FIGURE 4.

Gel filtration profiles of products formed in a reaction containing 1 μm UDP-GlcUA. A, membranes (70 μg of protein) were pulse-labeled in a 2-ml reaction mixture containing 1 mm UDP-Glc, 1 μm UDP-GlcUA (10⁶ cpm), 50 mm Hepes, pH 7.5, and 10 mm MgCl₂ at 35 °C. Reactants (0.5 ml) were removed after pulse labeling for 7 (•), 12 (○), 18 (▪), and 24 min (□) and placed in an ice-water bath to terminate the reaction. Carrier membrane (40 μg of protein) was added, and the membranes were centrifuged and washed twice as described under “Experimental Procedures.” One-half of the membranes were saved on ice. B, the remaining pulse-labeled products were chased by incubating the membranes in 200-μl reaction mixtures containing 0.5 mm UDP-Glc, 0.5 mm UDP-GlcUA, 50 mm Hepes, pH 7.5, and 10 mm MgCl₂, for 10 min at 35 °C. Carrier membrane (20 μg of protein) was added to all samples, and they were washed once as above. Pulse (A) and chase (B) products were solubilized and analyzed by chromatography on Sephacryl S-400 as described under “Experimental Procedures.” The numbers above the graph represent cellubiuronan polysaccharide size standards in kDa. OL, the elution position of the oligosaccharide-lipid micelle.