FIG. 1.

The type 3 synthase forms a hydrophobic product when incubated with a single nucleotide sugar. Membranes from type 3 S. pneumoniae strain WU2 were incubated for 20 min at 35°C in 200-μl reaction mixtures containing 100 mM HEPES (pH 8.0), 10 mM MnCl₂, and either (A) 1 μM UDP-[¹⁴C]Glc (•) or 1 μM UDP-[¹⁴C]GlcUA (▪) or (B) 1 μM UDP [¹⁴C]Glc and 1 μM UDP-GlcUA (○) or 1 μM UDP-[¹⁴C]GlcUA and 1 μM UDP-Glc (□). Membranes were sedimented by centrifugation and washed twice with 100 mM HEPES (pH 7.5) containing 10% glycerol. Aliquots (50 μl) were spotted on paper and chromatographed for 2 h in butanol:acetic acid:water (44:16:40). The chromatogram was cut into 1-cm strips, and the amount of radioactivity was determined by liquid scintillation counting. The circled Glc indicates the migration of the Glc standard.