FIGURE 6.

Increasing Lys-48-Ub chain length on a substrate results in decreased conjugation rates. A) Comparison of Cdc34-catalyzed elongation of β-cat-Ub₁ with β-cat-Ub₄, ∼0.1 μm each, using approach I (Fig. 5A) with heat denaturation. The quantification of β-cat-Ub₁ or β-cat-Ub₄ was based on phosphorimaging analysis of experiment shown in Fig. 5E. B, same as in A, but comparing β-cat-Ub₄ with β-cat-Ub₈. β-cat-Ub₈ was prepared as described for reaction shown in Fig. 5C, lane 5. Note that the graph shown in panels A and B integrates the results of three independent experiments, with error bars for the calculated S.D. The experiment shown in panel C was carried out using approach II without heat denaturation (Fig. 5D).