FIGURE 6.

Both native and denatured T-synthase can cause release of the reconstituted active T-synthase from Cosmc. A, schematic diagram of in vitro release experiment. B, both native and denatured T-synthase can alter the equilibrium between Cosmc and reconstituted active T-synthase but not of denatured BSA or buffer alone. Reactivated T-syn associated with Cosmc beads were incubated with native, heat-denatured His-sT-syn, heat-denatured BSA, or buffer alone. Supernatant and bound fractions were analyzed by Western blotting (WB) against HPC4. The silver-stained gel shows the amount of T-synthase and BSA used in the experiment. A vertical line separates the data taken from different parts of the same gel and the same experiment except the vertical line between 1 and 2, which separates the experiment done at different times. C, quantification of eluted fraction (Released) of Western blot data as compared with Input from B. Data are an average of three independent experiments. Error bars ± 1 S.D. from the average of three independent experiments. D, denatured His-sT-syn releases reactivated HPC4-sT-syn from Cosmc in a concentration-dependent manner. Reactivated T-syn associated with Cosmc beads was incubated with different concentrations of DT-syn. Eluted fractions (Released) and the amount of protein remained on the beads (Bound) were analyzed by Western blotting for HPC4. The silver-stained gel shows the amount of heat-denatured His-sT-syn used. F, native His-sT-syn releases reactivated HPC4-sT-syn from Cosmc in a concentration-dependent manner. As described in D, in vitro reconstitution followed by pulldown experiments were carried out and analyzed by Western blot to determine the amount released and the silver-stained gel to show the amount of His-sT-syn used for elution. E and G, quantification of eluted fraction (Released) as compared with Input. Data are an average of two independent experiments. Error bars ± 1 S.D. from the average of two independent experiments.