Figure 2.

Refolding of denatured luciferase by SYS and YSY. (A) Kinetics of luciferase refolding by Ssa1 and Hsp40 proteins. Guanidinium HCl denatured luciferase was incubated with Ssa1 (0.5 μM) and different Hsp40 proteins (1 μM) at 30°C. At the indicated times, aliquots of the refolding reaction were removed and luciferase activity was determined with a Turner TD-20/20 luminometer. Luciferase activity is expressed in arbitrary units as percentage of total activity observed when it was refolded by Ydj1 and Ssa1. (B) Stimulation of Ssa1 ATPase activity by SYS and YSY. Ssa1 (0.5 μM) and Hsp40 (1.0 μM) were mixed in reaction buffer (20 mM HEPES, pH 7.4, 80 mM KCl, 10 mM DTT, 1 mM MgCl₂, 50 μM [α-³²P]ATP; ICN Biomedicals, Costa Mesa, CA) at 30°C for 10 min. Then, 2-μl aliquots of reaction cocktails were loaded on PEI cellulose plates that were developed with a LiCl mobile phase. ADP formation was then measured by liquid scintillation counting. The results shown represent the average of two assays. (C) Complex formation between SYS and YSY and denatured luciferase. Aliquots (100 μl) of Hsp40 at 100 nM in PBS were immobilized in the wells of 96-well microtiter plates. Then, chemically denatured luciferase (0.4 μg) was added to wells. ELISA was then used to detect the luciferase that was retained in washed wells. Values are expressed as a percentage of the level of complex formation between immobilized Ydj1 and denatured luciferase. Results are average of three trials ± SD. The asterisk (^*) denotes values that were significantly different from the control with a P > 0.05.