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特性

• 批准号: 04670154
• 负责人: SANAI Yutaka
• 金额: $ 1.34万
• 依托单位: TOKYO METROPOLITAN INSTITUTE OF MEDICAL SCIENCE
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04670154/
• 关键词: GANGLIOSIDE; SIALIC ACIDE; SIALYLTRANSFERASE; BIOSYNTHESIS; GD1ALPHA

(1)Enzymatic Assay of Glycosphinogolipid Sialyltransferase Using Reverse-Phase Thin-Layr Chromatography : A rapid assay method for glycosphingolipid sialyltransferase was developed using reverse-phase thin-layr chromatography(RP-TLC). An acceptro glycolipid and a donor radioactive nucleotide sugar, CMP-[^<14>C]-N-acetylneuraminic acid(NeuAc), were incubated with the enzyme. After reaction, the enzymatic product was separated from unreacted CMP-[^<14>C]-NeuAc by C_<18>RP-TLC, developed in water for 10 min. CMP-NeuAc migrated with solvent front. Radioactivity of reaction product, remaining at the origin, was visualized and quantified using computed radiography which utilized photp-stimulated luminescence. We have used the assay method using RP-TLC to determine the activities of MCP-NeuAc : lactosylceramide alpha2-3 sialyltransferase(GM3 synthase) of porcine submaximally gland and CMP-NeuAc : Gm3 alpha2-8 sialyltransferase(GD3 synthase) of rat liver Golgi membranes. The assay was shown to … More be dependent on reaction time and concentration of the enzyme, CMP-NeuAc, and acceptors, respectively. The procedure is relaible to simultaneous and rapid mesurement of a large number os samples.[2]In Virto Synthesis of Disialoganglioside (GD1alpha) from asialo-GM1 using Sialyltransferases in Rat Liver Golgi Vesicles : Two gangliosides were efficiently synthesized from asialo-GM1 and CMP-NeuAc by using sialyltransferases in rat liver Golgi vesicles in vitro. These gangliosides were rapidly purified by a combination of anion exchange and revers-phase column chromatographies. The ganglioside structures were determined by TLC analysis, treatment with a sialidase from Salmonella typhimurium LT2, which spesifically hydrolyzes alpha2-3 N-acetylneuraminic acid linkages, TLC immunostaining, and ^1H-NMR spectroscopy. One of the gangliosides was identified as GD1alpha. The other ganglioside was determined to be GM1b. Finally, GM1b and GD1alpha were obtained from asialo-GM1 as starting material in 8.1% and 1.2% overall yields, respectively. This study also suggests that the novel synthetic pathway asialo-GM1 -> GM1b -> GD1alpha may exist in a rat liver. Less

（1）使用反相薄层色谱法的鞘糖脂唾液酸转移酶的酶法测定：使用反相薄层色谱法（RP-TLC）开发了鞘糖脂唾液酸转移酶的快速测定方法。将受体糖脂和供体放射性核苷酸糖CMP-[^<14>C]-N-乙酰神经氨酸（NeuAc）与酶一起孵育。反应后，通过C_ RP-TLC将酶产物与未反应的CMP-[3C<14>]-NeuAc<18>分离，在水中展开10 min。使用计算机X射线摄影（利用光激发发光）对残留在原点的反应产物的放射性进行可视化和定量。本文采用RP-TLC法测定了猪次最大腺MCP-NeuAc：乳糖神经酰胺α 2 -3唾液酸转移酶（GM 3合成酶）和大鼠肝高尔基体膜CMP-NeuAc：GM 3 α 2 -8唾液酸转移酶（GD 3合成酶）的活性。证实试验 ...更多信息 分别取决于酶、CMP-NeuAc和受体的反应时间和浓度。该方法适用于大量样品的同时快速测定。[2]In在大鼠肝高尔基体囊泡中使用唾液酸转移酶从无唾液酸-GM 1体外合成二唾液酸神经节苷脂（GD 1 α）：在大鼠肝高尔基体囊泡中使用唾液酸转移酶，在体外有效地从无唾液酸-GM 1和CMP-NeuAc合成两种神经节苷脂。这些神经节苷脂通过阴离子交换和反相柱色谱的组合快速纯化。神经节苷脂的结构通过TLC分析、用鼠伤寒沙门氏菌LT 2的唾液酸酶处理、TLC免疫染色和^1H-NMR光谱测定。其中一种神经节苷脂被鉴定为GD 1 α。另一种神经节苷脂被确定为GM 1b。最后，以去唾液酸-GM 1为起始原料，分别以8.1%和1.2%的总收率得到GM 1b和GD 1alpha。这项研究还表明，新的合成途径去唾液酸-GM 1-&gt; GM 1b-&gt; GD 1 α可能存在于大鼠肝脏中。少

项目成果

Kasahara,K.: "Enzymatic Assay of Glycosphingolipid Sialyltransferase Using Reverse-Phase Thin-Layer Chromatography" Anal.Biochem.(印刷中). (1984)

Kasahara, K.：“使用反相薄层色谱法对鞘糖脂唾液酸转移酶进行酶法测定”Anal.Biochem.（出版中）。

Hidari,K: "In vitro synthesis of disialoganglioside from asialo-GM1 using sialyltransferases in rat Iiver Golgi vesides" Eur.J.Biochem.(印刷中). (1984)

Hidari, K：“在大鼠高尔基体中使用唾液酸转移酶从 asialo-GM1 体外合成二唾液酸神经节苷脂”Eur.J.Biochem.（出版中）。

K.Kasahara, L.Guo, Y.Nagai&Y.Sanai: "In Vitro Synthesis of Disialoganglioside(GD1alpha)from asialo-GM1 using Sialyltransferases in Rat Liver Golgi Vesicles" Anal.Biochem.in press. (1994)

K.笠原、L.Guo、Y.Nagai

Kasahara,K.: "En_2ymatic Assay of Glycosphingolipid Sialyltransferase Using Reverse-Phase Thin-Layer Chromatography" Anal.Biochem.(印刷中). (1984)

Kasahara, K.：“使用反相薄层色谱法测定糖鞘脂唾液酸转移酶”Anal.Biochem.（出版中）。

Hidari,K.: "In vitro synthesis of disialoganglioside from asialo-GM1 using Sialyltransferases in rat liver Golgi vesicles" Eur.J.Biochem.(印刷中). (1984)

Hidari, K.：“在大鼠肝高尔基体小泡中使用唾液酸转移酶从 asialo-GM1 体外合成二唾液酸神经节苷脂”Eur.J.Biochem.（出版中）。