PURIFICATION AND CHARACTERIZATION OF UDP-N-ACETYLGLUCOSAMINE - ALPHA-6-D-MANNOSIDE BETA-1-6N-ACETYLGLUCOSAMINYLTRANSFERASE (N-ACETYLGLUCOSAMINYLTRANSFERASE-V) FROM A HUMAN LUNG-CANCER CELL-LINE
PURIFICATION AND CHARACTERIZATION OF UDP-N-ACETYLGLUCOSAMINE - ALPHA-6-D-MANNOSIDE BETA-1-6N-ACETYLGLUCOSAMINYLTRANSFERASE (N-ACETYLGLUCOSAMINYLTRANSFERASE-V) FROM A HUMAN LUNG-CANCER CELL-LINE
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DOI：
10.1093/oxfordjournals.jbchem.a124091
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发表时间：
1993-05-01
影响因子：
2.7
通讯作者：
TANIGUCHI, N
中科院分区：
文献类型：
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作者：
GU, JG;NISHIKAWA, A;TANIGUCHI, N
一种β1 - 6N - 乙酰氨基葡萄糖基转移酶（GnT - V）[EC 2.4.1.155]，它催化N - 乙酰氨基葡萄糖从UDP - N - 乙酰氨基葡萄糖转移到α - D - 6 - 甘露糖苷，已从QG小肺癌细胞系的培养上清液中纯化了20,000倍，产率为37%。分离过程包括在苯基 - 琼脂糖、羟基磷灰石、UDP - 己醇胺琼脂糖以及与活化的CH - 琼脂糖4B偶联的双天线糖底物（GnGn - bi - Asn）上进行色谱分析。在非还原条件下的十二烷基硫酸钠凝胶电泳显示出一条73 kDa的单条带。然而，在还原条件下，可看到一个60 kDa的额外组分。肽图谱分析表明这两种蛋白质基本相同，这表明60 kDa的组分可能是73 kDa蛋白质的蛋白水解切割形式。对该酶针对多种吡啶氨基化糖类的活性研究表明，该酶对三天线（GnGnGn - tri - PA）和双天线（GnGn - bi - PA）糖类活性最高。GnGn - bi - PA和UDP - GlcNAc的Km值分别为133 μM和3.5 mM。这些研究首次报道了高度纯化的人GnT - V的酶学特性。
A beta1-6N-acetylglucosaminyltransferase (GnT-V) [EC 2.4.1.155] which catalyzes the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to alpha-D-6-mannoside has been purified up to 20,000-fold from the cultured supernatant of the QG small lung cancer cell line with a 37% yield. The isolation procedure included chromatography on phenyl-Sepharose, hydroxylapatite, UDP-hexanolamine Sepharose, and a biantennary sugar substrate (GnGn-bi-Asn) coupled to activated CH-Sepharose 4B. Sodium dodecyl sulfate gel electrophoresis under non-reducing conditions showed a single band of 73 kDa. Under reducing conditions, however, an additional component of 60 kDa was seen. Peptide mapping analysis indicated that both of these proteins were essentially identical, indicating that the 60-kDa component is probably a proteolytically cleaved form of the 73-kDa protein. Studies on the activity of the enzyme toward a variety of pyridylaminated sugars showed that the enzyme is most active toward triantennary (GnGnGn-tri-PA) and biantennary (GnGn-bi-PA) sugars. The K(m) values for GnGn-bi-PA and UDP-GlcNAc were 133 muM and 3.5 mM, respectively. These studies represent the first report of the enzymatic properties of a highly purified human GnT-V.