Purification and properties of an altered form of elongation factor 2 from mutant cells resistant to intoxication by diphtheria toxin.
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 and EF-2 was purified from tissue homogenates by column chromatography. Both forms of EF-2 chromatograph identically in Whatman DE-52 DEAE-cellulose, Sephadex DEAE-A50 and Sephacryl S-200 resins. However, wild-type and the mutant form of EF-2 elute from phosphocellulose at 0.16 M and 0.24 M KCl respectively. Both forms of EF-2 migrate in sodium dodecyl sulfate/polyacrylamide gels as a single band with an Mr of 93000 and produce identical 125I-labeled tryptic peptide maps. However, additional labeled tryptic peptides are seen when wild-type EF-2 is ADP-ribosylated by fragment A diphtheria toxin. The purified mutant protein is totally resistant to ADP-ribosylation and cannot be 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.
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002472:Cell Transformation, Viral; D006224:Cricetinae; D004167:Diphtheria Toxin / Q000633:toxicity*; D004351:Drug Resistance; D007668:Kidney / Q000032:analysis; D009154:Mutation; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000302:isolation & purification* / Q000378:metabolism; D011120:Polyomavirus
D004167:Diphtheria Toxin; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D000246:Adenosine Diphosphate Ribose