Inhibition of initiation of protein synthesis by tosyl-L-lysyl chloromethyl ketone

1981 ◽  
Vol 59 (5) ◽  
pp. 321-327 ◽  
Author(s):  
Majambu Mbikay ◽  
Michael D. Garrick
Keyword(s):  
Protein Synthesis ◽  
Initiation Complex ◽  
Intact Cells ◽  
Chloromethyl Ketone ◽  
Chain Initiation ◽  
High Concentrations

Tosyl-L-lysyl chloromethyl ketone (TLCK) inhibits protein synthesis in intact cells and lysates. Its presence leads to polyribosome disaggregation. This inhibition is probably at the level of chain initiation because it does not slow poly(U)-dependent elongation of poly(F). This drug activates an inhibitor similar to the heme-controlled repressor in the postribosomal supernatant. High concentrations of ATP and GTP affect inhibitions of translation by the heme-controlled repressor and TLCK in a similar fashion. The latter also interferes with formation of the 40S initiation complex. Its action on translation can be prevented by preliminary incubation with thiols.

Hypoxanthine-xanthine oxidase-related defect in polypeptide chain initiation by endothelium

1989 ◽  
Vol 66 (1) ◽  
pp. 450-457 ◽  
Author(s):  
L. Jornot ◽  
A. F. Junod
Keyword(s):  
Protein Synthesis ◽  
Xanthine Oxidase ◽  
Rna Synthesis ◽  
Polypeptide Chain ◽  
Total Proteins ◽  
Initiation Complex ◽  
Chain Initiation ◽  
Oxidase System ◽  
Initiation Factors ◽  
Related Defect

After exposure to O2 intermediates generated by the hypoxanthine-xanthine oxidase system (HX-XO), the rate of [3H]phenylalanine incorporation into total proteins in cultured endothelial cells was markedly reduced. This reduction, which was prevented by catalase, could not be explained by 1) changes in amino acid pools, 2) increased rate of degradation of newly synthesized proteins, 3) impaired poly(A)+ RNA synthesis and efficiency, 4) decreased rate of amino acylation. On the other hand, the increase in the monoribosome-to-polyribosome ratio suggested that translation was affected at the level of chain initiation. Further analysis indicated that 40S initiation complex formation was normal, whereas the assembly of 80S initiation complex was inhibited. Results from reconstitution experiments showed that both normal and treated ribosomes could support normal protein synthesis in the presence of normal initiation factors (IFs). In contrast, IFs from HX-XO lysates did not support normal protein synthesis with ribosomes from either source. Thus, the effect of XO treatment on protein synthesis appears to be an initiation defect related to decreased IF activity and/or availability.

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 osmolarity, ions and compatible osmolytes on cell-free protein synthesis

2003 ◽  
Vol 369 (2) ◽  
pp. 369-374 ◽  
Author(s):  
Maurizio BRIGOTTI ◽  
Pier Giorgio PETRONINI ◽  
Domenica CARNICELLI ◽  
Roberta R. ALFIERI ◽  
Mara A. BONELLI ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Gradient Centrifugation ◽  
Inorganic Ions ◽  
Globin Mrna ◽  
Initiation Complex ◽  
Free Protein ◽  
Reticulocyte Lysate ◽  
Compatible Osmolytes ◽  
High Concentrations ◽  
Cell Free Protein Synthesis

