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At least 19 recordsLinked to original sources
PubMed1988

Purification and characterization of peptide-elongation factor 2 (aEF-2) from an extremely halophilic archaebacterium Halobacterium halobium.

A procedure is described for the purification of the archaebacterial peptide-elongation factor 2 (aEF-2) from an extremely halophilic archaebacterium Halobacterium halobium. The enrichment was about 530-fold, the obtained preparation practically homogeneous as judged by SDS-PAGE. The poly(U)-dependent poly(Phe) synthesis was completely dependent on aEF-2 in the presence of partially purified aEF-1, and the activity was equivalent to a poly(Phe)-synthesizing system containing unfractionated S-100 enzymes. aEF-2 consists of a single peptide with a relative molecular mass of 125,000 +/- 3000 and 100,000 +/- 3000 as determined by SDS-PAGE and gel filtration on Sephadex G-200 respectively. The isoelectric point was 5.7. The amino acid composition analysis indicated the predominance of acidic amino acids (aspartic acid and glutamic acid) and the low content of hydrophobic amino acid (phenylalanine) as compared with those of eukaryotes and prokaryotes. The factor was stable in a pH range from 6 to 8. 2-Mercaptoethanol and GTP but not GDP markedly protected aEF-2 from heat denaturation at 52 degrees C. aEF-2 became inactivated and insensitive to ADP-ribosylation by diphtheria toxin at low ionic strength but could be renatured by increasing ionic strength. Obviously higher concentrations of salts contribute to the conformational stability of aEF-2.

Adenosine Diphosphate Ribose↗
PubMed1989

Sequence analysis of the peptide-elongation factor EF-2 gene, downstream from those of ribosomal proteins H-S12 and H-S7, from the archaebacterial extreme halophile, Halobacterium halobium.

The gene for the peptide-elongation factor 2 (EF-2) was cloned from the archaebacterial extreme halophile Halobacterium halobium and sequenced. The 1013 nucleotides upstream from this gene was two open reading frames similar to ribosomal proteins S12 and S7 from Escherichia coli. Sequence alignment studies showed the halobacterial elongation factor 2 to be equivalent to eukaryotic EF-2 and eubacterial EF-G. Sequence similarity to the eukaryotic elongation factor was much higher than to the eubacterial factor. Conserved sequence regions were present within the factor and are likely to constitute functionally important domains. These include the sites of GTP binding and ADP ribosylation by diphtheria toxin.

Animals↗
PubMed1994

Role of elongation factor 2 in regulating peptide-chain elongation in the heart.

Cardiac muscles of experimentally induced diabetic rats show a progressive decrease in the rate of protein synthesis. The decline in protein synthesis is associated with decreases in both the number and efficiency of cardiac ribosomes. In hearts from 48 h diabetic rats, the decrease in protein synthesis was accounted for solely by a 28% reduction in the ribosome content. In contrast, the inhibition of protein synthesis in hearts from 72 h diabetic rats resulted from a reduction in both the ribosome content (28%) and the translational efficiency (30%). The decreased translational efficiency was not associated with an increase of RNA in ribosomal subunits, indicating the defect resulted from an inhibition of peptide-chain elongation/termination. Diabetes of 72 h duration resulted in a 37% inhibition in the rate of peptide-chain elongation. The decreased rate of peptide-chain elongation was associated with a 66% reduction in the amount of elongation factor 2 (EF-2). Treatment of diabetic rats with insulin for 3 days was sufficient to reverse the effects of 72 h diabetes on protein synthesis, RNA content, and translational efficiency. Also, insulin therapy increased the EF-2 content of diabetic rats to control values. These studies suggest that decreased EF-2 content is a molecular mechanism for the impaired rates of peptide-chain elongation in diabetes.

Animals↗
PubMed1990

Phosphorylation of elongation factor 2: a key mechanism regulating gene expression in vertebrates.

Elongation factor 2 (eEF-2) is a 100-kD protein that catalyzes the ribosomal translocation reaction, resulting in the movement of ribosomes along mRNA. eEF-2 is the target for a very specific Ca2+/calmodulin-dependent eEF-2 kinase. Phosphorylation of eEF-2 makes it inactive in translation, which suggests that protein synthesis can be regulated by Ca2+ through eEF-2 phosphorylation. Recent data demonstrate that eEF-2 phosphorylation can be involved in cell-cycle regulation and other processes where changes of intracellular Ca2+ concentration induce a new physiological state of a cell. The main role of eEF-2 phosphorylation in these processes is temporary inhibition of overall translation in response to transient elevation of the Ca2+ concentrations in the cytoplasm. Temporary inhibition of translation may trigger the transition of a cell from one physiologic state into another because of the disappearance of short-lived repressors and thus the activation of expression of new genes.

