FIG. 1.
Recombinant IRP1S138E fails to function as aconitase. (A) SDS-PAGE results of purified human recombinant (rec.) His-tagged wild-type IRP1 (wt), IRP1S138A, and IRP1S138E. The proteins were visualized by staining with Coomassie brilliant blue. (B) Purified recombinant proteins (80 ng) were analyzed by EMSA with a 32P-labeled IRE probe in the absence (top) or presence (bottom) of 2% 2-mercaptoethanol (2-ME). The positions of specific His-IRP1/IRE complexes and excess free probe are indicated by arrows. (C) Aconitase assay prior to (white bars) and after (black bars) treatment with ferrous sulfate-cysteine. Values correspond to results from triplicate samples; the aconitase activity is expressed as milliunits per microgram of recombinant protein. **, P < 0.01 versus values for the control (Student's t test).