N‐acetylglucosaminyl transferases from the pupal instar of the stable fly, Stomoxys calcitrans

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Abstract
N‐acetylglucosaminyl transferases from pupae of Stomoxys calcitrans (L.) were studied in 10,000g pellet suspensions. Characterization of these enzymes was based on formation of glycolipids (ie, Dol·PP‐GlcNAc and Dol·PP‐(GlcNAc)2), oligosaccharide lipids, and giycoproteins. Studies on transferase activity during the pupal instar showed that there were two peaks of activity; the first peak was on day 0 (prepupae) and the second at 3 days after pupation. Subcellular fractionation indicated that 10,000g and 100,000g pellets contained most of the transferase activities. The transferases required divalent cations (either Mn2+ or Mg2+). The pH optimum, which varied for each of the products formed, was 7.5 for glycolipids, 7.0 for oligosaccharide lipids, and 6.5 for glycoprotein. Inclusion of dolichol monophosphate doubled the amount of Dol·PP‐GlcNAc and Dol·PP·(GlcNAc)2 formed, but had little effect on oligosaccharide lipid and glycoprotein formation.Tunicamycin was a potent inhibitor of glycolipid formation with an I50 of 1.8–4.8 nM. It was confirmed that tunicamycin acts by preventing the transfer of GlcNAc‐1‐P from UDP‐GlcNAc to Dol·P. UMP reverses glycolipid formation, yielding UDP‐GlcNAc. Some characterization of the products was performed. Glycolipids were shown to be Dol·PP‐GlcNAc and Dol·PP‐(GlcNAc)2. Glycoprotein was rapidly solubilized by protease and detergent treatments, whereas oligosaccharide lipids appeared to be acid‐labile, pyrophosphate‐containing lipids. The apparent kinetic constants for the formation of glycolipids were as follows: UDP‐GlcNAc Km = 1.55 ± 0.47 μM, Vmax = 0.66 ± 0.21 pmol·min−1·mg−1; Dol·P Km = 2.08 ± 0.85 μM, Vmax = 0.13 ± 0.06 pmol·min−1·mg−1 protein.