scholarly journals Purification of elongation factor 2 from human placenta and evidence of its fragmentation patterns in various eukaryotic sources

1987 ◽  
Vol 244 (2) ◽  
pp. 337-344 ◽  
Author(s):  
A Giovane ◽  
L Servillo ◽  
L Quagliuolo ◽  
C Balestrieri
Keyword(s):  
Affinity Chromatography ◽  
Human Placenta ◽  
Degradation Product ◽  
Diphtheria Toxin ◽  
Intact Cell ◽  
Elongation Factor ◽  
Purification Step ◽  
Elongation Factor 2 ◽  
Fragmentation Patterns ◽  
Chromatography Step

While preparing human placenta elongation factor 2 (EF-2), whose purification and some molecular properties are reported, we noticed the presence of numerous protein fractions which did not have EF-2 activity, but were ADP-ribosylated by diphtheria toxin in the presence of NAD+. All these proteins, like EF-2, were selectively retained by a heparin-Sepharose column, which we used as an affinity-chromatography step. This was also observed when EF-2 was prepared, by this purification step, from other sources, i.e. ox liver and two species of yeasts. In order to assess whether these proteins were a degradation product of EF-2, independent proteins or a mixture of both, they were analysed by subjecting them, after [14C]ADP-ribosylation, to exhaustive trypsinolysis. Only one radioactive peptide was found, thus suggesting that those proteins originate from EF-2 by some proteolytic process. Our findings indicate that this proteolysis does not occur after cell disruption, but is more or less active in the intact cell, depending on the system considered.

Interaction of diphtheria toxin fragment A and of elongation factor 2 with Cibacron blue

1987 ◽  
Vol 7 (9) ◽  
pp. 737-743 ◽  
Author(s):  
Fioretta Rambelli ◽  
Maurizio Brigotti ◽  
Simonetta Sperti ◽  
Lucio Montanaro
Keyword(s):  
Affinity Chromatography ◽  
Dissociation Constant ◽  
Diphtheria Toxin ◽  
Equilibrium Dialysis ◽  
Elongation Factor ◽  
Competitive Inhibitor ◽  
Cibacron Blue ◽  
Elongation Factor 2 ◽  
Blue Sepharose ◽  
Spectral Titration

Diphtheria toxin fragment A interacts with Cibacron blue in solution, although it is not retained by blue Sepharose columns. Difference spectral titration of fragment A with the dye gives a dissociation constant of the order of 10−5 M and a 1:1 stoichiometry for the complex. In equilibrium dialysis experiments Cibacron blue behaves as a competitive inhibitor of the binding of NAD to diphtheria toxin fragment A. The dye inhibits in a non-competitive way the fragment A-catalysed transfer of ADP-ribose from NAD to elongation factor 2 (EF2). By affinity chromatography on blue Sepharose a binding of EF2 and of ADP-ribosyl-EF2 with the dye is also demonstrated. GDP, GTP and GDP(CH2)P are able to displace EF2 from blue Sepharose.

In vitro biosynthesis of diphthamide, studied with mutant Chinese hamster ovary cells resistant to diphtheria toxin

1984 ◽  
Vol 4 (4) ◽  
pp. 642-650
Author(s):  
T J Moehring ◽  
D E Danley ◽  
J M Moehring
Keyword(s):  
Chinese Hamster Ovary ◽  
Diphtheria Toxin ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Elongation Factor ◽  
Synthetase Activity ◽  
Post Translational Modification ◽  
Ovary Cells ◽  
Elongation Factor 2

Diphthamide, a unique amino acid, is a post-translational derivative of histidine that exists in protein synthesis elongation factor 2 at the site of diphtheria toxin-catalyzed ADP-ribosylation of elongation factor 2. We investigated steps in the biosynthesis of diphthamide with mutants of Chinese hamster ovary cells that were altered in different steps of this complex post-translational modification. Biochemical evidence indicates that this modification requires a minimum of three steps, two of which we accomplished in vitro. We identified a methyltransferase activity that transfers methyl groups from S-adenosyl methionine to an unmethylated form of diphthine (the deamidated form of diphthamide), and we tentatively identified an ATP-dependent synthetase activity involved in the biosynthesis of diphthamide from diphthine. Our results are in accord with the proposed structure of diphthamide (B. G. VanNess, et al., J. Biol. Chem. 255:10710-10716, 1980).

