FIGURE 7.

Effect of GroES on reactivation rate of GroEL bound MSG. MSG reactivation was monitored by measuring regain of enzymatic activity by MSG upon GroES/ATP addition. a, influence of increasing GroEL concentration on the MSG reactivation rate in the presence and absence of GroES. GroEL-MSG complex was generated as described previously (GroEL/MSG, 0.5:0.25 μm). After 10 min, increasing amounts of GroEL (total concentrations of GroEL are indicated) were added in the presence (▴) or absence (●) of GroES. GroES was always added in a 2:1 molar ratio to GroEL. Reactivation was initiated by adding ATP. b, effect of casein on MSG reactivation rates. Casein (0–5 μm) was added 5 min after GroES addition (1 μm) in GroES-containing reactions. After further incubation for 5 min, ATP (2.5 mm) was added. Rates in each case were calculated from their corresponding activity curves just like rates in Fig. 3b. c, SDS-PAGE showing that GroES does not encapsulate GroEL-bound MSG. GroES was added in a 2-fold molar excess to GroEL-MSG complex (MSG/GroEL/GroES, 1:2:4 μm) in the presence of ADP followed by trypsin digestion after which samples were run on a 10% SDS-polyacrylamide gel. Trypsin treatment was performed in the same way as described previously. Lane 1, medium range protein molecular mass marker; lane 2, trypsin-treated native MSG used as control; lane 3, GroEL-MSG complex treated with trypsin; lane 4, GroEL-MSG-GroES and ADP treated with trypsin; lane 5, GroEL-MSG complex without trypsin.