An
N-acetylglucosaminyltransferase has been partially purified from Novikoff
tumor cell
ascites fluid by affinity chromatography on
concanavalin A-Sepharose. The
enzyme was obtained in a highly concentrated form after lyophilization. The
enzyme appeared to be highly specific for acceptor
oligosaccharides and
glycoproteins carrying a terminal Gal beta 1----4GlcNAc beta 1----R unit. Characterization of products formed by the
enzyme in vitro by methylation analysis and 1H NMR spectroscopy revealed that the
enzyme catalyzed the formation of a GlcNAc beta 1----3Gal beta 1----4GlcNAc beta-R sequence. The
enzyme therefore could be described as an
UDP-GlcNAc:Gal beta 1----4GlcNAc beta-R beta 1----3-N-acetylglucosaminyltransferase. Acceptor specificity studies with
oligosaccharides that form part of N-
glycans revealed that the presence of a Gal beta 1----4GlcNAc beta 1----2(Gal beta 1----4GlcNAc beta 1----6)Man pentasaccharide in the acceptor structure is a requirement for optimal activity. Studies on the branch specificity of the
enzyme showed that the branches of this pentasaccharide structure, when contained in tri- and tetraantennary
oligosaccharides, are highly preferred over other branches for attachment of the 1st and 2nd mol of GlcNAc into the acceptor molecule. The
enzyme also showed activity toward
oligosaccharides related to
blood group I- and i-active polylactosaminoglycans. In addition the
enzyme together with calf thymus
UDP-Gal:GlcNAc beta-R beta 1----4-galactosyltransferase was capable of catalyzing the synthesis of a series of oligomers of
N-acetyllactosamine. Competition studies revealed that all acceptors were acted upon by a single
enzyme species. It is concluded that the
N-acetylglucosaminyltransferase functions in both the initiation and the elongation of
polylactosaminoglycan chains of N-
glycoproteins and possibly other
glycoconjugates.