ISOLATION AND CELL BIOLOGICAL CHARACTERIZATION OF SIALYLTRANSFERASES INVOLVED IN GANGLIOSIDE BIOSYNTHESIS

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(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