ABSTRACIThe N-acetylglucosamine (GlcNAc) transferase that catalyzes the formation of dolichyl-pyrophosphoryl-GlcNAc-GlcNAc from UDPGIcNAc and dolichyl-pyrophosphoryl-G1cNAc was solubilized from the microsomal enzyme fraction of mung beans with 1.5% Triton X-100, and was purified 140-fold on columns of DE-52 and hydroxylapatite. The partially purified enzyme preparation was quite stable when stored in 20% glycerol and 0.5 millimolar dithiothreitol, and was free of GIcNAc-I-P transferase and mannosyl transferases. The GIcNAc transferase had a sharp pH optimum of 7.4 to 7.6 and the K,,for dolichyl-pyrophosphorylGlcNAc was 2.2 micromolar and that for UDP-GlcNAc, 0.25 micromolar.The enzyme showed a strong requirement for the detergent Triton X-100 and was stimulated somewhat by the divalent cation Mg2. Uridine -nucleotides, especially UDP and UDP-glucose inhibited the enzyme as did the antibiotic, diumycin. However, a variety of other antibiotics including tunicamycin were without effect. The product of the reaction was characterized as dolichyl-pyrophosphoryl-GlcNAc-GIcNAc.The biosynthesis ofthe oligosaccharide portion ofthe N-linked glycoproteins involves the formation of lipid-linked oligosaccharides and transfer of oligosaccharide to protein (7,18,28). The formation of the lipid-linked oligosaccharides requires a number of membrane-bound glycosyl transferases that catalyze the addition of GlcNAc, mannose, and glucose to a lipid carrier with the ultimate formation of the tetradecasaccharide-lipid, Glc3Mang(GlcNAc)2-pyrophosphoryl-dolichol (7,20,29 The Glc3Man9(GlcNAc)2-pyrophosphoryl-dolichol has also been isolated and/or synthesized in several different plant systems (15,22,27). However, considerably less information is available on the individual reactions in the lipid-linked saccharide pathway in plants. Previously, we showed that a particulate enzyme fraction from mung bean seedlings incorporated mannose from GDP-mannose into a number of lipid-linked oligosaccharides (16). The oligosaccharides were partially characterized as Man(GlcNAc)2-PP-lipid, Man2(GlcNAc)2-PP-lipid, Man3(GlcNAc)2-PP-lipid, Man5(GlcNAc)2-PP-lipid, and Man7-(GlcNAc)2-PP-lipid. Recently, the mannQsyl transferases from suspension-cultured soybean cells were solubilized (17). The solubilized enzyime preparation synthesized Man3- ' Supported by grants from the National Institutes of Health (AM 21800) and the Robert A. Welch Foundation.(GIcNAc)2-lipid, Man5(GIcNAc)2-lipid, Man6(GlcNAc)2-lipid, Man7(GIcNAc)2-lipid, and Man8(GlcNAc)2-lipid when incubated with lipid acceptors, dolichyl-phosphate and GDP-['4C] mannose. However, when the incubations were done in the presence of EDTA and amphomycin, both of which inhibit the formation of dolichyl-phosphoryl-mannose, only the Man3(GlcNAc)2-lipid and the Man5(GlcNAc)2-lipid were formed ( 17). That study suggested that the first five mannose residues in the lipid-linked oligosaccharides come directly from GDP-mannose, whereas the next four mannose units come from dolichylphosphoryl-mannose. Similar resu...