The separation by liquid chromatography (under elevated pressure) of phenyl, benzyl, and o-nitrophenyl glycosides of oligosaccharides. Analysis of substrates and products for four N-acetyl- d-glucosaminyl-transferases involved in mucin synthesis

Abstract

Liquid chromatography under elevated pressure (h.p.l.c.) has been applied to the separation of the phenyl, benzyl, and O-nitrophenyl glycosides of 2-acetamido-2-deoxy- d-galactopyranose and of various mucin-type, di-, tri-, and tetra-saccharides. The separations were carried out with a Whatman Partisil PXS 5/25 PAC column and various proportions of acetonitrile and water in the mobile phase. These methods were subsequently used to separate the substrates and products of the following N-acetylglucosaminyltransferase reactions: UDP-GlcNAc + β-Gal-(1→3)-GalNAc-R → β-Gal-(1→3)-[β-GlcNAc-(1→6)]-GalNAc-R + UDP ( 1); UDP-GlcNAc + β-Gal-(1→3)-[β-GlcNAc-(1→6)]-GalNAc-R → β-GlcNAc-(1→3)-β-Gal-(1→3)-[β-GlcNAc-(1→6)]-GalNAc-R + UDP ( 2); UDP-GlcNAc + GalNAc-R′ → β-GlcNAc-(1→3)-GalNAc-R′ + UDP ( 3); and UDP-GlcNAc + β-GlcNAc-(1→3)-GalNAc-R′ → β-GlcNAc-(1→6)-[β-GlcNAc-(1→3)]-GalNAc-R′ + UDP ( 4), where R = benzyl or o-nitrophenyl, and R′ = benzyl or phenyl α- d-glycoside. Reaction 1 is catalyzed by a transferase in canine submaxillary glands and porcine gastric mucosa, and reaction 2 by an enzyme in porcine gastric mucosa. Enzyme activities catalyzing reactions 3 and 4 have recently been demonstrated in rat colonic mucosa. Liquid chromatography can be used at the preparative level for the purification and identification of the transferase products, and at the analytical level in the assay of glycosyltransferases.