Fig. 5. Analysis of the postulated exchange mechanism. (A) Increasing concentrations of a Cdc20 synthetic peptide were incubated with Mad2, which had or had not been previously incubated with solid phase-bound GST–Mad1^523–550. In the pre-incubation experiment the complex is allowed to form for 60 min, and the competing peptide is added afterwards. If no pre-incubation is allowed, the three components are mixed at the same time and incubated for 15 or 300 min. A diagrammatic representation is shown on the right-hand side. After incubation, beads and bound proteins are collected by centrifugation, washed and analysed by SDS–PAGE. Molecular weight marker, input GST–Mad1^523–550 and Mad2 are shown on lanes 1–3. (B) Superimposed SEC profiles of isolated Mad1–Mad2, Mad2^R133A and Cdc20^111–150 synthetic peptide. The proteins/peptides run as single peaks by SEC. SDS–PAGE separation of collected fractions are also shown. Nine 100 µl fractions (1–9) were collected from 1.2 to 2.1 ml. (C) When Mad2^R133A is incubated with a 5-fold excess of Cdc20^111–150 and separated by SEC, a Mad2–Cdc20 complex forms [compare lanes 4–5 in (B) and (C)]. (D) When the Mad1–Mad2 complex is incubated with Cdc20^111–150, no binding is observed. Furthermore, the peptide is unable to disrupt the complex, as no Mad2–Cdc20 complex or free Mad1 is detected.