Localization of the sites of ADP-ribosylation and GTP binding in the eukaryotic elongation factor EF-2.
Tryptic cleavage of EF-2, molecular mass 93 kDa, produced an 82-kDa polypeptide and a 10-kDa fragment, which was further degraded. By a slower reaction the 82-kDa polypeptide was gradually split into a 48-kDa and a 34-kDa fragment. Similarly, treatment with chymotrypsin resulted in the formation of an 82-kDa polypeptide and a small fragment. In contrast to the tryptic 82-kDa polypeptide the corresponding chymotryptic cleavage product was relatively resistant to further attack. The degradation of the 82-kDa polypeptide with either trypsin or chymotrypsin was facilitated by the presence of guanosine nucleotides, indicating a conformational shift in native EF-2 upon nucleotide binding. No effect was observed in the presence of ATP, indicating that the effect was specific for guanosine nucleotides. After affinity labelling of native EF-2 with oxidized [3H]GTP and subsequent trypsin treatment the radioactivity was recovered in the 48-kDa polypeptide showing that the GTP-binding site was located within this part of the factor. Correspondingly, tryptic degradation of EF-2 labelled with [14C]NAD+ in the presence of diphtheria toxin showed that the site of ADP-ribosylation was within the 34-kDa polypeptide. By cleavage with the tryptophan-specific reagent N-chlorosuccinimide the site of ADP-ribosylation could be located at a distance of 40-60 kDa from the GTP-binding site and about 4-11 kDa from the nearest terminus.
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000345:Affinity Labels; D001665:Binding Sites; D002918:Chymotrypsin; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D009702:Nucleoside Diphosphate Sugars / Q000378:metabolism*; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000302:isolation & purification*; D010446:Peptide Fragments / Q000302:isolation & purification; D013329:Structure-Activity Relationship; D013388:Succinimides; D014357:Trypsin
D000345:Affinity Labels; D009702:Nucleoside Diphosphate Sugars; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D010446:Peptide Fragments; D013388:Succinimides; C015752:N-chlorosuccinimide; D000246:Adenosine Diphosphate Ribose; D006160:Guanosine Triphosphate; D002918:Chymotrypsin; D014357:Trypsin