FIG. 4.

Labeling of prodrug hydrolase and recombinant CatA with ¹⁴C-labeled GS-7340 and [³H]DFP. Labeling of prodrug hydrolase, recombinant CatA, and concanavalin A with ¹⁴C-labeled GS-7340 and [³H]DFP was performed with both native (A) and denatured (B) enzymes as described in the text. Proteins were analyzed by SDS-PAGE and visualized by exposure to X-ray film. Incubation mixtures contained 1.0 mM ¹⁴C-labeled GS-7340 and prodrug hydrolase (lanes 1 and 7), recombinant CatA (lanes 2 and 8) or concanavalin A (lanes 5 and 11); 25 μM [³H]DFP or prodrug hydrolase (lanes 3 and 9), and recombinant CatA (lanes 4 and 10) or concanavalin A (lanes 6 and 12). (C) Aliquots of recombinant CatA (50 ng; lane 2) and 1 μg each of the Q-Source (lane 3), HIC (lane 4), and ConA (lane 5) pools were resolved using SDS-PAGE, transferred to a PVDF membrane, incubated with human cathepsin-A antibody, and visualized with a fluorescently labeled secondary antibody. M, molecular mass marker.