IDENTIFICATION OF A NOVEL UDP-GALNAC-GLCNAC-BETA-R BETA-1-4 N-ACETYLGALACTOSAMINYLTRANSFERASE FROM THE ALBUMIN GLAND AND CONNECTIVE-TISSUE OF THE SNAIL LYMNAEA-STAGNALIS

Both the albumen gland, one of the female accessory sex glands, and co nnective tissue of the freshwater snail Lymnaea stagnalis contain N-ac etylgalactosaminyltransferase activity, capable of transferring GalNAc from UDP-GalNAc in beta 1-4 linkage to the terminal GlcNAc residue of GlcNAc beta-R. The albumin gland enzyme was partially purified by aff inity chromatography on UDP-hexanolamine-Sepharose 4B. Using GlcNAc be ta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc be ta 1-4GlcNAc or GlcNAc beta 1-OMe as substrates, the enzyme showed an absolute requirement for Mn2+ with an optimum concentration of 12.5-50 mM. The optimal pH was approximately pH 7.0. The enzyme activity was independent of the Triton X-100 concentration in the range 0.25-2.5%, and no activation effect was found. The more labile connective tissue microsomal enzyme, subjected to the same optimization procedure, gave comparable results. Both enzyme activities have similar substrate spec ificities towards GlcNAc or GlcNAc beta 1-OMe, and towards oligosaccha rides or glycopeptides with a non-reducing terminal beta-GlcNAc unit, but cannot act on GlcNAc alpha 1-OMe. Saccharides with non-reducing te rminal Gal or GalNAc residues, and free GalNAc, Gal or Glc residues ar e not acceptors. Product analysis was carried out for albumen gland N- acetylgalactosaminyltransferase and four acceptors having GlcNAc beta 1-R as the terminal non-reducing unit, and for connective tissue N-ace tylgalactosaminyltransferase with GlcNAc beta 1-OMe as acceptor. In al l instances, products with GalNAc beta 1-4-linked to GlcNAc were obtai ned, showing that the connective tissue and the albumen gland activiti es are probably from one enzyme. This enzyme activity can be identifie d as UDP-GalNAc:GlcNAc beta-R beta 1-4 N-acetylgalactosaminyltransfera se, and is probably involved in the biosynthesis of N,N'-diacetyllacto sediamine-containing glycoproteins, like hemocyanin, in the snail L. s tagnalis.