PARTITIONING OF RHODANESE ONTO GROEL - CHAPERONIN BINDS A REVERSIBLY OXIDIZED FORM DERIVED FROM THE NATIVE PROTEIN

The mammalian mitochondrial enzyme, rhodanese, can form stable complex es with the Escherichia coli chaperonin GroEL if it is either refolded from 8 as urea in the presence of chaperonin or is simply added to th e chaperonin as the folded conformer at 37 degrees C, In the presence of GroEL, the kinetic profile of the inactivation of native rhodanese followed a single exponential decay. Initially, the inactivation rates showed a dependence on the chaperonin concentration but reached a con stant maximum value as the GroEL concentration increased. Over the sam e time period, in the absence of GroEL, native rhodanese showed only a small decline in activity. The addition of a non-denaturing concentra tion of urea accelerated the inactivation and partitioning of rhodanes e onto GroEL, These results suggest that the GroEL chaperonin may faci litate protein unfolding indirectly by interacting with intermediates that exist in equilibrium with native rhodanese. The activity of GroEL -bound rhodanese can be completely recovered upon addition of GroES an d ATP, The reactivation kinetics and commitment rates for GroEL-rhodan ese complexes prepared from either unfolded or native rhodanese were i dentical, However, when rhodanese was allowed to inactivate spontaneou sly in the absence of GroEL, no recovery of activity was observed upon addition of GroEL, GroES, and ATP, Interestingly, the partitioning of rhodanese and its subsequent inactivation did not occur when native r hodanese and GroEL were incubated under anaerobic conditions. Thus, ou r results strongly suggest that the inactive intermediate that partiti ons onto GroEL is the reversibly oxidized form of rhodanese.