FIGURE 5.

Purified H6-YopD associates with 30 S ribosomal particles. A–C, immunoblot analysis of sucrose density ultracentrifugation fractions (12 indicates top of supernatant, 1 indicates bottom of supernatant) collected after mixing class II factors with the indicated ribosomal particles and analyzing mobility through 30% sucrose. Either purified H6-YopD/LcrH (A and B) or GST-YscM1 (C) were mixed with 30, 50, or 70 S ribosomal particles for 15 min at 37 °C before loading on 30% sucrose cushion and ultracentrifugation. YopD was found in sediment after mixing with 30 S ribosomes from Y. enterocolitica (A), not from E. coli (B). Using quantitative immunoblotting, the dissociation constant for the YopD/30 S ribosomal particle complex was determined: K_d 2.1 × 10⁴ μm (±0.5 × 10⁴). D, amount of YopD bound for a given concentration of 30 S particles as determined in A. Data are representative of four independent determinations.