FIGURE 2.
A direct assay for NGT activity. A, schematic overview of assay set up. A fluorescently labeled peptide (CF, 5-carboxyfluorescein) is incubated with purified ApNGT and a sugar donor substrate. Product (glycopeptide) is separated from educt (peptide) using RP-HPLC, and both peptides are quantified separately. B, representative chromatogram of analysis of glycosylation of peptide COK_139457–76. The peptide was incubated for 30 min with UDP-Glc and purified ApNGT. Samples were analyzed before (2) and after incubation time (1). The following controls were included: peptide with UDP-Glc and ApNGT-K441A (3), peptide with ApNGT alone (4), and peptide with UDP-Glc alone (5). The unmodified peptide elutes after 11.75 min, glycosylation decreases the retention time to 9.3 min. Glycosylation was further confirmed by MALDI mass spectrometry. Contaminants are marked with an asterisks. C, determination of the reaction speed of glycosylation. D and E, determination of the reaction speeds of NGT reaction at different salt concentrations and different pH values.