Fig. 3.
OGT activities of Giardia nucleocytosolic extract and of the recombinant GlOGT expressed in yeast. (A) The OGT activity of the Giardia nucleocytosolic extract with itself showed a Km of 5.8 μM UDP-GlcNAc and a Vmax of 5.1 pgm/min/mg protein. (B) The activity of recombinant GlOGT (catalytic domain only), again with itself, showed a Km of 0.9 μM UDP- GlcNAc and a Vmax of 5.6 pgm/min/mg protein. (C) Transfer of a single, O-linked GlcNAc to recombinant GlOGT was confirmed by performing a reductive β-elimination on the precipitated pellet following the OGT assay. Analysis of the products on a Bio-Gel P2 showed a single GlcNAcitol. The negative control in B and C is the empty vector. In each case, representative data are shown from three experiments performed on separate days.