Figure 7.

Cyclosporin A Inhibition of Cyp5 PPIase and Protein Refolding Activities.

(A) A Coomassie blue–stained SDS–polyacrylamide gel with purified GST and GST–Cyp5_19–201 fusion used in PPIase and protein refolding assays. Numbers indicate molecular weight markers in kilodaltons.

(B) Inhibition of Cyp5 PPIase activity by different cyclosporin A concentrations. GST–Cyp5_19–201 (3 nM) was preincubated with varying concentrations of cyclosporin A, and PPIase activity was measured in 35 mM Hepes buffer, pH 7.8, at 10°C.

(C) Cyp5 catalysis of slow protein refolding of RNAse T1 (0.7 μM) and inhibition by cyclosporin A. The increase of fluorescence at 320 nm is shown as a function of the time of protein refolding. Curve 1 shows 77 nM Cyp5 without cyclosporin A; curve 2, 77 nM Cyp5 with 50 nM cyclosporin A; curve 3, 77 nM Cyp5 with 100 nM cyclosporin A; and curve 4, without Cyp5 and cyclosporin A. GST did not have an effect on refolding (not shown). Measurements were performed in 35 mM Hepes buffer, pH 7.8, at 10°C.