Adrenal Gland Neoplasms↗
PubMed1998

Identification of elongation factor 2 as the essential protein targeted by sordarins in Candida albicans.

The target for sordarins in Candida albicans has been elucidated. Kinetic experiments of sordarin inhibition as well as displacement experiments showed that the formation of a sordarin-target complex follows a reversible mechanism. Binding of tritiated drug to the target is enhanced in the presence of ribosomes. Isolation of the target by classical protein purification methods has allowed us to identify it as elongation factor 2. This is in agreement with the nature of sordarin derivatives as specific inhibitors of the elongation cycle within protein synthesis in yeasts.

Amino Acid Sequence↗
PubMed1983

Yeast ribosome core particles deficient in acidic proteins L44 and L45 and their activity in reconstitution experiments.

The yeast ribosome core particles partly depleted of acidic proteins, L44 and L45, were isolated and their activity was examined using a highly purified protein synthesizing system. It was shown that both the phenylalanine polymerization reaction and the elongation factor 2-dependent GTP hydrolysis were stimulated by the addition of the extracted acidic protein fraction. The results obtained clearly indicate a functional role of these proteins in the elongation step of eukaryotic protein synthesis. The essential parameters of ribosome reconstitution experiments are discussed.

Fungal Proteins↗
PubMed1988

Inhibition of protein synthesis by a Vero toxin (VT2 or Shiga-like toxin II) produced by Escherichia coli O157:H7 at the level of elongation factor 1-dependent aminoacyl-tRNA binding to ribosomes.

A Vero toxin (VT2 or Shiga-like toxin II) from Escherichia coli O157:H7 was shown to inhibit protein synthesis in a rabbit reticulocyte lysate, but not in wheat germ or Ercherichia coli lysates. The toxin, VT2, inactivated 60S ribosomal subunits of rabbit reticulocytes. The site of inhibition of protein synthesis by VT2 was shown to be elongation factor 1-dependent aminoacyl-tRNA binding to ribosomes. VT2 did not affect Met-tRNAf binding to ribosomes, non-enzymatic binding of aminoacyl-tRNA to ribosomes, peptide bond formation or translocation.

Animals↗
PubMed1982

The activity of the acidic phosphoproteins from the 80 S rat liver ribosome.

The selective removal of acidic phosphoproteins from the 80 S rat liver ribosome was accomplished by successive alcohol extractions at low salt concentration. The resulting core ribosomes lost over 90% of their translation activity and were unable to support the elongation factor 2 GTPase reaction. Both activities were partially restored when the dialyzed extracts were added back to the core ribosome. The binding of labeled adenosine diphosphoribosyl-elongation factor 2 to ribosomes was also affected by extraction and could be reconstituted, although not to the same extent as the GTPase activity associated with elongation factor 2 in the presence of the ribosome. The alcohol extracts of the 80 S ribosome contained mostly phosphoproteins P1 and P2 which could be dephosphorylated and rephosphorylated in solution by alkaline phosphatase and protein kinase, respectively. Dephosphorylation of the P1/P2 mixture in the extracts caused a decrease in the ability of these proteins to reactivate the polyphenylalanine synthesis activity of the core ribosome. However, treatment of the dephosphorylated proteins with the catalytic subunit of 3':5'-cAMP-dependent protein kinase in the presence of ATP reactivated the proteins when compared to the activity of the native extracts. Rabbit antisera raised against the alcohol-extracted proteins were capable of impairing both the polyphenylalanine synthesis reaction and the elongation factor 2-dependent GTPase reaction in the intact ribosomes.

Animals↗
PubMed1983

Translational barrier in central region of encephalomyocarditis virus genome. Modulation by elongation factor 2 (eEF-2).

A fractionated cell-free system from Krebs-2 cells was prepared which contained ribosomes and a high-speed supernatant. When this system was programmed with encephalomyocarditis virus RNA, the synthesis of a precursor of capsid proteins, polypeptide preA, proceeded at a rate not very different from that observed in unfractionated extracts, whereas the synthesis of more distally encoded proteins, in particular polypeptide F, was greatly retarded, if not abolished. A protein was purified from the cytoplasmic extracts of Krebs-2 cells which greatly enhanced production of polypeptide F as well as other noncapsid proteins in the fractionated system. By several criteria, this protein was identified as eukaryotic elongation factor 2 (eEF-2). By using the ADP-ribosylation assay, it was found that the fractionated system contained about 15% of the amount of eEF-2 present in the unfractionated extracts. The results suggest that changes in the eEF-2 content may affect the elongation rate differently at different regions of the RNA template.

Cell-Free System↗
PubMed1990

Monoclonal antibodies against acidic phosphoproteins P0, P1, and P2 of eukaryotic ribosomes as functional probes.

