酶需要非离子洗涤剂Tergitol NP-10和二价阳离子Mn2+或Mg2+才能发挥其活性，在Tris-Maleate缓冲液中的最适pH为7.2-7.5。动力学研究表明，供体N-乙酰氨基葡萄糖的Kmof为24μM，人工受体α-甲基甘露糖苷的Kmof为117 mM。GlcNAcPTase被UDP和UDP-葡萄糖以及带负电荷的磷脂(包括磷脂酰丝氨酸、磷脂酰甘油和磷脂酸)抑制。表观摩尔分数。人淋巴母细胞GlcNAcPTase的WT为∼1000 kDa，与已报道的大鼠肝脏部分纯化的酶相似(Waheedet al.，1982)。
N-Acetylglucosamine-1-phosphotransferase (GlcNAcPTase) was solubilized with 2% Tergitol NP-10 from cultured human lymphoblast cells and purified 3840-fold with 14% recovery using lentil lectin-Sepharose 4B, DEAE-Sephacel and Sephacryl S-400 chromatographies. The partially purified enzyme requires the non-ionic detergent Tergitol NP-10 and a divalent cation, Mn2+or Mg2+, for its activity and exhibits an optimal pH at 7.2–7.5 in Tris-maleate buffer. Kinetic studies demonstrated an apparentKmof 24 μM for the donor UDP-N-acetylglucosamine and of 117 mM for the artificial acceptor α-methylmannoside. The GlcNAcPTase is inhibited by UDP and UDP-glucose, and by negatively charged phospholipids including phosphatidylserine, phosphatidylglycerol and phosphatidic acid. The apparent mol. wt of the human lymphoblast GlcNAcPTase is ∼1000 kDa, which is analogous to that reported for the partially purified enzyme from rat liver (Waheedet al., 1982).