To mimic what might happen in cells exposed to hypertonicity, the effects of increased osmolarity and ionic strength on cell-free protein synthesis have been examined. Translation of globin mRNA by rabbit reticulocyte lysate decreased by 30—60% when osmolality was increased from 0.35 to 0.53osmol/kg of water by the addition of NaCl, KCl, CH3CO2Na or CH3CO2K. In contrast, equivalent additions of the compatible osmolytes betaine or myo-inositol caused a 40—50% increase in the rate of translation, whereas amino acids (50—135mM) that are transported via system A had no significant effect. Addition of 75mM KCl caused a dramatic fall in the amount of the 43S pre-initiation complex, whereas it was totally preserved when osmolarity was similarly increased by the addition of 150mM betaine. The formation of a non-enzymic initiation complex between rabbit [3H]Phe-tRNA, poly(U) and the 80S ribosomes was unaffected by the addition of 75mM NaCl or KCl, but translation of the complex decreased by 70%. Density-gradient centrifugation of reticulocyte extracts translating endogenous mRNA revealed that addition of 150mM betaine had no effect, whereas addition of 75mM KCl caused a marked decrease in the polysome peak, concomitant with an increase in the proportion of 80S ribosomes and ribosomal subunits, even when elongation was inhibited with fragment A of diphtheria toxin. These results are consistent with the notion that both initiation and elongation are inhibited by unusually high concentrations of inorganic ions, but not by the compatible osmolytes betaine or myo-inositol.

scholarly journals Hemin reversal of benzene-induced inhibition of reticulocyte protein synthesis

Blood ◽  
1976 ◽  
Vol 47 (1) ◽  
pp. 145-154 ◽  
Author(s):  
FJ Forte ◽  
HS Cohen ◽  
J Rosman ◽  
ML Freedman
Keyword(s):  
Protein Synthesis ◽  
Sickle Cell ◽  
Clinical Implications ◽  
Intact Cells ◽  
Free Protein ◽  
Translational Repressor ◽  
Heme Synthesis ◽  
Dependent Site ◽  
Cell Free Protein Synthesis ◽  
Rabbit Reticulocytes

Abstract Benzene (0.056–0.113 M) rapidly and reversibly inhibited protein synthesis in anucleate human sickle cell and rabbit reticulocytes. Hemin (50 muM) both prevented and reversed this effect of benzene. The inhibition in rabbit reticulocytes was accompanied by a conversion of polyribosomal disaggregation required ribosomal movement along mRNA and was also prevented and reversed by 50 muM hemin. Benzene was also shown to inhibit heme synthesis in rabbit reticulocytes while neither ATP nor GSH levels were altered. A translational repressor (HCR) of reticulocyte cell-free protein synthesis was isolated from intact cells incubated with benzene, while no significant amount of HCR was found in cells incubated with both benzene and hemin. These results indicated that benzene inhibits translation at the heme-dependent site of initiation. The clinical implications of these experiments remain to be elucidated.

Insulin-like growth factor I accelerates protein synthesis in skeletal muscle during sepsis

1995 ◽  
Vol 269 (5) ◽  
pp. E977-E981 ◽  
Author(s):  
C. V. Jurasinski ◽  
T. C. Vary
Keyword(s):  
Protein Synthesis ◽  
Growth Factor ◽  
Peptide Chain ◽  
Translational Efficiency ◽  
Insulin Like Growth Factor ◽  
Recombinant Human Insulin ◽  
Chain Initiation ◽  
Factor I ◽  
Igf I ◽  
Growth Factor I

Sepsis causes an inhibition of protein synthesis in gastrocnemius that is resistant to the anabolic effects of insulin. The purpose of the present studies was to investigate the effect of recombinant human insulin-like growth factor I (IGF-I) on protein synthesis during a 30-min perfusion of the isolated rat hindlimb from septic rats. Inclusion of IGF-I (1 or 10 nM) in the perfusate stimulated protein synthesis in gastrocnemius of septic rats 2.5-fold and restored rates of protein synthesis to those observed in control rats. The stimulation of protein synthesis did not result from an increase in the RNA content but was correlated with a 2.5-fold increase in the translational efficiency. The enhanced translational efficiency was accompanied by a 33 and 55% decrease in the abundance of free 40S and 60S ribosomal subunits, respectively, indicating that IGF-I accelerated peptide-chain initiation relative to elongation/termination. These studies provide evidence that IGF-I can accelerate protein synthesis in gastrocnemius during chronic sepsis by reversing the sepsis-induced inhibition of peptide-chain initiation.

scholarly journals The Oxazolidinone Linezolid Inhibits Initiation of Protein Synthesis in Bacteria

