Effect of oxidative stress on in vivo ADP-ribosylation of eukaryotic elongation factor 2.
Different lines of evidence indicate that eukaryotic elongation factor 2 (eEF2) can be ADP-ribosylated endogenously. The physiological significance of this reaction has, however, remained unclarified. In order to address this issue we investigated the in vivo ADP-ribosylation of eEF2 and the effect of oxidative stress thereon. The investigation revealed that the endogenous ADP-ribosylation of eEF2 is complex and can take place in K562 cell lysates either under the action of endogenous transferase from [adenosine-14C]NAD or by direct binding of free [14C]ADP-ribose. These two types of ADP-ribosylation were distinguished by use of different treatments based on the chemical stability of the respective bonds formed. Under standard culture conditions, in vivo labeling of eEF2 in the presence of [14C]adenosine was reversed to about 65% in the presence of diphtheria toxin and nicotinamide. This finding implied that the modification that took place under physiological circumstances was, mainly, of an enzymic nature. On the other hand, H2O2-promoted oxidative stress gave rise to a nearly two-fold increase in the extent of in vivo labeling of eEF2. This was accompanied by a loss of eEF2 activity in polypeptide chain elongation. Oxidative stress specifically inhibited the subsequent binding of free ADP-ribose to eEF2. The results thus provide evidence that endogenous ADP-ribosylation of eEF2 can also take place by the binding of free ADP-ribose. This nonenzymic reaction appears to account primarily for in vivo ADP-ribosylation of eEF2 under oxidative stress.
Bektaş
Muhammet
M
Department of Biophysics, Istanbul Faculty of Medicine, Istanbul University, 34390 Capa, Istanbul, Turkey. muhbektas@hotmail.com
Akçakaya
Handan
H
Aroymak
Aysin
A
Nurten
Rüstem
R
Bermek
Engin
E
D036002:ADP Ribose Transferases / Q000378:metabolism*; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D001426:Bacterial Proteins / Q000494:pharmacology; D002250:Carbon Radioisotopes / Q000378:metabolism / Q000494:pharmacology; D004167:Diphtheria Toxin / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D020014:K562 Cells; D009243:NAD / Q000378:metabolism*; D018384:Oxidative Stress; D020652:Peptide Elongation Factor 2 / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects
D001426:Bacterial Proteins; D002250:Carbon Radioisotopes; D004167:Diphtheria Toxin; D020652:Peptide Elongation Factor 2; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D006861:Hydrogen Peroxide; D036002:ADP Ribose Transferases