Purification and Properties of an Altered Form of Elongation Factor 2 from Mutant Cells Resistant to Intoxication by Diphtheria Toxin

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Abstract
Elongation factor 2 (EF-2) was isolated from wild type and mutant polyoma virus-transformed baby hamster kidney cells resistant to intoxication by diphtheria toxin. Cells were grown as tumors in hamsters; EF-2 was purified from tissue homogenates by column chromatography. The forms of EF-2 chromatographed identically in Whatman DE-52 DEAE-cellulose, Sephadex DEAE-A50 and Sephacryl S-200 resins. Wild-type and mutant forms of EF-2 eluted from phosphocellulose at 0.16 and 0.24 M KCl, respectively. Both forms of EF-2 migrated in sodium dodecyl sulfate/polyacrylamide gels as a single band with a MW of 93,000 and produced identical 125I-labeled tryptic peptide maps. Additional labeled tryptic peptides were seen when wild-type EF-2 was ADP-ribosylated by fragment A of diphtheria toxin. The purified mutant protein was totally resistant to ADP-ribosylation and was not transformed into an ADP-ribosylatable form in a posttranslational modification system in vitro, indicating that resistance to ADP-ribosylation results from a mutation in the structural gene for EF-2.