scholarly journals Inhibition by ricin of protein synthesis in vitro. Inhibition of the binding of elongation factor 2 and of adenosine diphosphate-ribosylated elongation factor 2 to ribosomes

1975 ◽  
Vol 146 (1) ◽  
pp. 127-131 ◽  
Author(s):  
L Montanaro ◽  
S Sperti ◽  
A Mattioli ◽  
G Testoni ◽  
F Stirpe
Keyword(s):  
Protein Synthesis ◽  
Binding Sites ◽  
Polypeptide Chain ◽  
Elongation Factor ◽  
Adenosine Diphosphate ◽  
Chain Elongation ◽  
Elongation Factor 2 ◽  
In Vitro Inhibition ◽  
Liver Ribosomes

The binding of EF2 (elongation factor 2) and of ADP-ribosyl-EF 2 to rat liver ribosomes is inhibited by ricin. This result suggests that the native enzyme and its ADP-ribose derivative have the same or closely related binding sites on the ribosome. The inhibition by ricin of the binding of EF 2 to ribosomes is consistent with the previous observation that ricin affects EF 2-catalysed translocation during polypeptide chain elongation.

scholarly journals Inhibition of protein synthesis in vitro by crotins and ricin. Effect on the steps of peptide chain elongation

1976 ◽  
Vol 156 (1) ◽  
pp. 7-13 ◽  
Author(s):  
S Sperti ◽  
L Montanaro ◽  
A Mattioli ◽  
G Testoni ◽  
F Stirpe
Keyword(s):  
Protein Synthesis ◽  
Rat Liver ◽  
Elongation Factor ◽  
Artemia Salina ◽  
Chain Elongation ◽  
Gtp Hydrolysis ◽  
Elongation Factor 2 ◽  
The Individual ◽  
Liver Ribosomes

The effects of crotin I and crotin II on the partial reactions of polypeptide chain elongation were investigated and compared with the known effects of ricin. Crotin II was a more powerful inhibitor than crotin I, but no qualitative differences between the two crotins were found. Rat liver ribosomes, preincubated with crotins and washed through sucrose gradients, remained inactive in protein synthesis. Among the individual steps of elongation, the peptidyltransferase reaction was unaffected by crotins, but some of the reactions that involve the interaction of elongation factors 1 and 2 with ribosomes were modified. A strong inhibition of the binding of elongation factor 2 to ribosomes and a stimulation of the elongation factor2-dependent GTP hydrolysis were observed; this indicates the formation of a very unstable elongation factor 2-GDP-ribosome complex, which, however, allows a single round of translocation to take place in the presence ofelongation factor 2 and added GTP. The elongation factor 1-dependent GTP hydrolysis was inhibited by crotins, whereas the enzymic binding of aminoacyl-tRNA, to both rat liver and Artemia salina ribosomes, was scarcely affected. In a protein-synthesizing system the inhibition by crotins and by ricin leads to a block of the nascent peptides on the ribosomal aminoacyl-tRNA site, an effect consistent with inhibition at the level of translocation. The mechanism of action of crotins appears to be very similar to that of ricin.

scholarly journals Regulation of protein-synthesis elongation-factor-2 kinase by cAMP in adipocytes

1998 ◽  
Vol 336 (3) ◽  
pp. 525-529 ◽  
Author(s):  
Tricia A. DIGGLE ◽  
Nicholas T. REDPATH ◽  
Kate J. HEESOM ◽  
Richard M. DENTON
Keyword(s):  
Protein Synthesis ◽  
Total Protein ◽  
Polypeptide Chain ◽  
Elongation Factor ◽  
Chain Elongation ◽  
Translation Elongation Factor ◽  
Translation Elongation ◽  
Eukaryotic Translation ◽  
Elongation Factor 2 ◽  
Fold Activation

