FIGURE 4.

Activity of expressed PfGMD and PfFS. A, Coomassie Blue-stained SDS-polyacrylamide gel of purified, GST-tagged, recombinantly expressed PfGMD (lane 1) and PfFS (lane 2) proteins. B, the purified recombinant enzymes were assayed using GDP-Man as a substrate. Sugar nucleotides were analyzed by multiple-reaction monitoring LC-MS/MS. Multiple-reaction monitoring (MRM) transitions are indicated in the panels. C, effect of time and GDP-Man and enzyme concentration on the yield of GDP-Fuc by PfGMD and PfFS. Conditions tested are: 1 mm GDP-Man, 3 h of 1.5 mg/ml PfGMD, and 2 h of 1.5 mg/ml PfFS (1) (control); 0.5 mm GDP-Man, 3 h of 1.5 mg/ml PfGMD, and 2 h of 1.5 mg/ml PfFS (2); 1 mm GDP-Man, 1 h of 1.5 mg/ml PfGMD, and 0.5 h of 1.5 mg/ml PfFS (3); 1 mm GDP-Man, 3 h of 1 mg/ml PfGMD, and 2 h of 1 mg/ml PfFS (4); and 1 mm GDP-Man, 3 h of 1.5 mg/ml PfGMD, and 2 h of 1 mg/ml PfFS (5).