P C Dunlop

[38] Diphthamide in elongation factor 2: ADP-ribosylation, purification, and properties

Publisher Summary 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.

Derepression of asparaginase II during exponential growth of Saccharomyces cerevisiae on ammonium ion

Abstract The biosynthesis of asparaginase II in Saccharomyces cerevisiae is sensitive to nitrogen catabolite repression. In cell cultures growing in complete ammonia medium, asparaginase II synthesis is repressed in the early exponential phase but becomes derepressed in the midexponential phase. When amino acids such as glutamine or asparagine replace ammonium ion in the growth medium, the enzyme remains repressed into the late exponential phase. The three nitrogen compounds permit a similar rate of cell growth and are assimilated at nearly the same rate. In the early exponential phase the internal amino acid pool is larger in cells growing with glutamine or asparagine than in cells growing with ammonium sulfate as the sole source of nitrogen.

Anion-exchange chromatography of proteins on AG MP-1 using high-performance liquid chromatography equipment☆

That the macroporous anion-exchange resin AG MP-1 can be used with HPLC equipment and common aqueous buffers for the chromatography of proteins is shown. The utility of this system is illustrated by the partial purification and complete resolution of the three protein synthesis elongation factors from each other, starting with a crude extract of Escherichia coli. The factors were purified 10- to 30-fold in a yield of 50 to 90% with a single 60-min chromatographic program of increasing NaCl concentration. Other proteins from various biological sources were purified with similar results. Thus, it appears that AG MP-1 is useful, at least in some applications, for the rapid, reproducible, and economical purification of proteins using HPLC equipment.