Localization of the sites of ADP-ribosylation and GTP binding in the eukaryotic elongation factor EF-2

Eur J Biochem. 1985 Apr 15;148(2):299-304. doi: 10.1111/j.1432-1033.1985.tb08839.x.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Diphosphate Ribose / metabolism*
  • Affinity Labels
  • Binding Sites
  • Chymotrypsin
  • Guanosine Triphosphate / metabolism*
  • Hydrolysis
  • Nucleoside Diphosphate Sugars / metabolism*
  • Peptide Elongation Factor 2
  • Peptide Elongation Factors / isolation & purification*
  • Peptide Fragments / isolation & purification
  • Structure-Activity Relationship
  • Succinimides
  • Trypsin

Substances

  • Affinity Labels
  • Nucleoside Diphosphate Sugars
  • Peptide Elongation Factor 2
  • Peptide Elongation Factors
  • Peptide Fragments
  • Succinimides
  • N-chlorosuccinimide
  • Adenosine Diphosphate Ribose
  • Guanosine Triphosphate
  • Chymotrypsin
  • Trypsin