TY - JOUR
T1 - [38] Diphthamide in Elongation Factor 2
T2 - ADP-Ribosylation, Purification, and Properties
AU - Bodley, James W.
AU - Dunlop, Patricia C.
AU - Vanness, Brian G.
PY - 1984/1/1
Y1 - 1984/1/1
N2 - 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.
AB - 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.
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U2 - 10.1016/0076-6879(84)06040-7
DO - 10.1016/0076-6879(84)06040-7
M3 - Article
C2 - 6387379
AN - SCOPUS:0021161567
SN - 0076-6879
VL - 106
SP - 378
EP - 387
JO - Methods in enzymology
JF - Methods in enzymology
IS - C
ER -