FIGURE 6.

PgtA is a Skp1 GalT and FucT. A, GalT activity directed toward GlcNAc-Skp1 was assayed as described for GlcNAcT activity in Fig. 5A, except that GlcNAc-Skp1 and UDP-[³H]Gal were used in place of HO-Skp1 and UDP-[³H]GlcNAc. The reaction time, inclusion of GlcNAc-Skp1 and GDP-Fuc, and source of the extract (RHΔΔ or RH, or RHpgtAΔ or pgtAΔ) were varied as indicated. B, entire lane from a parallel 3-h reaction (RH, +Gn-Skp1) from A was analyzed for incorporation of ³H. Incorporation was only detected at the migration position of DdSkp1. C, [³H]DdSkp1 from the 3-h GalT reaction in A was isolated as in Fig. 5D and hydrolyzed in 4 m TFA. The hydrolysate was chromatographed on a Dionex PA-1 column with internal standards of Gal, Glc, Man, and Fuc, and the elution of ³H was monitored by scintillation counting of collected fractions. D–F, FucT activity assays. Reactions were conducted as above except that GDP-[³H]Fuc replaced UDP-[³H]Gal, and the dependence of incorporation on a 10-fold concentration excess of UDP-Gal, GlcNAc-Skp1, and PgtA in the extract and time was examined.