Isolation and Cell Biological Characterization of Sialyltransferases Involved in Ganglioside Biosynthesis

参与神经节苷脂生物合成的唾液酸转移酶的分离和细胞生物学表征

• 批准号: 02680128
• 负责人: SANAI Yutaka
• 金额: $ 1.28万
• 依托单位: Tokyo Metropolitan Institute of Medical Science (1991)The University of Tokyo (1990)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02680128/
• 关键词: Ganglioside; sialic acid; glycosyltransferase; tumor

To elucidate the role of sugar chain of ganglioside in cell recognition and signal transduction, isolation and characterization of sialyltransferases which synthesize bioactive gangliosides, was investigated.[1]Solubilization of membrane-bound sialyltransferase from rat liver Golgi apparatus using novel cationic detergent : For effective identification of sialyltransferase, novel cationic detergent, sodium polyoxyethylenelaulylethersulfate/ laulyldimethylamineoxide mixture (7 : 3) was used for solubilization of the enzyme. One of the sialyltransferase, GD3 synthase(alpha2-8 sialyltransferase) succeeded in solubilization as active form. This detergent was also useful for evaluation of molecular weight and subunit structure by polyacrylamide gel electrophoresis.[2]Photo-affinity labelling of sialyltransferases : Photoactive di-azilin derivatives of lactosylceramide which was the substrate of GM3 synthase, was synthesized. This compound was well-substrate and had high affinity to GM3 synthase from rat liver Golgi apparatus. This compound had biotin-residue in molecule and could be utilized for identification and isolation of the labelled sialyltransferases.[3]Structural characterization of gangliosides in Xenopus laevis oocyte : To evaluate Xenopus oocyte as the host cells for the expression of foreign gene involved in the expression of sugar chain such as glycosyltransferases, glycosphigolipids of Xenopus oocyte were analyzed. From the results of sugar composition analysis, enzymatic hydrolysis, pennethylation analysis, and negative ion fast atom bombardment mass spectrometry, the structure of one of the major ganglioside was determined to be as follows :

为了阐明神经节苷脂糖链在细胞识别和信号转导中的作用，对合成生物活性神经节苷脂的唾液酸转移酶进行了分离和表征。[1]使用新型阳离子洗涤剂溶解大鼠肝脏高尔基体膜结合唾液酸转移酶：为了有效识别唾液酸转移酶，新型阳离子洗涤剂 使用聚氧乙烯月桂基醚硫酸钠/月桂基二甲胺氧化物混合物(7:3)来溶解酶。唾液酸转移酶之一，GD3合酶（α2-8唾液酸转移酶）成功以活性形式溶解。该去污剂还可用于通过聚丙烯酰胺凝胶电泳评估分子量和亚基结构。[2]唾液酸转移酶的光亲和标记：合成了乳糖神经酰胺的光活性二阿齐林衍生物，乳糖神经酰胺是 GM3 合酶的底物。该化合物是良好的底物，并且与大鼠肝脏高尔基体的 GM3 合酶具有高亲和力。该化合物分子中具有生物素残基，可用于标记的唾液酸转移酶的鉴定和分离。 [3]非洲爪蟾卵母细胞神经节苷脂的结构表征：评估非洲爪蟾卵母细胞作为宿主细胞表达参与糖基转移酶、糖脂等糖链表达的外源基因的表达。 分析了非洲爪蟾卵母细胞。根据糖成分分析、酶水解、五甲基化分析和负离子快原子轰击质谱的结果，确定一种主要神经节苷脂的结构如下：

项目成果

K. Hidari, S. Itonori, Y. Sanai, M. Ohashi, K. Kasama & Y. Nagai: "Isolation and characterization of monosialosylganglio-pentaosyl ceramide from Xenopus laevis oocyte" J. Biochem. 11. 412-416 (1991)

K. Hidari、S. Itonori、Y. Sanai、M. Ohashi、K. Kasama

K.Hidari,Y.Sanai T.Tai,F.Inagaki & Y.Nagai: "In vitro synthesis of ganglioside from asialo-glycolipid"

K.Hidari,Y.Sanai T.Tai,F.Inagaki

Y.Sanai,K.Oda and Y.Nagai: "Induction of GD3 ganglioside by adenovirus E1A gene 13Sーand 12SーmRNA products in rat 3Y1 cells" J.Biochem.107. 740-742 (1990)

Y.Sanai、K.Oda 和 Y.Nagai：“腺病毒 E1A 基因 13S 和 12S mRNA 产物在大鼠 3Y1 细胞中诱导 GD3 神经节苷脂”J.Biochem.107（1990）。

K.Hidari,S.Itonori Y.Sanai,M.Ohashi,K.Kasama & Y.Nagai: "Isolation and characterization of a monosialosylgang-liopentaosyl Ceramide from Xenopus laevis Oocyte" J.Biochemistry. 110. 412-416 (1991)

K.Hidari,S.Itonori Y.Sanai,M.Ohashi,K.Kasama

K. HIDARI, Y. SANAI, & Y. NAGAI: "Enzymatic Basis of Accumulation of Monosialosylgangliopentaosylceramide in Xenopus Laevis Oocyte"

K.希达里，Y.萨奈，