Mice were immunized against ribosomal acidic proteins P1 and P2 from Artemia salina, and three kinds of monoclonal antibodies were isolated. One recognized P0 in addition to both P1 and P2 (anti-P). The other two recognized either P1 (anti-P1) or P2 (anti-P2) specifically and did not recognize P0. The anti-P antibody, but not anti-P1 or anti-P2, recognized a 22-amino acid peptide corresponding to the carboxyl-terminal sequence common to P1 and P2. This antibody, but not the others, inhibited poly(U)-directed polyphenylalanine synthesis. The anti-P1 bound to ribosomes but failed to inhibit polyphenylalanine synthesis: the anti-P2 did not bind to ribosomes at all. The anti-P and its Fab fragments inhibited the elongation step of protein synthesis, namely, the binding of elongation factors 1 alpha and 2 to ribosomes as well as their ribosome-coupled GTPase activities. Anti-P had little effect on the nonenzymatic phenylalanyl-tRNA binding to ribosomes and on peptidyltransferase activity. These results suggest the functional importance of the homologous carboxyl-terminal region of the three P proteins for the interaction of the ribosome with the two elongation factors. The epitope of anti-P1 must reside in a region of the protein which is not directly involved in its function.

Animals↗
PubMed1996

Effect of oxidative stress, produced by cumene hydroperoxide, on the various steps of protein synthesis. Modifications of elongation factor-2.

We have studied the effect of oxidative stress on protein synthesis in rat liver. Cumene hydroperoxide (CH) was used as an oxidant agent. The approach used was to determine the ribosomal state of aggregation and the time for assembly and release of polypeptide chains in the process of protein synthesis in rat liver in vivo. The results suggest that the elongation step is the most sensitive to CH treatment. The measurement of both carbonyl groups content and ADP-ribosylatable elongation factor 2 (EF-2), the main protein involved in the elongation step, indicates that under CH treatment EF-2 is oxidatively modified and a lower amount of active EF-2 is present. These results are corroborated by in vitro oxidation of EF-2 and could explain for the decline in the elongation step.

Adenosine Diphosphate Ribose↗
PubMed1995

Dephosphorylation of the phosphorylated elongation factor-2 in the livers of calorie-restricted and freely-fed rats during ageing.

Slowing down of the rate of protein synthesis during ageing is accompanied by alterations in the amounts and activities of elongation factors, eEF-1 and eEF-2. Since the activity of eEF-2 is regulated by phosphorylation, we have determined the changes in the activities of eEF-2-specific phosphorylating and dephosphorylating enzymes during ageing. Previously, we have reported an age-related increase in the activity of eEF-2 kinase (BBRC, 192, 1210, 1993). We have now compared the activities of a dephosphorylating enzyme protein phosphatase 2A (PP2A) in young and old liver extracts from freely-fed or calorie-restricted rats. The activity of PP2A remain unaltered during ageing. Furthermore, there was no change in the kinetics and extent of PP2A-dependent and PP2A-independent dephosphorylation of eEF-2 during ageing.

Aging↗
PubMed1990

Kinetic determination of the effects of ADP-ribosylation on the interaction of eukaryotic elongation factor 2 with ribosomes.

The effect of ADP-ribosylation on the function of eukaryotic elongation factor 2 (EF-2) was investigated by kinetic analysis of the EF-2-catalyzed hydrolysis of GTP in the presence of ribosomes and by direct determination of the affinity of the modified factor for the ribosome. Under conditions where the concentration of EF-2 was rate-limiting, the ADP-ribosylation reduced the maximum rate of GTP hydrolysis and the second order rate constant Kcat/Km by approximately 50%. A similar decrease in Kcat and Kcat/Km was observed when the concentration of ribosomes were kept rate-limiting. The affinity of EF-2 for the pretranslocation type of ribosomes was reduced by 2 orders of magnitude after ADP-ribosylation. No effect was observed in the interaction with the post-translocation type of ribosomes, the ribosomal conformation responsible for activation of the EF-2-dependent GTPase. We conclude that the ADP-ribosylation affects both the association of the modified factor with pretranslocation ribosomes and the hydrolytic capacity of the factor.

Adenosine Diphosphate Ribose↗
PubMed1998

Analysis of translation elongation factors from wheat during development and following heat shock.

Translational activity in plants undergoes rapid changes during developmental stages such as seed formation and germination, and during abiotic stresses such as heat shock, hypoxia and wounding. We examined the protein levels and isoelectric state of two components of the translation machinery, elongation factor (EF) 1 alpha and 2, to determine their roles in the regulation of translation. We found that the apparent protein levels of EF1 alpha increase relative to the EF2 levels which decline slightly during the development of the wheat seed. During germination, high levels of these factors are present in seedling tissues known to be actively engaged in translation; however, no differences in isoelectric state were observed during germination. As an example of abiotic stress, heat shock had little impact on the apparent levels of EF1 alpha or EF2 present in wheat leaves, nor were changes in the number or levels of isoforms observed.

