FIGURE 5.

TgGnt1 is a Skp1 GlcNAcT. A, GlcNAcT activity in S100 cytosolic parasite was assayed based on transfer of ³H from 0.5 μm UDP-[³H]GlcNAc to exogenous HO-DdSkp1 for 1–3 h as described under “Experimental Procedures.” Reactions were loaded onto and separated on an SDS-polyacrylamide gel, and the Coomassie Blue-stained DdSkp1 bands were excised and subjected to liquid scintillation spectroscopy. The reaction time, presence of HO-Skp1, and source of the extract (RHΔΔ or RH, or RHgnt1Δ or gnt1Δ) were varied as indicated. B, entire lane from a parallel 3-h reaction (RH, +HO-Skp1) from A was analyzed for incorporation of ³H. Incorporation was only detected at the migration position of DdSkp1. C, donor substrate specificity of the GlcNAcT activity was examined by including a 9-fold excess of unlabeled UDP-GlcNAc or UDP-GalNAc to reactions containing 10 μm UDP-[³H]GlcNAc. Incorporation was measured as in A. Error bars show standard deviations of the mean of two replicates from each of two independent reactions. D, analysis of incorporated ³H. The reacted Skp1 band was excised from a PVDF membrane electroblot of the SDS-polyacrylamide gel, subjected to acid hydrolysis in 6 n HCl, and analyzed by high pH anion exchange chromatography. The hydrolysate was supplemented with GlcNH₂ and GalNH₂ and chromatographed on a Dionex PA-1 column. Elution of the sugar standards was monitored by a pulsed amperometric detector (nC), and fractions were collected to monitor the elution of ³H by scintillation counting (dpm).