1998 ◽  
Vol 42 (12) ◽  
pp. 3251-3255 ◽  
Author(s):  
Steve M. Swaney ◽  
Hiroyuki Aoki ◽  
M. Clelia Ganoza ◽  
Dean L. Shinabarger
Keyword(s):  
Protein Synthesis ◽  
Complex Formation ◽  
Antimicrobial Agents ◽  
Ribosomal Subunit ◽  
Multidrug Resistant ◽  
Binary Complex ◽  
Initiation Complex ◽  
Ribosomal Subunits ◽  
Bacterial Translation

ABSTRACT The oxazolidinones represent a new class of antimicrobial agents which are active against multidrug-resistant staphylococci, streptococci, and enterococci. Previous studies have demonstrated that oxazolidinones inhibit bacterial translation in vitro at a step preceding elongation but after the charging ofN-formylmethionine to the initiator tRNA molecule. The event that occurs between these two steps is termed initiation. Initiation of protein synthesis requires the simultaneous presence of N-formylmethionine-tRNA, the 30S ribosomal subunit, mRNA, GTP, and the initiation factors IF1, IF2, and IF3. An initiation complex assay measuring the binding of [3H]N-formylmethionyl-tRNA to ribosomes in response to mRNA binding was used in order to investigate the mechanism of oxazolidinone action. Linezolid inhibited initiation complex formation with either the 30S or the 70S ribosomal subunits fromEscherichia coli. In addition, complex formation withStaphylococcus aureus 70S tight-couple ribosomes was inhibited by linezolid. Linezolid did not inhibit the independent binding of either mRNA or N-formylmethionyl-tRNA toE. coli 30S ribosomal subunits, nor did it prevent the formation of the IF2–N-formylmethionyl-tRNA binary complex. The results demonstrate that oxazolidinones inhibit the formation of the initiation complex in bacterial translation systems by preventing formation of theN-formylmethionyl-tRNA–ribosome–mRNA ternary complex.

Changes in protein metabolism of ovine primary muscle cultures on treatment with growth hormone, insulin, insulin-like growth factor I or epidermal growth factor

1987 ◽  
Vol 112 (1) ◽  
pp. 87-96 ◽  
Author(s):  
J. M. M. Harper ◽  
J. B. Soar ◽  
P. J. Buttery
Keyword(s):  
Growth Hormone ◽  
Protein Synthesis ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Insulin Like Growth Factor ◽  
Factor I ◽  
Muscle Cultures ◽  
Epidermal Growth ◽  
Growth Factor I ◽  
High Concentrations

ABSTRACT Methods for the primary culture of muscle cells from fetal sheep were developed which gave high yields of cells. Myoblasts were grown in vitro, and allowed to fuse to form contractile multinucleate myotubes; these could be maintained in a good condition for at least 2 weeks. Protein turnover in these differentiated cultures was examined for sensitivity to each of four potentially anabolic peptide hormones and growth factors: insulin, insulin-like growth factor I (somatomedin C), epidermal growth factor and growth hormone. Insulin was found to have no effect except at high concentrations (1 μmol/l), compatible with its role as a somatomedin analogue. Insulin-like growth factor I was active at lower levels (1 nmol/l) but the cultures were not as responsive to it as were primary rat muscle cultures or differentiated L6 cells, which were tested in similar experiments. The maximum stimulation of protein synthesis observed with the ruminant system was only 16%. Epidermal growth factor was highly anabolic for primary cultures from sheep muscle, and the cells were very sensitive to it, half-maximal stimulation of protein synthesis being seen with concentrations as low as 20 pmol/l. No effects of bovine growth hormone were seen in the ovine system. However, an inhibition of protein breakdown was found with high concentrations (0·1 μmol/l) in the L6 rat myoblast cell line. It was found that the culture conditions used could affect the observed responses of protein synthesis and degradation, despite withdrawal of serum from the incubation media 22 h before testing. J. Endocr. (1987) 112, 87–96

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