Treatment of primary rat epididymal adipocytes or 3T3-L1 adipocytes with various agents which increase cAMP led to the phosphorylation of eukaryotic translation elongation factor-2 (eEF-2). The increase in eEF-2 phosphorylation was a consequence of the activation of eEF-2 kinase (eEF-2K), which is a Ca2+/calmodulin-dependent kinase. eEF-2K was shown to be essentially inactive at less than 0.1 µM free Ca2+ when measured in cell-free extracts. Treatment of adipocytes with isoproterenol induced Ca2+-independent eEF-2K activity, and an 8–10-fold activation of eEF-2K was observed at Ca2+ concentrations of less than 0.1 µM. Increased cAMP in 3T3-L1 adipocytes led to the inhibition of total protein synthesis and decreased the rate of polypeptide-chain elongation. We also show that the phosphorylation of eEF-2 and the activity of eEF-2K are insulin-regulated in adipocytes. These results demonstrate a novel mechanism for the control of protein synthesis by hormones which act by increasing cytoplasmic cAMP.

scholarly journals Translating Ribosomes Inhibit Poliovirus Negative-Strand RNA Synthesis

1999 ◽  
Vol 73 (12) ◽  
pp. 10104-10112 ◽  
Author(s):  
David J. Barton ◽  
B. Joan Morasco ◽  
James B. Flanegan
Keyword(s):  
Protein Synthesis ◽  
Polypeptide Chain ◽  
Rna Replication ◽  
Viral Rna ◽  
In Vitro Translation ◽  
Chain Elongation ◽  
Protein Synthesis Inhibitors ◽  
Positive Strand ◽  
Negative Strand

ABSTRACT Poliovirus has a single-stranded RNA genome of positive polarity that serves two essential functions at the start of the viral replication cycle in infected cells. First, it is translated to synthesize viral proteins and, second, it is copied by the viral polymerase to synthesize negative-strand RNA. We investigated these two reactions by using HeLa S10 in vitro translation-RNA replication reactions. Preinitiation RNA replication complexes were isolated from these reactions and then used to measure the sequential synthesis of negative- and positive-strand RNAs in the presence of different protein synthesis inhibitors. Puromycin was found to stimulate RNA replication overall. In contrast, RNA replication was inhibited by diphtheria toxin, cycloheximide, anisomycin, and ricin A chain. Dose-response experiments showed that precisely the same concentration of a specific drug was required to inhibit protein synthesis and to either stimulate or inhibit RNA replication. This suggested that the ability of these drugs to affect RNA replication was linked to their ability to alter the normal clearance of translating ribosomes from the input viral RNA. Consistent with this idea was the finding that the protein synthesis inhibitors had no measurable effect on positive-strand synthesis in normal RNA replication complexes. In marked contrast, negative-strand synthesis was stimulated by puromycin and was inhibited by cycloheximide. Puromycin causes polypeptide chain termination and induces the dissociation of polyribosomes from mRNA. Cycloheximide and other inhibitors of polypeptide chain elongation “freeze” ribosomes on mRNA and prevent the normal clearance of ribosomes from viral RNA templates. Therefore, it appears that the poliovirus polymerase was not able to dislodge translating ribosomes from viral RNA templates and mediate the switch from translation to negative-strand synthesis. Instead, the initiation of negative-strand synthesis appears to be coordinately regulated with the natural clearance of translating ribosomes to avoid the dilemma of ribosome-polymerase collisions.

scholarly journals Lack of inhibition by l-1-chloro-4-phenyl-3-toluene-p-sulphoamidobutan-2-one (l-1-tosylamido-2-phenylethyl chloromethyl ketone) of the formation of bacterial polypeptide chain initiation complex (Short Communication)

1973 ◽  
Vol 136 (2) ◽  
pp. 433-436 ◽  
Author(s):  
J. Jonák ◽  
B. F. C. Clark
Keyword(s):  
Short Communication ◽  
Polypeptide Chain ◽  
Protein Biosynthesis ◽  
Elongation Factor ◽  
Initiation Factor ◽  
Chain Elongation ◽  
Initiation Complex ◽  
Chloromethyl Ketone ◽  
Chain Initiation

