[38] Diphthamide in Elongation Factor 2: ADP-Ribosylation, Purification, and Properties

This chapter describes ADP-ribosylation, purification, and properties of diphthamide in elongation factor 2 (EF-2). Diphthamide is a posttranslational derivative of histidine which occurs in a single location in protein synthesis EF-2. The occurrence of diphthamide was first found through the study of a second post translational modification reaction, the ADP-ribosylation of EF-2 by diphtheria toxin. The purification and study of EF-2 and diphthamide revolve around the specificity and nature of the diphtheria toxin reaction. With the aid of toxin and radioactive NAD⁺ (either [adenine-2,8-³H]NAD⁺ or [³²P]NAD⁺) it is possible to specifically radiolabel EF-2 either in crude extracts or in purified protein preparations. The reaction is specific in that only a single eukaryotic polypeptide is labeled and there are no toxinspecific ADP-ribose acceptors in eubacterial extracts. The toxin reaction also provides a direct measure of the quantity of EF-2 because the reaction is both irreversible and stoichiometric. The preparation of large quantities of ADP-ribosyl EF-2 is facilitated by the partial purification of the protein prior to its modification by diphtheria toxin. When performed on a small scale, a single chromatography step is sufficient to purify the protein approximately 10- to 15-fold and to remove interfering substances so that EF-2 can be ADP-ribosylated without dilution.