FIG. 7.

Type 3 polysaccharide is synthesized on a glycerophosphate lipid. Type 3 polysaccharide was labeled in a 600-μl-volume pulse reaction containing 100 mM HEPES (pH 7.0), 10 mM MnCl₂, 1 μM UDP-[¹⁴C]Glc, 1 μM UDP-GlcUA, and membranes from type 3 strain JD614 for 1 min at 35°C. A 300-μl aliquot was taken and placed on ice. The reaction was then chased for 5 min at 35°C with 500 μM of UDP-Glc and UDP-GlcUA. The membranes from the pulse and chase samples were sedimented by centrifugation, washed twice, and resuspended in 100 μl of wash buffer. Samples were solubilized and treated with PLD, as described in the legend to Fig. 2. (A) Pulse samples, untreated (•) and PLD digested (○), and (B) chase samples, untreated (▪) and PLD digested (□), were chromatographed on a Sephacryl S-300 column and eluted as described above. (C) A portion of the untreated chase sample (▪) was digested with type 3 polysaccharide-specific depolymerase (▵) in a 200-μl-volume reaction containing 0.1 M morpholineethanesulfonic acid (pH 6.0) for 15 min at 35°C. For all samples, 1-ml fractions were collected and the amount of radioactivity present in the even-numbered fractions was determined.