Immobilization of reticulocyte elongation factor EF-2.
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Conditions are described whereby the ADP-ribosylation (from NAD+) of reticulocyte elongation factor EF-2, catalyzed by diphtheria toxin, is essentially complete and whereby the reverse of this process may be carried out with recovery of 60--70% of the original EF-2 activity. Both reactions proceed well at room temperature. The reverse reaction is much slower than the ADP-ribosylation process and requires high nicotinamide concentrations. For the reverse reaction to occur at a significant rate it is necessary to lower the pH to 6.5 (from the 7.5 used for the forward reaction). NAD+ covalently linked to agarose may replace NAD+ in the diphtheria toxin reaction. The characteristics of this reaction are similar to those of the reaction employing free NAD+ except that the velocity is reduced and the concentration of NAD+ moieties greatly increased. NAD+ immobilized on agarose through the C-8 of the adenine ring is a superior substrate compared with NAD+ linked to agarose via its periodate-oxidized ribose moieties. Preliminary experiments indicate that reversal of this latter reaction with recovery of biological activity may be possible. (+info)
Isolation of adenosine 5'-diphosphate-D-glycero-D-mannoheptose. An intermediate in lipopolysaccharide biosynthesis of Shigella sonnei.
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From a Shigella sonnei R mutant which incorporates into its cell wall lipopolysaccharide D-glycero-D-mannoheptose and contains no L-glycero-D-mannoheptose, a nucleotide-linked sugar was isolated and identified as adenosine 5'-diphosphate-D-glycero-d-mannoheptose by chemical and chromatographic analysis. This intermediary compound is assumed to play a role in heptose biosynthesis of Enterobacteria. (+info)
Isolation and properties of the trypsin-derived ADP-ribosyl peptide from diphtheria toxin-modified yeast elongation factor 2.
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We have developed a method for the purification in micromole amounts of the trypsin-derived ADP-ribosyl peptide from diphtheria toxin-modified yeast elongation factor 2 (EF-2). EF-2 was partially purified (15 to 20% purity) by ammonium sulfate precipitation and DEAE-Sephadex chromatography. After [3H]ADP-ribosylation by [3H]nad+ and diphtheria toxin, EF-2 was digested with trypsin and a homogeneous [3H]ADP-ribosyl peptide was isolated by chromatography on DEAE-Sephadex and dihydroxyboryl-substituted cellulose. Based on the amount of ADP-ribose acceptor activity in the crude extract, the overall yield of the peptide was 35%. The yeast peptide contains an unusual amino acid (X) which is the site of ADP ribosylation and is apparently identical to the amino acid reported from rat liver EF-2 by Robinson et al. (Robinson, E. A., Hendriksen, O., and Maxwell, E.S. (1974) J. Biol. Chem. 249, 5088-5093). The sequence of the 15-residue yeast peptide was determined to be: Val-Asn-Ile-Leu-Asp-Val-Thr-Leu-His-Ala-Asp-Ala-Ile-X-Arg. The 11 COOH-terminal residues of this peptide and of the homologous 15-residue peptide reported by Maxwell and co-workers from rat liver EF-2 are identical. (+info)
Interactions of adenosine diphosphate-ribosylated elongation factor 2 with ribosomes.
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The binding of adenosine diphosphate-ribosylated elongation factor 2 (ADPRib-EF-2) to ribosomes was inhibited both in the presence and absence of GTP in proportion to the amounts of unmodified EF-2 added. Concomitant with this inhibition, an increase in the activity of ribosome-bound EF-2 in polyphenylalanine synthesis was observed. On the other hand, the addition of ADPRib-EF-2 reduced the rate of poly(Phe) synthesis observed in the presence of a saturating amount of EF-2 and increased the amount of EF-2 required for the half-maximal rate of poly(Phe) synthesis. Phe-tRNA, nonenzymatically bound to the ribosome in the presence of poly(U), inhibited the subsequent binding of ADPHRib-EF-2. The same ribosomal population appeared to preferentially bind either aminoacyl-tRNA or ADPRib-EF-2. The Scatchard plot of the binding of ADPRib-EF-2 to the ribosome in the presence of GTP revealed the presence of two ribosomal binding sites (or ribosomal populations) with apparent different affinities for the modified factor (K371 degrees d,1 = 6.6 nM and K37 degrees d,2 = 126 nM). At saturating concentrations of ADPRib-EF-2, a maximum of about 1 molecule of the factor was bound per ribosome. The binding of ADPRib-EF-2 to the ribosome was stimulated by GTP. The binding of radioactive GTP to the ribosome was observed concomitantly with the binding of ADPRib-EF-2. One mole of GTP was bound per mole of ADPRib-EF-2. No significant difference could be found in the binding of GTP to ribosome required in the presence of either EF-2 or ADPRib-EF-2. The binding of ADPRib-EF-2 to the ribosome required the presence of Mg2+ and reached a maximum at 5 mM. The binding was greatest at K+ concentrations below 20 mM. ADPRib-EF-2 was bound primarily to the large ribosomal subunit. A slight, but reproducible binding to the 40 S subunit was also observed. The addition of 40 S to 60 S subunits stimulated the binding of ADPRib-EF-2. GTP displayed a stimulatory effect on the binding only in the presence of recombined subunits. Human ADPRib-EF-2 was bound to rat liver ribosomes as efficiently as to human tonsil ribosomes, while the binding to Escherichia coli ribosomes was insignificant. (+info)