Abstract
Apparent kinetic parameters have been measured for the transfer of N-acetyl-D-neuraminic acid (Neu5Ac) from CMP-Neu5Ac to analogues of the Gal(beta 1-4)GlcNAc (type II) and Gal(beta 1-3)GlcNAc (type I) substrates by the rat liver Gal(beta 1-4)GlcNAc alpha 2,6-sialyltransferase and the Gal(beta 1-3/4)GlcNAc alpha 2,3-sialyltransferase. In these acceptor analogues, the substituents of the pyranose rings were modified, particularly by deoxygenation, to identify (i) the key polar groups required for efficient transfer and (ii) the substituents that can be removed or modified. A topography including the 6-hydroxyl of the beta Gal and the 2-acetamido of the GlcNAc unit is required for transfer to a terminal type II disaccharide by the alpha 2,6-sialyltransferase. The other hydroxyls can be replaced by hydrogen without a substantial decrease in activity. The alpha 2,3-sialyltransferase requires the 3-, 4-, and 6-hydroxyls of the terminal beta Gal and some contribution from the subterminal sugar. This may explain the cross-reactivity of this enzyme for the type I and type II acceptors. For both enzymes, an influence of the hydrophobic nature of the aglycone is noticed. The results allow an evaluation of the efficiency of the transfer of Neu5Ac to modified substrates.
Highlights
Apparent kinetic parameters have been measured thepresence of polylactosaminyl corestructures(3),the for the transfer of N-acetyl-D-neuraminic acid expression of sialylated and fucosylated determinants recog
A topography including the 6-hydroxyl of the #?Galand the 2acetamido of the GlcNAc unit is required for transfer to a terminal type I1 disaccharide by the a2,6-sialylnized by selectins may have important biological functions (4)
The results of this study indicatethat thespecificity of the a2,6-ST for the Gal(Pl-4)GlcNAc terminal disaccharide is a result of the requirement for the 6-hydroxyl of the terminal @Galand for the 2-amido group of the neighboring GlcNAc for the transfer of Neu5Ac (Fig. 2)
Summary
Incubation mixtures contained 9nmol of CMP-[14C] Neu5Ac (3,340cpm/nmol), 2mM acceptor substrate, 1mg/ml bovine. U. Lemieux as blocked the a2,6-STfor synthetic acceptors were determined under the above materials that were deprotected using standard procedures, providing standard assay conditions using a saturating concentrationof CMP-. Compounds 25 and 27 were prepared by [14C]Neu5Ac(37). Trisaccharide 9 was prepared as reported whereas the concentration of CMP-[14C]Neu5Ac waskept constant (29). Compounds 5, 1a2n,d 30 were prepared following a method at 560 p M (893 cpm/nmol). The corresponding 2-azido-2-deoxy disaccharides which were both ob- radiolabeled product was isolated by the Sep-Pak method for acceptained by reacting @-D-galactopyranosypl entaacetate with 6-0-acetyl-2-azido-2-deoxy-@-~-galactopyranosidei-nOtRh~e presence of tritors with the hydrophobic 8-methoxycarbonyloctyl aglycone (37) or by ion-exchange chromatography on a Dowex 1x8-200
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