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

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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.