The chymotrypsin inhibitor l-1-chloro-4-phenyl-3-toluene-p-sulphonamidobutan-2-one does not inhibit the function of the initiation factor during the formation of the polypeptide chain initiation complex in vitro. Since the inhibitor has been shown previously to inhibit polypeptide chain elongation by reacting with elongation factor EF-Tu, the inhibitor can be used to investigate the initiation and elongation steps of protein biosynthesis separately.

scholarly journals Effects of antibody to 5 S-RNA-binding protein on protein synthesis in Artemia salina ribosomes

1989 ◽  
Vol 259 (1) ◽  
pp. 277-281 ◽  
Author(s):  
N Kenmochi ◽  
Y Takahashi ◽  
N L Sato
Keyword(s):  
Protein Synthesis ◽  
Rna Binding ◽  
Rna Binding Protein ◽  
Elongation Factor ◽  
Artemia Salina ◽  
Globin Mrna ◽  
Ribosomal Subunits ◽  
Elongation Factor 2 ◽  
Subunit Interface

The effects of an affinity-purified polyclonal antibody to Artemia salina ribosomal protein L5 on protein synthesis in vitro were examined. The antibody interacted with 60 S subunits more strongly than with 80 S ribosomes, and inhibited reassociation of ribosomal subunits to some extent at 5 mM-Mg2+ but not at 10 mM. Polyphenylalanine synthesis in vitro at 10 mM-Mg2+ was significantly inhibited, especially when the antibody was first preincubated with 60 S subunits prior to the assay. The incorporation of amino acid directed by globin mRNA was inhibited only when the preincubation with 60 S subunits was carried out. On the other hand, no effect was detected on elongation factor 2- and 60 S subunit-dependent uncoupled GTPase activity. These results suggest that L5 is probably located at or near the subunit interface and may play an important role in protein synthesis.

scholarly journals Effect of elongation factor 2 and of adenosine diphosphate-ribosylated elongation factor 2 on translocation

1976 ◽  
Vol 156 (1) ◽  
pp. 15-23 ◽  
Author(s):  
L Montanaro ◽  
S Sperti ◽  
G Testoni ◽  
A Mattioli
Keyword(s):  
Rat Liver ◽  
Bond Formation ◽  
Elongation Factor ◽  
Adenosine Diphosphate ◽  
Peptide Bond ◽  
Native Enzyme ◽  
Peptide Bond Formation ◽  
Elongation Factor 2 ◽  
Hydrolysis Of ◽  
Liver Ribosomes

1. The effect of elongation factor 2 (EF 2) and of adenosine diphosphate-ribosylated elongation factor 2 (ADP-ribosyl-EF 2) on the shift of endogenous peptidyl-tRNA from the A to the P site of rat liver ribosomes (measured by the peptidyl-puromycin reaction) and on the release of deacylated tRNA (measured by aminoacylation) was investigated. 2. Limiting amounts of EF2, pre-bound or added to ribosomes, catalyse the shift of peptidyl-tRNA in the presence of GPT; when the enzyme is added in substrate amounts GMP-P(CH2)P [guanosine (beta, gamma-methylene)triphosphate] can partially replace GTP. ADP-ribosyl-EF 2 has no effect on the shift of peptidyl-tRNA when present in catalytic amounts, but becomes almost as effective as EF 2 when added in substrate amounts together with GTP; GMP-P(CH2)P cannot replace GTP. 3. The release of deacylated tRNA is induced only by substrate amounts of added EF 2 and also occurs in the absence of guanine nucleotides. In this reaction ADP-ribosyl-EF 2 is only 25% as effective as EF 2 in the absence of added nucleotide, but becomes 60-80% as effective in the presence of GTP or GMP-P(CH2)P. 4. The results obtained on protein-synthesizing systems are consistent with the hypothesis that ADP-ribosyl-EF 2 can operate a single round of translocation followed by binding of aminoacyl-tRNA and peptide-bond formation. 5. From the data obtained with the native enzyme it is concluded that the two moments of translocation require different conditions of interaction of EF 2 with ribosomes; it is suggested that the shift of peptidyl-tRNA is catalysed by EF 2 pre-bound to ribosomes, and that the release of tRNA is induced by a second molecule of interacting EF 2. The hydrolysis of GTP would be required for the release of pre-bound EF 2 from ribosomes. 5. The inhibition of the utilization of limiting amounts of EF 2 on ADP-ribosylation is very likely the consequence of a concomitant decrease in the rate of association and dissociation of the enzyme from ribosomes.

