Hyaluronan Synthase: The Mechanism of Initiation at the Reducing End and a Pendulum Model for Polysaccharide Translocation to the Cell Exterior

Figure 3

Glycosidase treatment converts larger (GlcNAc)_n-UDP oligomers to GlcNAc-UDP. SeHAS membranes were incubated for 30 min with UDP-GlcNAc alone, Folch extracted, fractionated over a size exclusion column, and samples were either untreated (0 min) or treated (120 min) with jack bean hexosaminidase. The samples were then analyzed by MALDI-TOF MS to identify and quantify m/z signals of candidate oligomeric chitin-UDP fragments corresponding to (boldface white or black numbers). The presence of chitin linkages was confirmed by the ability of glycosidase treatment to shift species with 3 or 4 sugars to products with 1 (GlcNAc-UDP) or 2 sugars. Additional MS/MS analysis of the starting sample ions (not shown) revealed smaller members of the expected oligomer series, including GlcNAc-UDP.