A novel HPLC assay system for sialyltransferase activity based on the use of fluorescein-labelled acceptor oligosaccharides is described. The fluorescein-labelled disaccharides PGal-(1+4)-PGlc-OR (4), PGal-(lG+4)-PGlcNAc-OR (17), and PGal-(1+3)-~GlcNAc-OR (22) where OR consists of a six carbon spacer with fluorescein attached, were synthesised. Synthetic standard products were produced chemo-enzymatically on a preparative scale to yield fluorescein-labelled trisaccharides. The use of reverse phase HPLC with an ionpairing agent allowed the separation of starting materials from products and separation of the two isomeric trisaccharides aNeu5Ac-(2-+3/6)-PGal-( 1-+4)-PGlcNAc-OR (24 and 25), so that the assay could be used to measure the different silalyltransferase activities in a mixture. The assay was successfully applied to the detection of sialyltransferase activity of commercially available enzymes and a crude preparation of bovine colostrum. The predominant sialyltransferase activity in bovine colostrum adds sialic acid a(2-6) to 17.Sialyltransferases belong to the family of glycosyltransferases which are responsible for the biosynthesis of the carbohydrate components in glycoproteins and glycolipids[lJ. Glycosyltransferases transfer nucleotide-activated monosaccharides to sugar residues already bound to a protein or lipid moiety, or linked to an artificial spacer which resembles these There are a number of different sialyltransferases from different sources that differ in their acceptor specificities, but all use cytidine monophosphate (CMP)-activated N-acetylneuraminic acid (Neu5Ac) as the donor substrater3I. These enzymes are becoming increasingly important as analytical as well as catalysts in the syntheses of complex oligosa~charides [~], and considerable effort has been directed at developing simple, reliable and inexpensive assays for them. Common assays for sialyltransferases rely upon monitoring the fate of the donor CMP-Neu5Ac by using radioactiveL61 or fluorescently labelled N e~5 A c [~] , or detecting the equimolar amount of liberated CMP[*]. A new assay based on isolation of the sialylated product, its acid hydrolysis and measurement of free NeuSAc released with a colourimetric assay was reported Our intention in carrying out this work was a reinvestigation of the sialyltransferases present in bovine colostrum, a cheap and readily available source of glycosyltransferases. To our knowledge the only sialyltransferase isolated from colostrum so far is an a2,6-sialyltransferase (a2,6-ST)[6.101.However, early studies by Bartholomew et al.["] on partially purified bovine colostrum showed evidence of formation both a(2 + 6)-and a(2 + 3)-sialylated lactose. No a(2 + 3) activity was detected for the purified a2,6-ST isolated from bovine c o l~s t r u m [~~~~'~] .We reasoned that a putative bovine a2,3-sialyltransferase could have been lost during purification of the other enzyme. We therefore required an assay system that was able to detect small amounts of sialyltransferase activity in crude protein prepa...
As a precursor for the chemical synthesis of sialylated oligosaccharides, the trisaccharide glycoside Neu5Ac alpha (2-8)Gal beta (1-4)GlcNAc beta (1-O)-pent-4-ene was synthesized starting from GlcNAc beta (1-O)-pent-4-ene, UDP-glucose and N-acetylneuraminic acid in a one pot reaction employing galactosyltransferase and alpha (2-6)sialyltransferase in a complete cofactor regeneration system.
The preparative formation of cytidine 5′‐triphosphate by an enzymatic process with adenylate kinase and pyruvate kinase is described. The enzymes may either be used free, in the “MEEC” technique, or immobilized on a synthetic copolymer of vinyl acetate and N,N′‐divinylethyleneurea (“VA‐Epoxy”). Cytidine 5′‐monophosphosialate synthase (preferably isolated from calf brain) and inorganic pyrophosphatase were used to prepare the activated neuraminic acid cytidine 5′‐monophosphosialate (CMP‐Neu5Ac) from CTP and neuraminic acid in good yield. Finally, both processes could be integrated and led to an in situ approach which, owing to moderate yields, requires further development.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.