Biosynthesis of fucose containing lacto-series glycolipids in human colonic adenocarcinoma Colo 205 cells.
Biosynthesis of fucose containing lacto-series glycolipids in human colonic adenocarcinoma Colo 205 cells.
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人结肠腺癌细胞 Colo 205 细胞中含岩藻糖的乳系列糖脂的生物合成。
DOI：
10.1016/0003-9861(89)90479-7
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发表时间：
1989
影响因子：
3.9
通讯作者：
Levery,SB
中科院分区：
文献类型：
--
作者：
Holmes,EH;Levery,SB
已在人结肠腺癌细胞 Colo 205 细胞中研究了含有乳糖系列糖脂的岩藻糖的生物合成。用 Triton X-100 溶解的膜组分证明了岩藻糖在 α1 → 3 连接到 2 型链受体和 α1 → 4 连接到 1 型链受体上的转移。该酶被发现在 6.0 至 7.5 的广泛 pH 范围内具有高度活性。研究了转移反应的动力学，结果表明该酶与受体 nLc4 和 Lc4 的 GDP 岩藻糖表观 Km 分别为 53 和 49 μm。受体 Lc4、nLc4 和 IV3NeuAcnLc4 的表观 Km 值分别确定为 42、18 和 26 μm。单独将岩藻糖转移至 1 型链受体 Lc4，并在浓度增加的 2 型链受体 IV3NeuAcnLc4 或 Gb3 存在下，表明 1 型和 2 型受体都是单一酶的替代受体。 IV3NeuAcnLc4 相对于 Lc4 表现为岩藻糖转移的竞争性抑制剂，这一发现进一步证实了这一点。定义了由 Colo 205 细胞酶催化的 nLc6 岩藻糖基化产物体外制备规模合成的条件。从 5 mg 原始 nLc6 获得 2.5 mg (46%) 的单岩藻糖基衍生物和 1 mg (17%) 二岩藻糖基衍生物的产率。通过 1 H NMR、+FAB-MS 和甲基化分析仔细研究了这些生物合成产物的结构。这些研究揭示了由 III3FucnLc6 和 III3V3Fuc2nLc6 组成的极高纯度产物。讨论了这些产品的性质和酶性质的重要性。
Biosynthesis of fucose containing lacto-series glycolipids has been studied in human colonic adenocarcinoma Colo 205 cells. Transfer of fucose in both α1 → 3 linkage to type 2 chain acceptors and α1 → 4 linkage to type 1 chain acceptors was demonstrated with a Triton X-100 solubilized membrane fraction. The enzyme was found to be highly active over a broad pH range between 6.0 and 7.5. Kinetics of the transfer reactions were studied and indicated that the enzyme had an apparentKmfor GDPfucose of 53 and 49 μmwith acceptors nLc4and Lc4, respectively. The apparentKmvalues for acceptors Lc4, nLc4, and IV3NeuAcnLc4were determined to be 42, 18, and 26 μm, respectively. Transfer of fucose to the type 1 chain acceptor Lc4alone and in the presence of increasing concentrations of the type 2 chain acceptor IV3NeuAcnLc4or Gb3suggested that both type 1 and 2 acceptors were alternate acceptors for a single enzyme. This was further established by the finding that IV3NeuAcnLc4behaved as a competitive inhibitor of fucose transfer with respect to Lc4. Conditions were defined for preparative scalein vitrosynthesis of fucosylated products of nLc6catalyzed by the Colo 205 cell enzyme. Yields of the monofucosyl derivative of 2.5 mg (46%) and 1 mg (17%) of the difucosyl derivative were obtained from 5 mg of original nLc6. The structures of these biosynthetic products were carefully studied by1H NMR,+FAB-MS, and methylation analysis. These studies revealed extremely high purity products composed of III3FucnLc6and III3V3Fuc2nLc6. The significance of the nature of these products and enzymatic properties is discussed.