Lipid-linked oligosaccharides were synthesized with the particulate enzyme preparation from mung bean (Phaseols aureus) seedlngs in the presence of GDP-1J4C1 mannose. The oligosaccharides were released from the lipids by mild acid hydrolysis and purified by several passages on Biogel P4 columns. Five different oUgosacchardes were purified in this way.Based on their relative elution constants (Kd) compared to a variety of standard oligosaccharides, they were sized as (mannose-acetylglucosamine) Man7GlcNAc2, Man5GIcNAc2, Man3GlcNAc2, Man2GlcNAc2, and ManGlcNAc2. These oUgosaccharides were treated with endoglucosaminidase H and a-and 8i-mannosidase, and the products were examined on Biogel P4 columns. They also were subjected to a number of chemical treatments including analysis of the reducing sugar by NaB3H4 reduction, methylation analysis, and in some cases acetolysis. From these data, the likely structures of these oligosaccharides are as foUows: E, Man,BGlcNAc-GlcNAc; D, Mana1-*3Manfi-GlcNAc-GlcNAc; C, Manal-+ 2Mana-1 3Man#-GlcNAc-GlcNAc; B, Manal-2Mana1-*2Mana1--* 3(ManaI-.6)Man,8-GlcNAc-GlcNAc; and A, Manal-2Mana1--+ 2Manal-3(Manal-3lManal-6I Manal--.6)Man8-GkcNAc-GlcNAc.The synthesis of the Man7GlcNAc2 was greatly diminished when tunicamycin (10 pg/ml) was added to the incubation mixtures.Many plant and animal glycoproteins have an oligosaccharide composed of mannose, N-acetylglucosamine (GlcNAc) and possibly other sugars, that is attached to the protein in a GlcNAcasparagine bond (10,17,32). The biosynthetic pathway for the formation of this oligosaccharide involves a series of membranebound glycosyl transferases that utilize dolichol-linked saccharide intermediates (10, 32). In the animal systems, it is now wellestablished that these enzymes catalyze the transfer of GlcNAc, mannose, and glucose to dolichyl-phosphate to form a dolichylpyrophosphoryl-oligosaccharide having the composition, Glc3Man9GlcNAc2 (5,20,22). This oligosaccharide is then transferred to the protein (6,7,24,28). Several of the intermediate lipid-linked oligosaccharides have been isolated from animal tissues and the oligosaccharides have been characterized (4,15,32,33). Plant cells also contain glycoproteins having oligosaccharides attached in GlcNAc--asparagine bonds (10), and the structure of the oligosaccharide of soybean lectin has been shown to be a highmannose type (21).Although the general pathway of assembly of the lipid-linked saccharides of plant cells is known (1-3, 8, 13, 19) The reactions were terminated by the addition of 2 ml of CHC13:CH30H (1:1) and 0.5 ml of H20. The mixture was stirred vigorously, and the layers were separated by centrifugation. The lower CHC13 layer was removed and saved, and the aqueous phase and interface were extracted with another 1 ml of CHC13. The combined CHC13 phases, which contained lipid-linked monosaccharides and lower mol wt lipid-linked saccharides, were extracted with CHC13:CH30H:H20 (3:48:47) before being placed in scintil-12 www.plantphysiol.org on May 9, 2018 -Published by Dow...