Eukaryotic elongation factor-2 (eEF-2) activity in bovine mammary tissue in relation to milk protein synthesis

2002 ◽  
Vol 69 (2) ◽  
pp. 205-212 ◽  
Author(s):  
CLAUS T. CHRISTOPHERSEN ◽  
JAKOB KARLSEN ◽  
METTE O. NIELSEN ◽  
BENT RIIS
Keyword(s):  
Skeletal Muscle ◽  
Protein Synthesis ◽  
Mammary Gland ◽  
Diphtheria Toxin ◽  
Milk Protein ◽  
Elongation Factor ◽  
Mammary Tissue ◽  
Limiting Factor ◽  
Elongation Factor 2 ◽  
High Level

The amount of protein synthesis translational elongation factor 2 (eEF-2) was estimated employing diphtheria toxin-dependent ADP-ribosylation in samples prepared from small amounts of tissue from mammary gland, skeletal muscle and liver from lactating dairy cows. A very high level of ADP-ribosylatable eEF-2 was found in mammary gland, amounting to 20-times the level found in liver and 50-times the level found in skeletal muscle. This obviously reflects the high protein synthesis activity in mammary tissue. To our knowledge, similar high activities have previously been reported only for cancer cells. A close linear relationship was found between the amount of diphtheria-toxin catalysed ADP-ribosylated eEF-2 and protein and casein output in milk from cows in late lactation. This strongly suggests that eEF-2 may be a limiting factor in milk protein synthesis.

scholarly journals Diphtheria toxin-resistant mutants of Saccharomyces cerevisiae.

1985 ◽  
Vol 5 (12) ◽  
pp. 3357-3360 ◽  
Author(s):  
J Y Chen ◽  
J W Bodley ◽  
D M Livingston
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Diphtheria Toxin ◽  
Elongation Factor ◽  
Selection Procedure ◽  
Prolonged Storage ◽  
Elongation Factor 2 ◽  
Resistant Phenotype ◽  
Complementation Groups ◽  
Mendelian Character

We developed a selection procedure based on the observation that diphtheria toxin kills spheroplasts of Saccharomyces cerevisiae (Murakami et al., Mol. Cell. Biol. 2:588-592, 1982); this procedure yielded mutants resistant to the in vitro action of the toxin. Spheroplasts of mutagenized S. cerevisiae were transformed in the presence of diphtheria toxin, and the transformed survivors were screened in vitro for toxin-resistant elongation factor 2. Thirty-one haploid ADP ribosylation-negative mutants comprising five complementation groups were obtained by this procedure. The mutants grew normally and were stable to prolonged storage. Heterozygous diploids produced by mating wild-type sensitive cells with the mutants revealed that in each case the resistant phenotype was recessive to the sensitive phenotype. Sporulation of these diploids yielded tetrads in which the resistant phenotype segregated as a single Mendelian character. From these observations, we concluded that these mutants are defective in the enzymatic steps responsible for the posttranslational modification of elongation factor 2 which is necessary for recognition by diphtheria toxin.

scholarly journals Nucleotide binding to elongation factor 2 inactivated by diphtheria toxin

FEBS Letters ◽  
1986 ◽  
Vol 208 (2) ◽  
pp. 217-220 ◽  
Author(s):  
Gary Burns ◽  
Abraham K. Abraham ◽  
Anni Vedeler
Keyword(s):  
Diphtheria Toxin ◽  
Elongation Factor ◽  
Nucleotide Binding ◽  
Elongation Factor 2

Diphtheria toxin-resistant mutants of Saccharomyces cerevisiae

1985 ◽  
Vol 5 (12) ◽  
pp. 3357-3360
Author(s):  
J Y Chen ◽  
J W Bodley ◽  
D M Livingston
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Diphtheria Toxin ◽  
Elongation Factor ◽  
Selection Procedure ◽  
Prolonged Storage ◽  
Elongation Factor 2 ◽  
Resistant Phenotype ◽  
Complementation Groups ◽  
Mendelian Character

We developed a selection procedure based on the observation that diphtheria toxin kills spheroplasts of Saccharomyces cerevisiae (Murakami et al., Mol. Cell. Biol. 2:588-592, 1982); this procedure yielded mutants resistant to the in vitro action of the toxin. Spheroplasts of mutagenized S. cerevisiae were transformed in the presence of diphtheria toxin, and the transformed survivors were screened in vitro for toxin-resistant elongation factor 2. Thirty-one haploid ADP ribosylation-negative mutants comprising five complementation groups were obtained by this procedure. The mutants grew normally and were stable to prolonged storage. Heterozygous diploids produced by mating wild-type sensitive cells with the mutants revealed that in each case the resistant phenotype was recessive to the sensitive phenotype. Sporulation of these diploids yielded tetrads in which the resistant phenotype segregated as a single Mendelian character. From these observations, we concluded that these mutants are defective in the enzymatic steps responsible for the posttranslational modification of elongation factor 2 which is necessary for recognition by diphtheria toxin.

Sign in / Sign up

Export Citation Format

Share Document