Isolation and Cell Biological Characterization of Sialyltransferases Involved in Ganglioside Biosynthesis

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，对合成活性神经节苷脂的唾液酸基转移酶的分离和性质进行了研究。[1]用新型阳离子清洗剂溶解大鼠肝脏高尔基体膜上唾液酸基转移酶：为了有效鉴定唾液酸基转移酶，新型阳离子清洗剂，聚氧乙烯醚硫酸钠/十二烷基二甲胺混合物(7：3)溶解该酶。唾液酸转移酶中的GD3合成酶(α2-8唾液酸转移酶)以活性形式成功溶解。[2]唾液酸基转移酶的光亲和标记：合成了GM3合成酶的底物--乳糖基神经酰胺的光活性二氮杂环衍生物。该化合物与大鼠肝脏高尔基体GM3合成酶有较高的亲和力。该化合物分子中含有生物素残基，可用于标记唾液酸基转移酶的鉴定和分离。[3]非洲爪哇卵母细胞中神经节苷脂的结构特征：分析非洲爪哇卵母细胞糖基转移酶、糖磷脂等参与糖链表达的外源基因作为宿主细胞。根据糖组成分析、酶解、羟乙基化分析和负离子快原子轰击质谱仪的结果，确定了主要的神经节苷脂之一的结构如下：

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 :