Phosphorylation of elongation factor-2 kinase on serine 499 by cAMP-dependent protein kinase induces Ca2+/calmodulin-independent activity

2001 ◽  
Vol 353 (3) ◽  
pp. 621-626 ◽  
Author(s):  
Tricia A. DIGGLE ◽  
Tatiana SUBKHANKULOVA ◽  
Kathryn S. LILLEY ◽  
Nita SHIKOTRA ◽  
Anne E. WILLIS ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Kinase ◽  
Elongation Factor ◽  
Luciferase Expression ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Elongation Factor 2 ◽  
Fold Reduction ◽  
Dependent Protein

Elongation factor-2 kinase (eEF-2K) negatively regulates mRNA translation via the phosphorylation and inactivation of elongation factor-2 (eEF-2). We have shown previously that purified eEF-2K can be phosphorylated in vitro by cAMP-dependent protein kinase (PKA) and that this induces significant Ca2+/calmodulin (CaM)-independent eEF-2K activity [Redpath and Proud (1993) Biochem. J. 293, 31–34]. Furthermore, elevation of cAMP levels in adipocytes also increases the level of Ca2+/CaM-independent eEF-2K activity to a similar extent, providing a mechanistic link between elevated cAMP and the inhibition of protein synthesis [Diggle, Redpath, Heesom and Denton (1998) Biochem. J. 336, 525–529]. Here we describe the expression of glutathione S-transferase (GST)-eEF-2K fusion protein and the identification of two serine residues that are phosphorylated by PKA in vitro. Endoproteinase Arg-C digestion of GST-eEF-2K produced two phosphopeptides that were separated by HPLC and sequenced. 32P Radioactivity release from these peptides indicated that the sites of phosphorylation were Ser-365 and Ser-499, both of which lie C-terminal to the catalytic domain. Mutation of these sites to non-phosphorylatable residues indicated that both sites need to be phosphorylated to induce Ca2+/CaM-independent eEF-2K activity in vitro. However, expression of Myc-tagged eEF-2K in HEK 293 cells, followed by treatment with chlorophenylthio-cAMP (CPT-cAMP), showed that Ser-499 phosphorylation alone induced Ca2+/CaM-independent eEF-2K activity in cells. Co-expression of wild-type eEF-2K with luciferase resulted in a 2–3-fold reduction in luciferase expression. Expression of eEF-2K S499D resulted in a 10-fold reduction in luciferase expression despite the fact that this mutant was expressed at very low levels. This indicates that eEF-2K S499D is constitutively active when expressed in cells, thus leading to the suppression of its own expression. Our data demonstrate an important role for the phosphorylation of Ser-499 in the activation of eEF-2K by PKA and the inhibition of protein synthesis.

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

1994 ◽  
Vol 266 (4) ◽  
pp. E628-E634 ◽  
Author(s):  
T. C. Vary ◽  
A. Nairn ◽  
C. J. Lynch
Keyword(s):  
Protein Synthesis ◽  
Elongation Factor ◽  
Diabetic Rats ◽  
Peptide Chain ◽  
Chain Elongation ◽  
Translational Efficiency ◽  
Ribosomal Subunits ◽  
Elongation Factor 2 ◽  
Cardiac Muscles

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.

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