FIG. 5.
Pulse-labeling and immunoprecipitation analysis of HemA[WT] and HemA[KK] expressed from F′ plasmids in S. typhimurium. The positions of native HemA, the truncated HemA protein from hemA60, and HemA1–416-LacZ are all indicated. Cultures were the same as those analyzed by Western blotting in Fig. 4. One milliliter of each culture (OD600 = 0.4) was pulse-labeled with Tran35S-label (ICN) for 5 min and then chased for 2 min with unlabeled l-methionine (1.3 mM) and l-cystine (0.6 mM). Protein extracts were prepared, immunoprecipitated with anti-HemA MAb H17, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results were quantitated by using a PhosphorImager and ImageQuant software. Lanes: a, LT-2 wild type; b, TE 518 hemA60 recA; c, TE7619 hemA60 recA/F′ hemA1–416[WT]-lac [pr]; d, TE7620 hemA60 recA/F′ hemA[WT]; e, TE7621 hemA60 recA/F′ hemA1–416[KK]-lac [pr]; f, TE7622 hemA60 recA/F′ hemA[KK].