FIGURE 7.

Steady state proportion of synthase in the fully processive state. Washed E. coli membranes (66 μg of protein) were preloaded with polysaccharide by incubating for 6 min at 35 °C in 2-ml reaction mixtures containing 100 mm Hepes, pH 7.5, 10 mm MgCl₂, and either 0.1 (○) or 1 mm UDP-Glc (•), at the indicated concentrations of UDP-GlcUA. The reactions were terminated in an ice-water bath, and the membranes were washed twice as described in the legend to Fig. 4. The membranes were suspended in 0.1 ml of ice-cold 100 mm Hepes, pH 7.5. As explained under “Experimental Procedures,” the amount of fully processive activity was then determined by incubating the preloaded membranes (30 μl) in 0.2-ml reaction mixtures containing 10 μm UDP-[¹⁴C]GlcUA (10⁵ cpm), 10 μm UDP-Glc, 100 mm Hepes, pH 7.5, and 10 mm MgCl₂, previously brought to 35 °C. The reactions were incubated for 60 s at 35 °C, terminated by the addition of 5 μl of 12.5 m acetic acid, and the products were separated by paper chromatography. The formation of polysaccharide (origin product) is indicated as the percentage of fully processive activity of synthase in the membranes preincubated at the indicated concentration of UDP-GlcUA.