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Novikoff ascites tumor cells contain a UDP‐GlcNAc:β‐galactoside β1→6‐N‐acetylglucosaminyltransferase (β6‐GlcNAc‐transferase B) that acts on galactosides and N‐acetylgalactosaminides in which the accepting sugar is β1→3 substituted by a Gal or GlcNAc residue. Characterization of enzyme products by 1H‐NMR and methylation analysis indicates that an Rβ1→3(GlcNAcβ1→6)Gal‐ branching point is formed such as occurs in blood‐group‐I‐active substances. The enzyme does not show an absolute divalent cation requirement and 20 mM EDTA is not inhibitory. The activity is strongly inhibited by Triton X‐100 at concentrations of ≥ 0.2%. Competition studies suggest that a single enzyme acts on Galβ1→3Galβ1→4Glc, GlcNAcβ1→3Galβ1→4GlcNAc and GlcNAcβ1→3GalNAcα‐O‐benzyl (Km values 0.71, 0.83 and 0.53 mM, respectively). Galβ→3Galβ1→4Glc as an acceptor substrate for β6‐GlcNAc‐transferase B does not inhibit the incorporation of GlcNAc in β1→6 linkage to the terminal Gal residues of asialo‐α1‐acid glycoprotein catalyzed by a β‐galactoside β1→6‐N‐acetylglucosaminyltransferase (β6‐GlcNAc‐transferase A) previously described in Novikoff ascites tumor cells [D. H. Van den Eijnden, H. Winterwerp, P. Smeeman & W. E. C. M. Schiphorst (1983) J. Biol. Chem. 258, 3435–3437]. Neither is Triton X‐100 at a concentration of 0.8% inhibitory for the activity of β6‐GlcNAc‐transferase A. This activity is absent from hog gastric mucosa microsomes, which has been described to contain high levels of β6‐GlcNAc‐transferase B. [F. Piller, J. P. Cartron, A. Maranduba, A. Veyrières, Y. Leroy & B. Fournet (1984) J. Biol. Chem. 259, 13385–13390]. Our results show that Novikoff tumor cells contain two β‐galactoside β‐galactoside β6‐GlcNAc‐transferases, which differ in acceptor specificity and tolerance towards Triton X‐100. A role for these enzymes in the synthesis of branched polylactosaminoglycans and of O‐linked oligosaccharide core structures having blood‐group I activity is proposed.

Four acidic trisaccharides have been obtained by alkaline borohydride reductive cleavage (8-elimination) of a fraction of porcine submaxillary mucin precipitating at 60 -75% ethanol. Their structures have been investigated using the techniques of methylation analysis involving gas-liquid chromatography/mass spectrometry along with high-resolution 'H-NMR analysis. Two of the four oligosaccharides, B1-B4, contain N-acetylneuraminic acid (NeuAc) while two contain N-glycolylneuraminic acid (NeuGc). The following structures are proposed for the acidic trisaccharide fraction: (Bl) NeuAca(2-+3)Gal~(l-+3)GalNAcol, (B2) NeuGca(2+3)Galj?(1+3)GalNAcol,These oligosaccharides were present in a molar ratio of 69: 22: 4: 5. Although oligosaccharide B4 has previously been found in porcine submaxillary much B1, B2 and B3 have not. Furthermore, oligosaccharide B2 is a novel structure.The oligosaccharide structures of porcine submaxillary mucin and their biosynthesis have been the subject of many investigations [l -91. Some questions have, however, remained unanswered. For instance, Rearick et al. [q have reported the characterization of porcine submaxillary P-galactoside a2 +3-sialyltransferase, to form the sequence NeuAca(2-t 3)-Galfl(1+3)GalNAc, although this structural element had not been reported in porcine submaxillary mucin (PSM). In addition, a study by Sherblom and Dahlin [lo] showing that both NeuAc and NeuGc were present in tumour cell glycoproteins, along with the report of Buscher et al. [l 11 on the biosynthesis of NeuGc in porcine submaxillary glands and the considerable controversy which has surrounded the oligosaccharide structures of PSM [3 -51 prompted a further investigation.The mucin was precipitated from porcine submaxillary gland extracts using a higher alcohol concentration than that employed by earlier investigators. Oligosaccharides were released by P-elimination, and their structures were determined by high-resolution 'H-NMR spectroscopy and methylation analysis. Four sialic-acid-containing trisaccharides, three of Correspondence to A. V. Savage, Department of Chemistry, University College, Galway, IrelandAbbreviations. GalNAcul, reduced N-acetylgalactosamine; NeuAc, N-acetylneuraminic acid; NeuGc, N-glycolylneuraminic acid; HPLC, high-pressure liquid chromatography; GLC-MS, gasliquid chromatography/mass spectrometry; PSM, porcine submaxillary mucin.Enzymes. P-Galactoside ct2-+3-sialyltransferase (EC 2.4.99.4); NeuAc : a-N-acetylgalactosaminide a 2 +6-sialyltransferase (EC 2.4.99.3); N-acetylneuraminate monooxygenase (EC 1.14.99.18). which had not previously been described in PSM and one of which is novel, were found. MATERIALS AND METHODS Isolation of oligosaccharidesMucin was isolated from porcine submaxillary glands according to de Salegui and Plonska [12] except that additional precipitates were obtained: following the removal of the 48 -54% and 54 -60% ethanol precipitates by centrifugation, the mucin solution was brought to 75% (v/v) ethanol, allowed to stand at 4°C for 16 hand centrifuged at 270...

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