ISOLATION AND CELL BIOLOGICAL CHARACTERIZATION OF SIALYLTRANSFERASES INVOLVED IN GANGLIOSIDE BIOSYNTHESIS

(1)反相薄层色谱法测定糖鞘脂唾液基转移酶：建立了反相薄层色谱法快速测定糖鞘脂唾液基转移酶的方法。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。反应结束后，用C_<18>的RP-TLC将酶促产物从未反应的CMP-[^<14>C]- neuac中分离出来，在水中发育10 min。CMP-NeuAc沿溶剂前沿迁移。反应产物的放射性，留在原点，可视化和量化使用计算机放射成像，利用光刺激发光。本研究采用RP-TLC法测定了猪亚最大腺体MCP-NeuAc：乳糖神经酰胺alpha2-3唾液基转移酶（GM3合成酶）和大鼠肝脏高尔基膜CMP-NeuAc: GM3 alpha2-8唾液基转移酶（GD3合成酶）的活性。结果表明，该检测结果更多地依赖于酶、CMP-NeuAc和受体的反应时间和浓度。该方法对大量样品的同时快速测量是可靠的。利用大鼠肝脏高尔基囊胞唾液基转移酶从asialo-GM1合成双神经节苷脂（GD1alpha）：体外利用大鼠肝脏高尔基囊胞唾液基转移酶从asialo-GM1和CMP-NeuAc高效合成两种神经节苷脂。这些神经节苷通过阴离子交换和反相柱色谱的组合快速纯化。通过TLC分析、鼠伤寒沙门氏菌LT2唾液酸酶处理（特异性水解α - 2-3 n -乙酰神经氨酸键）、TLC免疫染色和^1H-NMR波谱测定神经节苷脂的结构。其中一种神经节苷脂被鉴定为GD1alpha。另一种神经节苷脂确定为GM1b。最后，以asialo-GM1为原料得到GM1b和GD1alpha，总产率分别为8.1%和1.2%。本研究还提示大鼠肝脏中可能存在新的合成途径asialo-GM1 -> GM1b -> GD1alpha。少

(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