Germination↗
PubMed1988

Evidence of a yeast proteinase specific for elongation factor 2.

Two proteinases active on elongation factor 2 have been found in yeast. The former hydrolyzes the factor producing a single ADP-ribosylatable fragment, whereas it does not produce any fragment when incubated with different proteins. The latter, less specific, is active in cleaving both EF-2 and other proteins giving rise to a noticeable number of fragments. Moreover, when native EF-2 is incubated with the most specific of the two proteinases, the amount of the ADP-ribosylatable fragment increases with time, while no fragments are evident when ADP-ribosylation of EF-2 comes before its incubation with the proteolytic enzyme. A possible regulatory role of this proteinase on EF-2 turnover is hypothesized.

Adenosine Diphosphate Ribose↗
PubMed1990

Functional properties of phosphorylated elongation factor 2.

The effect of phosphorylation on the functional activity of eukaryotic elongation factor 2 (eEF-2) was studied using a purified phosphorylated factor. The modified factor was unable to stimulate protein synthesis in an eEF-2-dependent rabbit reticulocyte lysate. The functional alteration was further analyzed by measuring the effects of phosphorylation on the ability of the factor to catalyse the ribosome-dependent hydrolysis of GTP. Kinetic analysis showed that both phosphorylated and unmodified factor was able to hydrolyse GTP with approximately the same maximum rate, indicating that the rate of nucleotide exchange was not impaired by the modification. However, the phosphorylated factor showed a marked reduction in the second-order rate constant, suggesting that the phosphorylation interfered with ribosome.eEF-2 complex formation by reducing the affinity of eEF-2 for the ribosome. This assumption was confirmed by direct measurements of the dissociation constants for the ribosomal complexes containing unmodified and phosphorylated eEF-2.

Animals↗
PubMed1983

Biosynthetic labeling of diphthamide in Saccharomyces cerevisiae.

Diphthamide, the post-translational amino acid derivative in the diphtheria toxin-modification site of protein synthesis elongation factor 2 (EF-2), has the proposed structure 2-[3-carboxyamido-3-(trimethylammonio)propyl]histidine (Van Ness, B. G., Howard, J. B., and Bodley, J. W. (1980) J. Biol. Chem. 255, 10710-10716). The identification of the biosynthetic precursors of diphthamide would provide a means of evaluating its proposed structure and determining if the amino acid occurs in proteins other than EF-2. Toward this end, yeast were grown on potential radiolabeled precursors and the resulting radiolabeled protein was hydrolyzed in acid. The acid hydrolysates were subjected to amino acid analysis with a program optimized to resolve diphthine, the acid hydrolysis product of diphthamide, from the common amino acids. Radiolabel from [beta-3H]histidine, [alpha-3H]methionine, and [Me-3H] methionine was found to be incorporated into diphthine in a molar ratio of 1:1:3 while that of [35S]methionine was not incorporated. These results are in accord with the proposed structure of diphthamide and suggest that in its biosynthesis the backbone and 3 methyl groups of methionine are added to a histidine residue in the peptide chain of EF-2. These labeling experiments show that diphthine (diphthamide) constitutes approximately 6 X 10(-6) mol fraction of the total amino acids in yeast protein hydrolysates. Estimates of the amount of diphthamide present in the diphtheria toxin-modification site of EF-2 indicate that it constitutes from 4.5 to 9 X 10(-6) mol fraction of the total amino acids in yeast protein. The present evidence suggests that diphthamide occurs only in EF-2.

Amino Acids↗
PubMed1985

Primary structure of elongation factor 2 around the site of ADP-ribosylation is highly conserved from archaebacteria to eukaryotes.

Elongation factor 2 (EF-2) from eukaryotes and archaebacteria can be ADP-ribosylated by diphtheria toxin (DT) [(1977) Annu. Rev. Biochem. 46, 69-94; (1980) Nature 287, 250-251]. The primary structure of the ADP-ribose accepting region in EFs from the archaebacteria Thermoplasma acidophilum Halobacterium cutirubrum and Methanococcus vannielli was determined in order to elucidate the degree of conservation compared with 4 previously established eukaryotic sequences [(1971) FEBS Lett. 103, 253-255]. Within a 9-residue sequence including the site of ADP-ribosylation 5 positions were found to be occupied by the same amino acid in all the archaebacterial and eukaryotic factors studied. There were more differences among the 3 archaebacterial sequences than among the 4 eukaryotic ones.

Adenosine Diphosphate Ribose↗