Endogenous ADP-ribosylation of elongation factor 2 in polyoma virus-transformed baby hamster kidney cells.
Polyoma virus-transformed baby hamster kidney (pyBHK) cells were cultured in medium containing [32P]orthophosphate and 10% (vol/vol) fetal bovine serum. A 32P-labeled protein with an apparent molecular mass of 97 kDa was immunoprecipitated from cell lysates with antiserum to ADP-ribosylated elongation factor 2 (EF-2). The 32P labeling of the protein was enhanced by culturing cells in medium containing 2% serum instead of 10% serum. The 32P label was completely removed from the protein by treatment with snake venom phosphodiesterase and the digestion product was identified as [32P]AMP, indicating the protein was mono-ADP-ribosylated. HPLC analysis of tryptic peptides of the 32P-labeled 97-kDa protein and purified EF-2, which was ADP-ribosylated in vitro with diphtheria toxin fragment A and [32P]NAD, demonstrated an identical labeled peptide in the two proteins. The data strongly suggest that EF-2 was endogenously ADP-ribosylated in pyBHK cells. Maximum incorporation of radioactivity in EF-2 occurred by 12 hr and remained constant over the subsequent 12 hr. It was estimated that 30-35% of the EF-2 was ADP-ribosylated in cells cultured in medium containing 2% serum. When 32P-labeled cultures were incubated in medium containing unlabeled phosphate, the 32P label was lost from the EF-2 within 30 min.
Fendrick
J L
JL
Department of Microbiology and Immunology, University of Rochester, NY 14642.
Iglewski
W J
WJ
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D002461:Cell Line, Transformed; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media; D007700:Kinetics; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000378:metabolism*; D010455:Peptides / Q000032:analysis; D011120:Polyomavirus / Q000502:physiology*; D011233:Precipitin Tests; D011506:Proteins / Q000032:analysis
D003470:Culture Media; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D010455:Peptides; D011506:Proteins; D000246:Adenosine Diphosphate Ribose